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Michigan 3 rate
University

ABSTRACT

SELENIUM AND/OR VITAMIN E SUPPLEMENTATION OF
PRACTICAL SWINE DIETS

BY

Albert Wayne Groce

Three feeding trials with growing-finishing swine and
six selenium balance studies with young pigs were conducted
over a two year period. The purposes of the feeding trials
were to determine if the addition of sodium selenite or vita-
min E to practical corn-soybean meal diets would be effica-
cious against selenium and/or vitamin E deficiency in pigs
and if such a practice would produce harmful levels of
selenium in carcasses and edible organs of swine fed
selenite—supplemented diets. The feeding trials were con—
ducted with Yorkshire, Hampshire and crossbred pigs main—
tained in a modern confinement management situation. Dams
of these pigs were also maintained in confinement during
gestation and lactation. The balance trials were conducted
utilizing the same levels of added dietary selenium as were
used in the feeding trials. The effect of added dietary
vitamin E on selenium balance was evaluated in these balance
studies as were differences between the manner in which
selenite selenium and naturally-occurring selenium are uti-

lized by the young pig. The effect of an unstable selenite

Albert Wayne Groce
premix on selenium balance was also tested. Young pigs of
the same breeds and history as those in the feeding trials
were housed in stainless steel metabolism cages while re-
ceiving a near ad libitum portion of diet in three meal-
feedings a day. Since all diets in both the feeding and
balance studies were based on corn of local origin an auxil-
iary survey of the selenium levels of Michigan-grown corn
was included. In this survey, the selenium levels of 17
samples of Michigan 275-2X hybrid corn grown in 13 counties
in Michigan and 20 varieties of hybrid corn grown at one
location were determined. The intent of this survey was to
determine if there were significant location, soil pH, vari-
etal or yield effects on the selenium content of corn in
Michigan.

In feeding trials it was determined that the supplemen—
tation of selenium-deficient practical corn—soybean meal
diets with 0.1 ppm selenium from sodium selenite or 11 IU
vitamin E (as d-q-tocopheryl acetate) per kilogram of diet
prevented death losses, gross pathology and histopathological
lesions of nutritional muscular dystrophy or dietary hepatic
necrosis in growing-finishing swine. The period when death
losses were observed was in the early post-weaning and early
feeding segment of the life cycle. Pretreatment of the dams
with added dietary vitamin E did not seem to affect the sus-
ceptibility of their offspring to the deficiency although it
did cause minor alterations in SGOT activity of the pigs at

weaning. After 10 weeks of selenium and/or vitamin E

Albert Wayne Groce
supplementation, added vitamin E had depressed serum selenium
levels on the basal diet while increasing them on the diets
with added inorganic selenium. In a second study supple-
mental vitamin E depressed erythrocyte selenium levels with-
out showing any effect on serum or whole blood selenium
concentrations.

Selenium concentrations in skeletal muscle, myocardium,
liver and kidney were increased over levels observed on the
deficient basals by supplementation of the basal corn-soy-
bean meal diets with sodium selenite. The increase in
tissue selenium noted with graded levels of added selenite
was not directly proportional to the increments of added
selenium. Even the highest levels of tissue selenium that
resulted from feeding 0.05 to 1.0 ppm added selenium as
sodium selenite until slaughter were well below those re—
ported for the same tissues from swine receiving selenium—
adequate diets containing only naturally-occurring selenium.
The levels of tissue selenium in selenite-supplemented swine
had declined significantly 60 days after supplemental
selenite selenium was removed from the diet. Supplemental
vitamin E increased kidney selenium levels in one study, but
had no apparent effect on the selenium levels of other
tissues analyzed.

The data from the balance trials paralleled observations
of tissue selenium levels in the feeding trials. Supple-
mental selenite selenium was absorbed and retained quite

well when added to selenium-deficient practical corn—soybean

Albert Wayne Groce
meal diets at levels up to 0.1 ppm added selenium. Levels
of added selenium above 0.1 ppm (0.2 and 0.5 ppm) were
readily absorbed and then excreted in greater proportions in
the urine to give absolute selenium retentions very similar
to those observed on the 0.1 ppm added selenite selenium
diets. Serum selenium levels plateaued at about the same
level of supplemental dietary selenite selenium indicating
the possible existence of tissue and serum thresholds for
selenium of inorganic origin in the young pig. Erythrocyte
selenium levels tended to increase proportionately more than
serum selenium. This could have been due to the incorpora-
tion of seleno-amino acids into new red cells as there was
a definite lag followed by a marked increase in erythrocyte
selenium concentration in the one 35 day balance study in
which this effect was observed.

Over short-term balances, added dietary vitamin E de-
creased the retention of selenite selenium by increasing its
urinary excretion, an effect that may have been related to
its effect on serum selenium levels as discussed for the
feeding trial above. Additions of dietary vitamin E in-
creased the retention of selenium from seleniferous corn
primarily by decreasing the fecal excretion of selenium.
Urinary excretion of selenium was higher for selenite
selenium than for seleniferous corn. The higher serum
selenium levels on selenite selenium correlated well with
this finding. Fecal selenium losses were greater on seleni-

ferous corn than on selenite indicating poorer absorption or

Albert Wayne Groce
greater resecretion of selenium into the gastrointestinal
tract. The changes occurring in an unstable selenite
selenium premix had no consistent effect on selenium balance
in the young pig.

It seemed that the pig retains and stores physiological
levels of inorganic selenium in proportion to its require-
ments. Once the physiological stores are filled, the body
tends to excrete any excesses as long as excretory mechan-
isms are not overwhelmed by toxic levels of this nutrient.
It is fairly obvious that the resultant tissue levels are
affected by the form in which the dietary selenium is pro-
vided to the animal. This probably is due to selenium
serving both metabolic and structural roles in the tissues
of the body.

Michigan 275-2X corn was found to contain from 0.013 to
0.089 ppm selenium (on a dry basis) at the different loca-
tions sampled. Selenium content of these corn samples was
significantly (P < 0.01) correlated with soil pH (r = 0.73)
and the correlation of yield with selenium level (r = -0.475)
was very nearly significant (P < 0.05). The geographic dis-
tribution of selenium levels agreed reasonably well with
data from earlier surveys of the selenium content of forages
in the area. Even though the selenium levels of the 20
varieties of corn grown at one location were very low, there
was some evidence for varietal differences in selenium con-
tent since these samples ranged from 0.007 to 0.024 ppm

selenium on a dry basis. There was some indication that

Albert Wayne Groce
soil type and stress factors may play a role in determining
the selenium content of corn. If corn-soybean meal diets
for swine were compounded, using soybean meal from the mid-
western or eastern United States and any of the Michigan-
grown corns analyzed in this survey, diets very deficient to

borderline in selenium content would result.

SELENIUM AND/OR VITAMIN E SUPPLEMENTATION OF

PRACTICAL SWINE DIETS

BY

Albert Wayne Groce

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry
and

Institute of Nutrition

1972

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to Dr. D. E. Ullrey and Dr. E. R. Miller for their guidance
during the conduction of this research and for their con—
structive reviews of this manuscript. Appreciation is also
expressed for the helpful guidance and interest of my other
graduate committee members Dr. K. K. Keahey and Dr. R. W.
Luecke. A Special note of thanks is also extended to an
unofficial member of my committee, Dr. D. J. Ellis, for his
assistance in many facets of these studies and for his in-
spiration during my veterinary medical studies.

The assistance of the staff of the Veterinary Diagnos-
tic Laboratory was invaluable during these research projects.
The services of Mr. Roger Hale and his group at the

Swine Research Center with regard to animal care and the
mixing of experimental rations is gratefully acknowledged.

Appreciation is extended to fellow graduate students,
laboratory personnel and departmental secretaries for their
assistance during my stay at Michigan State University.
Special thanks are due Mrs. Rosemary Covert for her assist-
ance with laboratory procedures and Mrs. Dorothy Schmidt for
the skillful preparation of the histological specimens for
evaluation. The assistance of Dr. Pao Ku in performing some
of the selenium assays was appreciated.

ii

The financial support of the Department of Animal
Husbandry throughout the graduate program was much ap-
preciated. The author wishes to especially thank Dr. J. A.
Hoefer for having the faith to initially offer this support.

The cooperation of Dr. John P. Newman and the admis-
sions committee of the College of Veterinary Medicine in
allowing me to pursue this dual program of study was es-
sential to its completion and is gratefully acknowledged.

The assistance of selenium researchers and selenium
analysts at other institutions in fathoming some of the
mysteries hidden so well between the lines of some of the
literature is gratefully acknowledged.

Special thanks are due Mrs. Ann Brown and Mrs. Deborah
Wheaton for their assistance in typing, organizing and
printing this manuscript.

The encouragement and tolerance of my family during
these many years of college work have been most helpful.

Last, but certainly not least, the author is indebted
to his wife, Karen, for her support and uncomplaining sacri-
fice during the past five years. Her love and encouragement
have made these years of study tolerable and even very

rewarding at times.

iii

Albert Wayne Groce
Candidate for the degree of
Doctor of Philosophy

DISSERTATION:

Selenium and/or Vitamin E Supplementation of Practical
Swine Diets

OUTLINE OF STUDIES:

Main Area: Animal Nutrition, Department of Animal
Husbandry and Institute of Nutrition

Minor Areas: Biochemistry and Pathology

BIOGRAPHICAL ITEMS:

Born: May 5, 1944, Monticello, Arkansas

Undergraduate Studies: Arkansas A & M College,

1962-1964

University of Arkansas,
1964-1966

Graduate Studies: University of Arkansas, 1966—1967

Michigan State University,
1967-1972

Experience: Graduate Research Assistant, 1968-1972

MEMBER: American Society of Animal Science

American Veterinary Medical Association

iv

TABLE OF CONTENTS

I. INTRODUCTION . . . . . . . . . . . . . . . . .

II. REVIEW OF LITERATURE . . . . . . . . . . . . .

Selenium and/or Vitamin E Deficiency in Farm
Animals . . . . . . . . . . . . . . . . . .

Experimental diet—induced deficiencies . .
Poultry . . . . . . . . . . . . . . .
Cattle and sheep . . . . . . . . . .
Swine . . . . . . . . . . . . . . . .

Occurrence of deficiencies under practical
conditions . . . . . . . . . . . . . . .

Cattle and Sheep . . . . . . . . . .
SWine o o o o O o o o o o o o o o o 0

Pathology of selenium and/or vitamin E de-
ficiency in swine . . . . . . . . . . .

Gross and microscopic pathological
changes . . . . . . . . . . . . . .

Clinical pathology of selenium and/or
vitamin E deficiency in swine . . .

Page

11
15

21
21
21

25

25

27

Tissue and blood selenium levels in healthy,

deficient and supplemented animals . . .
Selenium Balance in Animals. . . . . . . . . .

Absorption and tissue distribution of
selenium . . . . . . . . . . . . . . . .

Excretion and secretion of selenium . . .

Selenium Content of Crops . . . . . . . . . .

Factors affecting selenium content of crops

Geographical considerations . . . . .
Species differences . . . . . . . . .
Soil and climatic effects . . . . . .

Selenium levels of common feedstuffs . . .

30
36

36
46
53

53
55
58
60

TABLE OF CONTENTS (Cont.)

III.

IV.

Introduction .

Experiment
Experiment
Experiment
Experiment
EXperiment
Experiment
Experiment
Experiment
Experiment

EXperiment

Hemoglobin

Hematocrit

Total serum protein

1
2
3
4
5
6
7
8

9
10

Experimental Details

Laboratory Procedures

EXPERIMENTAL PROCEDURE

General Conduct of Experiments

Serum glutamic—oxaloacetic

(SGOT) determinations

Electrophoresis of serum proteins

Histology

transaminase

Sample preparation for selenium analyses

Dry matter

Experiment
Experiment
Experiment
EXperiment
Experiment
Experiment

Experiment

determination

1

2
3
4
5
b
7

Statistical Analyses

RESULTS AND DISCUSSION

vi

Page
63
63
64
69
69
70
74
76
76
77
77
78
80
81
83
83
83
83

83
84
84
85
86
88

90
90
93
103
113
115
115
118

TABLE OF CONTENTS (Cont.)
Page

Experiment 8 . . . . . . . . . . . . . . . 124
EXperiment 9 . . . . . . . . . . . . . . . 124
Conclusions from Swine Studies . . . . . . . . 127
Experiment 10 . . . . . . . . . . . . . . 130

Conclusions from Corn Study . . . . . . . . . 137
V. BIBLIOGRAPHY . . . . . . . . . . . . . . . . . 141
VI. APPENDIX I: Fluorometric Selenium Analyses . 159

VII. APPENDIX II: Clinton County Corn Varieties . 172

vii

LIST OF TABLES

Table Page
1. COMPOSITION OF DIETS (EXPERIMENTS 1, 2 and 3) 67
2. DIETARY TREATMENTS (EXPERIMENTS 2 AND 3) . . . 72
3. PERFORMANCE AND HEMATOLOGICAL PARAMETERS

(EXPERIMENT 1) . . . . . . . . . . . . . . . 91
4. FINAL TISSUE SELENIUM LEVELS (EXPERIMENT 1) . 92

5. INITIAL HEMATOLOGICAL PARAMETERS. EFFECT OF
SEX AND sow PRETREATMENT (EXPERIMENT 2) . . 94

6. EFFECT OF SELENIUM AND/OR VITAMIN E SUPPLEMENTA-
TION ON 10-WEEK SERUM SELENIUM LEVELS
(EXPERIMENT 2) . . . . . . . . . . . . . . . 96

7. FINAL HEMATOLOGICAL PARAMETERS AND SELENIUM CON-
TENT OF LONGISSIMUS MUSCLE (EXPERIMENT 2) . 98

 

8. SUMMARY OF PATHOLOGICAL FINDINGS (EXPERIMENT 2)102

9. EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM
WITHDRAWAL ON BLOOD PARAMETERS (EXPERIMENT 3)104

10. EFFECT OF SUPPLEMENTARY DIETARY SELENIUM ON
BLOOD PARAMETERS (EXPERIMENT 3) . . . . . . . 104

11. EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM
WITHDRAWAL ON TISSUE SELENIUM (EXPERIMENT 3) 106

12. EFFECT OF SUPPLEMENTARY DIETARY SELENIUM ON
TISSUE SELENIUM (EXPERIMENT 3) . . . . . . 106

13. EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM
WITHDRAWAL ON TISSUE DRY MATTER CONTENT
(EXPERIMENT 3) . . . . . . . . . . . . . . 108

14. DAILY SELENIUM BALANCE (EXPERIMENT 4) .. . . 114

15. EFFECT OF SUPPLEMENTARY DIETARY SELENIUM AND

VITAMIN E ON SELENIUM BALANCE IN THE YOUNG
PIG (EXPERIMENTS 5 AND 6) . . . . . . . . 116

viii

LIST OF TABLES (Cont.)

Table
16. A COMPARISON OF THE EFFECT OF NATURAL gs,
SELENITE SELENIUM ON SELENIUM BALANCE AND
BLOOD SELENIUM IN THE YOUNG PIG (EXPERIMENT 7)
17, EFFECT OF UNSTABLE OR FRESH SODIUM SELENITE PRE-
MIXES AND VITAMIN E ON SELENIUM BALANCE IN THE
YOUNG PIG (EXPERIMENT 8) . . . . . . . . . . .
18. EFFECT OF UNSTABLE OR FRESH SODIUM SELENITE PRE-
MIXES AND VITAMIN E ON SELENIUM BALANCE IN THE
YOUNG PIG (EXPERIMENT 9) . . . . . . . . . .
19. SELENIUM CONTENT OF MICHIGAN 275-2X CORN IN 13
MICHIGAN COUNTIES (EXPERIMENT 10) . . . . .
20. SELENIUM CONTENT OF 20 VARIETIES OF CORN GROWN
IN CLINTON COUNTY, MICHIGAN (EXPERIMENT 10).
APPENDIX
1. CLINTON COUNTY CORN VARIETIES . . . . . . . .

ix

Page

119

125

126

133

139

172

LIS T OF FIGURES

Figure Page
1. Metabolic pathways of selenium in monogastrics 132

2. Selenium content (ppm dry) of Michigan 275-2X
corn . . . . . . . . . . . . . . . . . . . . 138

INTRODUCTION

In 1957 Schwarz reported that selenium was an integral
part of the Factor 3 that protected against dietary necrotic
liver degeneration occurring in rats fed Torula yeast diets
that he had previously described in 1951. Sodium selenite
in the feed or by injection also prevented the condition.
Prior to this time most of the research on selenium in nutri-
tion had been concerned with its toxic effects on the animal.
It had been recognized that selenium was the toxic factor
that accumulated in certain plants of the north central
United States and extensive efforts had been expended to
reduce the economic losses to livestock producers in the
affected areas.

Obel (1953) described a condition she termed hepatosis

 

diaetetica in young swine and suggested that a deficiency

 

of Schwarz's Factor 3 might be involved in this disease.
Her description of the pathology of the disease was excel-
lent and depicted very well the condition recognized today
as selenium and/or vitamin E deficiency in swine in the
United States. In her review of the literature She con-
cluded that this condition was probably described in Europe
as early as 1887. Most of the affected animals had been on
diets containing considerable levels of fish oils.

Schwarz and Foltz (1958), Schwarz (1961), and Schwarz

and Fredga (1969) reported extensive work concerning the

2

Factor 3 activity of selenium compounds, both inorganic and
organic. Isolated Factor 3 was more potent than any of the
inorganic compounds tested. Sodium selenite was quite
active while elemental selenium was inactive. Organic
selenium compounds varied widely in their bio-potencies
with some being inactive and others more active than
selenite selenium against dietary liver necrosis in rats.
The fact that vitamin E and synthetic antioxidants
protect animals against some of the lesions associated with
"selenium deficiency" has confused matters for many years.
Most of the experimental work in this area has involved the
use of Torula yeast diets which were low in selenium, but
were also deficient in sulfur amino acids and high in poly-
unsaturated fatty acids. Depending on the starting material
used, the Torula yeast diets may or may not be very low in
a-tocopherol content. The low level of sulfur amino acids
and the high levels of unsaturated fatty acids are recog-
nized as antagonizing factors in selenium and/or vitamin E
deficiency. The definitive work of Thompson and Scott
(1970), in which chicks receiving crystalline amino acid
diets containing high levels of vitamin E but less than
0.02 ppm selenium required selenium supplementation for
proper pancreatic function, established that selenium is
indeed an essential trace element. The observations of
McCoy and Weswig (1969) thatratscm aTtmula yeast diet with
adequate added vitamin E grew and reproduced normally,
while their offspring were hairless unless the diet was

supplemented with selenium added further evidence for a

3

function of selenium not related to vitamin E. Some re-
searchers have indeed begun to question the status of a—
tocopherol as a vitamin rather than the status of selenium
as an essential trace element. Diplock and co-workers have
extensively studied over several years the interrelation-
ship between selenium and vitamin E. They recently (1971)
proposed and presented evidence for the hypothesis that the
active form of selenium in the body may be selenide which
forms part of the active Center of an uncharacterized Class
of catalytically active non-heme-iron proteins that are pro-
tected from oxidation in yiyg'by vitamin E. In other words
many researchers now think that vitamin B may only serve
as a non-specific antioxidant in the body while selenium
may have a specific essential metabolic role. Other signi-
ficant studies have recently suggested a relationship of
selenium to glutathione(GSH) peroxidase in rats (Rotruck
-_E._l., 1972) and an alleviation of the toxic effects of
mercury in tuna for quail by dietary additions of inorganic
or naturally-occurring selenium (Ganther _E'al., 1972).
Although a rather high incidence of naturally—occur-
ring selenium and/or vitamin E deficiency disease had been
recognized in certain areas of the United States for many

years in ruminant farm animals,it was not until the late

1960's that field cases of selenium and/or vitamin E

deficiency were recorded in swine in this country. Al-
though several researchers in this country had produced

the condition in swine by fairly severe dietary manipula-

4

tion, Michel, Whitehair and Keahey (1969) were the first to
report the occurrence of the disease in commercial swine
herds in Michigan on practical diets. The condition prob-
ably existed prior to their first recorded cases (1967)
since a considerable differential diagnostic problem iS in-
volved as outlined by Trapp _E _l. (1970).

The studies presented in this dissertation were under-
taken to define more clearly the problem of selenium and/or
vitamin E deficiency in swine receiving practical corn-
soybean meal diets and to establish criteria for the ration—
al supplementation of such rations with selenium and/or
vitamin E. Our interest in selenium was heightened by the
fact that vitamin E supplementation of swine rations is
relatively expensive and supplementation of rations, on
some problem farms, with very high levels of vitamin E had
failed to prevent losses due to selenium and/or vitamin E
deficiency. The studies on tissue selenium levels and
selenium balance studies were conducted to elucidate how
natural and supplemental selenium are utilized by swine and
to establish if harmful levels of selenium in the tissues
would result from such supplementation. Since some work
reported (Nelson, Fitzhugh and Calvery, 1943; Schroeder
and Mitchener, 1971) had led the Food and Drug Administra-
tion to class selenium as a carcinogen, thus prohibiting
its addition to the diets of food-producing animals, the
tissue selenium level information was of prime interest.

An auxiliary survey of the selenium levels of corn grown in

Michigan is also included.

REVI EW OF LITERATURE
Selenium and/or Vitamin E Deficiency in Farm Animals

Experimental diet-induced deficiencies

 

Induced selenium and/or vitamin E deficiency is herein
defined as the deficiency as it occurs in animals fed semi-
purified diets and diets which contain abnormally high
levels of highly unsaturated fats or rancid fats. The
classic low selenium-low vitamin E diet employs Torula yeast
as the protein source. This material is deficient in sulfur
amino acids and very high in unsaturated fatty acids, how-
ever, some batches of Torula yeast are fairly rich in @-

tocopherol.

Poultry

Shortly after Schwarz and Foltz (1957) reported that
selenium was an integral part of Factor 3 and that inorganic
salts of selenium would prevent liver necrosis in rats, two
groups (Patterson, Mflstrey and Stokstad, 1957; Schwarz 33 31.,
1957) reported that selenium as well as vitamin E additions
to Torula yeast diets would prevent exudative diathesis in
chicks. Levels of added selenium as low as 0.1 ppm in the
form of sodium selenite or seleno-cystathionine were ef-

fective in these studies. Selenium and/or vitamin E

5

6
deficiency in the chick produced three related yet seemingly
distinct pathological changes. These were exudative di-
athesis, muscular dystrophy and encephalomalacia. Nesheim
and Scott (1961) stated that the most marked nutritional
effect of dietary selenium in chicks and turkeys was its
effectiveness in preventing exudative diathesis and that it
was only partially effective against Skeletal muscular
dystrophy in chicks. Much higher levels of selenium sup-
plementation (1 to 5 ppm) were required to Show any effect
on nutritional muscular dystrophy in this species. A
growth-stimulation effect of selenium in these studies was
apparent only when Torula yeast diets were used. Rahman 3E
31. (1960) reported that sodium selenate (0.05 or 0.1 ppm),
vitamin E, and dried brewers' yeast prevented exudative
diathesis in chicks on a Torula yeast diet while corn dis-
tillers solubles and condensed fish solubles were partially
effective. A growth response to selenium was apparent only
in the presence of added vitamin E. Tests on the dried
brewers' yeast indicated that acid-ashing destroyed its
activity, thus suggesting this activity was due to an acid
volatile component, e.g. selenium. Selenium, vitamin E, or
the use of starch as the carbohydrate source completely pre-
vented the development of exudative diathesis in turkey
poults in these studies. In a statistical treatment of the
combined results of many feeding trials Mathias and Hogue
(1971) reported the effectiveness of selenium and vitamin E

against exudative diathesis and a strong interaction between

7

selenium and vitamin E in the prevention of deaths in Chicks
on Torula yeast diets. Deaths without signs of exudative
diathesis in chicks on high levels of synthetic antioxidants
were interpreted as inability of these antioxidants to pre-
vent deaths due to selenium deficiency although they were
capable of preventing many of the gross lesions of exudative
diathesis.

Skeletal muscular dystrophy of the pectoral and leg
muscles tended to predominate in the chick while myopathy
of the gizzard and myocardium were the major muscle lesions
noted in turkey poults fed selenium and/or vitamin E defic-
ient diets. Machlin and Shalkop (1956) described a muscu-
lar degeneration manifested grossly as white striations of
the breast and leg muscles and microscopically as a hyaline-
type degeneration in chicks fed casein-gelatin diets low in
vitamin E and sulfur amino acids. In these studies the
addition of o-tocopheryl acetate, methionine, cystine or a
high level of an antioxidant to the diets completely pre-
vented muscular degeneration. Hintz and Hogue (1964b)
reported that raw kidney beans added to a vitamin E-supple-
mented Torula yeast diet increased the incidence of nutri-
tional muscular dystrophy in chicks from 5—6% to 45-100%.
It was determined that these beans contained two tocopherol
antagonists. One was alcohol-soluble and heat labile, while
the other was alcohol-insoluble and heat Stable. They con-
cluded that the alcohol-soluble antagonist was probably un—

saturated fat. Scott in 1966 suggested that d-a-tocopherol

8
may be involved in a selenium-tocopherol complex associated
with the y-globulin fraction of serum proteins that plays
some unknown role in cystine metabolism and thus in the
prevention of nutritional muscular dystrophy of chicks.
Hathcock and Scott (1966) used a casein-soy-Torula yeast
diet to study the role of cysteine in the prevention of
muscular dystrophy. This diet contained sodium selenite at
a low level to prevent exudative diathesis and ethoxyquin
to prevent encephalomalacia while it allowed the production
of muscular dystrophy in chicks from vitamin E-depleted
hens. The results of this study indicated that compounds
such as guanidoacetic acid which accelerated the conversion
of methionine to cysteine reduced the severity of muscular
dystrophy while compounds such as creatine, Choline, and
betaine, that inhibit this conversion, accentuated the mus-
cular dystrophy. These results led the authors to postulate
that cysteine was the metabolically active sulfur amino acid
in the prevention of nutritional muscular dystrophy in vita-
min E-deficient chicks. Hathcock, Hull and Scott (1968)
studied the effectiveness of cysteine analogs and some sulf-
hydryl compounds against muscular dystrOphy in the chick and
concluded similarly that cysteine was the functional com-
pound in one of the pathways involved in the prevention of
nutritional muscular dystrOphy in the chick and that vita-
min E may be concerned in another pathway. Hathcock, Scott
and Thompson (1968) utilized the fact that one of the nor—

mal metabolic fates of cysteine is oxidation and

9
decarboxylation to taurine, and that taurocholate is the
only bile salt produced by the chicken, to study the role
of cysteine in prevention of muscular dystrophy in this
species. Cholic acid additions to the diet increased the
rate of cysteine to taurine conversion and accentuated
muscular dystrophy. Taurocholic acid and taurine additions
decreased the rate of cysteine to taurine conversion and
partially alleviated muscular dystrophy. Vitamin E addi-
tions to the diet reduced the taurine excretion rate of
chicks fed the basal diet.

Walter and Jensen (1963) reported complete protection
of turkey poults against gizzard and Skeletal muscular
dystrophy by adding 1 ppm selenium (as selenious acid) or
20 IU/kg of q-tocopheryl acetate to Torula yeast diets
calculated to be only slightly deficient in sulfur amino
acids. Skeletal muscles were affected to a lesser extent
than the gizzard musculature. Additions of cystine (0.15%),
methionine (0.44), low levels of ethoxyquin (0.025%) or
selenium (0.01 and 0.1 ppm) were ineffective in preventing
the muscular dystrophy. Ethoxyquin at a high level (0.3%)
reduced the incidence but did not prevent gizzard and
skeletal muscular dystrophy. Selenium, vitamin E and the
high level of ethoxyquin prevented the anemia and reduced
plasma albumin to globulin ratios that accompany these mus-
cle lesions in several Species. Scott _E-_l. (1967),
utilizing a practical-type corn-soybean meal diet composed

of feedstuffs obtained from an area with known low selenium

10

soils and without supplemental vitamin E or methionine, con-
cluded that selenium iS the primary nutritional factor re-
quired to prevent myopathies in the turkey poult while vita-
min E is of less importance and sulfur amino acids were
ineffective. The treatments in this study only included
additions of methionine and methionine hydroxy analog and
did not include cystine additions. The authors gave the
order of prominence of "selenium responsive" diseases of

the young poult as: myopathy--first of the gizzard, second
of the myocardium, and third of the skeletal muscle. They
concluded that the selenium requirement for the poult on a
practical—type diet ranged from approximately 0.18 ppm in
the presence of added vitamin E to approximately 0.28 ppm

in the absence of added vitamin E. '

Thompson and Scott (1969) in an attempt to establish
the essentiality of selenium as a trace element found that,
with semipurified diets, exudative diathesis could be pre-
vented by 10 ppm d-g-tocopheryl acetate or 0.04 ppm selenium.
On crystalline amino acid diets (less than 0.005 ppm se-
lenium) poor growth and high mortality occurred even when
the diet contained up to 200 ppm d-a-tocopheryl acetate.
Higher levels of d-q-tocopheryl acetate prevented mortality
but even with 1000 ppm growth was inferior to that obtained
with supplemental selenium and no added vitamin E. With no
added vitamin E the selenium requirement on this diet was
approximately 0.05 ppm while with 10 ppm added d-q-tocopheryl

acetate it was 0.02 ppm and with 100 ppm added

11
d-m-tocopherfl_ametate it was less than 0.01 ppm. In further
studies with the crystalline amino acid diet (0.005 to 0.02
ppm selenium) these authors (1970) reported that with high
levels of vitamin E but without supplemental selenium this
diet resulted in poor growth, poor feathering and atrophy
of the pancreas with impairment of fat hydrolysis. Absorp-
tion of lipids (and Vitamin E) was poor. These studies thus
established that selenium is an essential trace element for
the chick and in one of its roles maintained the integrity
of the pancreas. The authors concluded from these studies
that the impairment of vitamin E absorption in severe
selenium deficiency did not explain the ability of both
selenium and vitamin E to prevent exudative diathesis and
that another more direct interrelationship was responsible

for this effect.

Cattle and Sheep

 

Muscular dystrophy or white muscle disease of young
ruminant farm animals has been recognized in many areas of
low selenium soils. In an effort to study methods of con-
trolling or preventing this problem many experiments have
been conducted. Some of these will be presented here.

Proctor, Hogue and Warner (1958), using a known dystro-
phogenic diet of mixed hay and raw cull kidney beans for
ewes one month prepartum, were able to control muscular
dystrophy in the lambs born to these ewes by adding 1 ppm

selenium as sodium selenite to the diet or by feeding 0.25

12
pound linseed meal or 100 IU d-tocopheryl acetate per head
per day. The effect of the linseed meal was attributed to
its relatively high selenium content (1.18 ppm). In other
studies reported by this group (Hogue, Proctor and Maplesden,
1959) 100 mg n-tocopheryl acetate or 1 mg selenium given to
lambs every other day controlled the problem. Vitamin E at
the rate of 100 IU/ewe/day was better than the hay and cull
kidney beans basal but not as effective as 1 ppm selenium
in controlling muscular dystrophy of lambs. Hintz and Hogue
(1964a), using a similar diet, imposed dietary treatments
on ewes or lambs at parturition. Selenium as sodium
selenite at the level of 0.17 ppm during lactation had no
effect on clinical incidence of nutritional muscular dys-
trophy but reduced the number of lambs with elevated serum
transaminase values. Dietary sulfur (0.33%) as sodium sul-
fate increased the clinical incidence of nutritional mus-
cular dystrophy and when fed with selenium prevented any
beneficial effect of selenium. Oral dosing of lambs, from
ewes fed the basal diet, with cystine or methionine was in-
effective against nutritional muscular dystrophy.

Welch _E 31, (1960) reported that feeding fish liver
oil to ewes during pregnancy and lactation lowered ewe and
lamb plasma vitamin E levels and increased the incidence of
muscular dystrophy in lambs. Vitamin E treatment of ewes
was effective in preventing muscular dystrophy in lambs and
in curing dystrophic lambs. Selenium in the ration of ewes

decreased but did not prevent the occurrence of muscular

13

dystrophy. Ewan, Pope and Baumann (1964) reported a lamb
feeding trial utilizing an artificial milk diet based on
Torula yeast and stripped lard. In this study the lambs
on the basal diet survived an average of 27 days. Selenium
improved total weight gains, rate of gain and survival time
while the effect of vitamin E on total weight gains and
survival time only approached significance. No interaction
was noted between vitamin E and selenium.

Buchanan-Smith et 31. (1969) reported that the develop-
ment of selenium and/or Vitamin E deficiency in sheep on a
urea-based diet was controlled by weekly injections of
sodium selenate (5 mg selenium) and/or Q-tocopheryl acetate
(700 IU vitamin E). In these studies selenium delayed, but
did not prevent, death. Growth was improved by selenium
injections but satisfactory reproductive performance of
ewes was obtained only in ewes given the combined treatment.

The group at Oregon State University has conducted
several studies in which they have fed dams forages and
grains from areas of known low soil selenium to produce the
deficiency under controlled conditions. Schubert et 31.
(1961) using this technique were able to produce the char-
acteristic clinical symptom of erratic locomotion and the
gross and histopathological lesions of skeletal and cardiac
muscle degeneration and calcification in preweaning lambs
and calves. Supplementation of the dams' ration with 0.1
ppm aS sodium selenite during gestation and lactation pro-

vided complete protection against the disease, while

14
moderate levels of vitamin E did not. Selenium and Vitamin
E therapy of the newborn animals prevented gross lesions
but was not entirely effective in preventing microscopic
lesions. They have suggested that a sulfur antagonism may
influence the biological availability of the selenium pres-
ent in soils and forages. This conclusion is based parti-
ally on an observation that gypsum treatment seemingly led
to an increased incidence of white muscle disease in grazing
animals. It is also possible that the lowered soil pH
effected by the gypsum treatment may have lowered the avail-
ability of the soil selenium to plants. Oldfield, Shubert
and Muth (1963) found that white muscle disease could be
prevented by raising the dietary selenium level to 0.06 ppm.
Selenium provided parenterally in a Slowly absorbed vehicle
was effective in a single dose for the duration of pregnancy
and lactation. Levels of selenium in the blood of lambs
were Similar to those of their dams. Whole blood selenium
levels of 0.11 ppm selenium in the ewes and 0.12 ppm selen-
ium in the lambs were established as compatible with the
prevention of white muscle disease in this study using an
alfalfa hay-oats basal containing less than 0.02 ppm selen-
ium. Burton E£.3l° (1962) and Hidiroglou 33 31. (1968)
have utilized this technique of feeding forages and grains
from enzootic areas to produce muscular dystrOphy in calves
and lambs and to determine the tissue levels of selenium
resulting from selenium deficiency and therapy. These data

will be discussed later.

15

Swine

The experiments conducted concerning selenium and/or
vitamin E deficiency in swine have generally utilized diets
based on Torula yeast, diets in which the grains have been
subjected to considerable physical or chemical damage in
storage or diets containing high levels of rancid or un-
saturated fat. Such diets are not commonly fed in commer—
cial swine feeding operations in this country. These
factors have and still do make it difficult for some to
accept the fact that selenium and/or vitamin E deficiency
can occur in swine fed corn-soybean meal rations in con-
finement.

Adamstone, Krider and James (1949) reported a study
utilizing a wheat flour-casein diet with 10% added rancid
lard or 50 mg d,l-q-tocopherol/head/day. The first two of
these dietary impositions had severe effects on the repro-
ductive performance of the gilts to which they were fed for
one gestation-lactation period. The pigs born to sows on
the basal diet and the basal plus rancid lard diet exhib-
ited wobbly gaits and incoordination of the hind legs.
Histologically the Skeletal muscles of these pigs showed
lesions very Similar to those recognized today as nutri—
tional muscular dystrophy of swine. The livers of these
same pigs were fatty and the livers of their dams were
fibrotic and fatty with pooling of blood in many lobules.
The sows fed the basal diet plus vitamin E had a superior

reproductive performance, and the pigs of these sows

16

exhibited none of the clinical Signs or histological lesions
noted in the other two lots. Hove and Seibold (1955) fed a
soybean meal-sucrose diet ( % crude protein) containing 6%
lard and 2% cod liver oil or this diet plus 0.01% d,1-n-
tocopheryl acetate to two litters of 6 pigs each with aver-
age initial weights of 10.5 and 15.9 kg. Three pigs died
during the study on the Vitamin E deficient diet and exhib-
ited acute hemorrhagic necrosis of the liver. Two more pigs
on this diet showed post-necrotic cirrhosis at slaughter
after 187 days on trial. No muscle lesions were noted in
any of these pigs. Reid et al. (1968) reported a study of
combined protein-Vitamin E deficiency using 5 kg pigs fed
the experimental diets for 8 weeks. The basal diet con-
tained 3% isolated soy protein and 25% corn oil while the
control diet contained 30% isolated soy protein and 25%
corn oil. The basal diet contained 7.60 IU vitamin E per
kg and 0.018 ppm selenium. Severe liver necrosis that de-
veloped on the basal diet was completely prevented by addi-
tions of vitamin E (100 IU/kg diet), selenium (0.5 ppm as
sodium selenite) or both. Choline supplementation aggra-
vated the liver damage while methionine additions provided
considerable protection. A second study indicated that the
methionine effect was not related to selenium contamination
of the methionine used.

Swedish workers have reported numerous studies in

which they have been able to induce selenium and/or vita-

min E deficiency in swine by feeding high levels of fish

17
oils or heat- or weather-damaged grains and fats. It iS a
fairly common practice for Scandfimvian swine feeders to

utilize fish oils in swine feeds. Obel (1953) described

the naturally occurring condition she named hepatosis

 

diaetetica in swine submitted for necropsy at the State

 

Veterinary Medical Institute of Sweden. This condition
accounted for 6.9 to 14.5% of all diagnoses in this labor-
atory from 1947 to 1951. She was able to consistently pro-
duce the condition experimentally using a starch-brewers'
yeast diet containing 6% cod liver oil. She concluded that
vitamin E deficiency was the major problem but suggested
that Schwarz's Factor 3 could also be involved. Lannek

_E El. (1961) reported studies with pigs of 20-25 kg initial
weight fed a casein-yeast-sucrose basal diet with fat addi-
tions. Vitamin E-stripped lard in this diet did not result
in the production of nucritional muscular dystrophy in pigs
while cod liver oil did, even though this fish oil was rich
in Q-tocopherol. Further additions of q-tocopherol pre-
vented the development of nutritional muscular dystrophy.
Injections of sodium selenite lowered plasma transaminase
levels and had a curative effect in affected pigs. Orstadius,
Nordstrom and Lannek (1963) reported that intramuscular in-
jections of Vitamin E or sodium selenite inhibited the
development of nutritional muscular dystrophy on a grain
diet containing heated cottonseed oil. These treatments

had a curative effect on overt disease and a synergism was

indicated between the Vitamin E and selenium treatments.

18
Grant and Thafvelin (1958) fed pigs a known hepatonecro-
genic soya-meal diet or this diet plus 0.2 ppm selenium as
sodium selenite. Pigs on the basal diet died between the
22nd and 45th days of the experiment. At necropsy these

animals Showed typical lesions of hepatosis diaetetica,

 

 

massive transudations, skeletal and myocardial muscle de-
generation and ceroid pigment in the adipose tissue. None
of the pigs receiving sodium selenite died. Three were
killed at various intervals and a laparotomy was performed
on the other two. Livers in all selenium-fed animals were
normal. No transudation was present but skeletal muscle
degeneration and ceroid deposition in adipose tissue were

observed. Thafvelin (1960) stated that when hepatosis

 

diaetetica, muscular degeneration and allied disorders

 

are seen in pigs fed on grain, the occurrence of these
lesions can be coupled with the properties of the cereal
fat. By heating grains he was able to lower the iodine
number and vitamin E levels of cereal fats in 2 of 3 cases.
This heating process also increased the peroxide value of
the cereal fats. In three feeding trials, the heated grains
or heated maize oil produced nutritional muscular dystrophy

and hepatosis diaetetica with increased serum transaminase

 

levels and several sudden deaths among the pigs. Swahn and
Thafvelin (1962) in a further report of this work concluded
that heating disrupted the natural antioxidative system of
the cereal fat and permitted oxidation of the fatty acids.

These changes were analogous with those that occur gradually

19
in ground grains stored under usual conditions. Vitamin E
and Sodium selenite were effective in preventing micro-

angiopathy, transudation and hepatosis diaetetica. Selenite

 

did not completely alleviate muscular degeneration although
it decreased its severity. Lannek et 31. (1960) purchased
moldy grain from a farm experiencing trouble with selenium
and/or vitamin E deficiency in pigs. This material pro-
duced muscular dystrophy in pigs of 20 to 25 kg initial
weight. While the addition of 6.5% casein had no effect,
additions of d,l-n-tocopheryl acetate (50 mg/kg diet)

or 0.2 ppm selenium as sodium selenite prevented the disease
and probably had a curative effect.

Most of the studies on selenium and/or vitamin E defic-
iency in North America have been conducted with Torula yeast
diets. Eggert _£.i£° (1957),using such a diet and 2 to 3
week old pigs, reported that 4 of 6 pigs on the basal diet
died suddenly within 53 days and exhibited typical lesions
of liver necrosis and yellow fat. No deaths occurred in
pigs receiving the basal plus 40 ppm o-tocopheryl acetate
or 1.0 ppm selenium as sodium selenite and no gross lesions

were observed in these animals when sacrificed. Wastell

l. (1968) and Ewan 3E.al. (1969) reported a series of

it.
studies where Torula yeast or isolated soy protein diets of
reasonable crude protein content were used to study the ef-
fects of selenium and/or vitamin E deficiency in young pigs.
These diets contained 5% fortified cod liver oil and thus

contained very high levels of vitamins A and D.

20
Supplementation of the deficient diets had no effect on
growth rate. These supplements, however, prevented the
development of the Characteristic pathology of selenium
and or vitamin E deficiency of pigs and reduced mortality
from 54% to 7%. Wastell _£._l. (1972) reported results of
these studies when the same pigs were maintained to growing—
finishing weights. On Torula yeast diets, supplementation
with vitamin E and selenium resulted in faster gains than
with either of these supplements alone. These effects did
not prevail when isolated soy protein diets were employed.
In these older pigs the lesions noted on Torula yeast diets
plus selenium were edema, hyalinization of Skeletal muscles
and a yellowish-brown discoloration of body fat and tissues.
Pigs fed Torula yeast diets supplemented with vitamin E and
selenium showed some liver fibrosis, hyalinized Skeletal
muscle fibers and degenerative heart myofibrils. This
doubly—supplemented group had significantly higher gains
than either singly-supplemented group. No lesions were ob-
served in pigs fed Torula yeast plus vitamin E alone or in
pigs fed isolated soy protein diets supplemented with vita-
min E and selenium alone or in combination. Michel, White-
hair and Keahey (1969) fed a 6% crude protein Torula yeast-
stripped lard diet to young pigs. Dietary hepatic necrosis
occurred with a high frequency on this basal or the basal
plus methionine or low levels of Vitamin E. Selenium, high
levels of vitamin E or additional protein prevented dietary

hepatic necrosis. Nutritional muscular dystrophy was

21
observed in some experimental pigs fed the basal diets plus
selenium, methionine or additional protein. This myopathy
did not occur in the groups fed vitamin E or ethoxyquin.
Erythrocytic cells in the bone marrow had nuclear abnormali—
ties in the pigs fed the basal diet. Sharp, Young and Van
Dreumel (1970) reported studies with growing pigs in which
Torula yeast diets and high moisture corn diets increased
the incidence of the disease. Supplementation of these
diets with vitamin E alone or in combination with selenium

increased pig survival on deficient diets.

Occurrence of deficiencies under practical conditions

 

Cattle and sheep

 

White muscle disease has been recognized in lambs and
calves for many years. Muth (1955) in a report describing
the incidence and Character of the malady in Oregon stated
that it was recognized in that state in lambs in 1920 and
in calves in 1939. Hartley and Grant (1961) presented an
excellent review of the problem in New Zealand. They
described both a congenital and a delayed white muscle dis-
ease in lambs and white muscle disease in hoggets (yearling
sheep). A beneficial effect of selenium treatments on
fertility in sheep and in curing ill thrift of Sheep and
cattle was also reported. White muscle disease was also
reported by these authors to be quite common in calves and
foals in New Zealand. The treatments applied and the re-

sults obtained in New Zealand, as outlined by these authors,

22
were good examples of the results attainable through the
rational use of selenium treatments and dietary supplements.
Wolf, Kollonitsch and Kline (1963) used a questionnaire
technique to conduct a survey of the incidence of selenium
deficiencies and their treatment in the United States and
principal overseas agricultural centers. Such data was use-
ful in the delineation of selenium-deficient areas.

Blaxter (1963) concluded that selenium deficiency in
Scottish soils is less severe than in New Zealand or the
northwestern United States and that the risk of toxicity
problems with selenium drenches or injections outweighed the
meager response in weight gains they obtained from selenium
treatment of several Scottish flocks. Shirley at 31. (1966)
found no effect of intramuscular sodium selenite injections
on weaning weights of calves or rate of gain of lambs in
Florida. The selenium content of the pastures were low and
in the range of those which gave problems with white muscle
disease in Oregon. No clinical Signs of white muscle dis-
ease were observed in any of the cattle or lambs. These
results have led subsequent investigators to postulate that
the increased tocopherol intake of these animals, due to the
year-round grazing season in Florida, may have prevented the
disease from developing even though selenium levels were

very low.

Swine

 

As mentioned earlier, Obel (1953) indicated that hepa-

tosis diaetetica accounted for a considerable proportion of

 

23

pig losses in Sweden. The incidence of the condition was
greater in the counties with poorer swine husbandry prac-
tices, was highest in the months of October and November
and occurred most fequently in 6-week old pigs. These ani-
mals would die suddenly without noticeable illness or after
a Short period of dullness. Other Swedish research groups
(Lannek St 31., 1960; and Lindberg and Siren, 1965) have
described nutritional muscular dystrophy on several farms.
Thomke, Dahl and Persson (1965) described trials in which
additions of Vitamin E and sodium selenite were made to
practical skim milk and barley diets for pigs from 20 to 90
kg. Neither of these supplements had any effect on growth
rate, feed conversion or carcass quality. Vitamin E addi-
tions did improve the "keeping" quality of depot fat.

Hartley and Grant (1961) described the occurrence of

hepatosis diaetetica on at least 20 swine farms in New Zea-

 

land. The condition was usually seen around the time of
weaning in pigs fed skim milk and barley with liberal amounts
of cod liver oil. Most outbreaks occurred in areas where
selenium-reSponsive diseases were common in sheep. Discon-
tinuing the feeding of cod liver oil and giving the pigs 5
mg of oral inorganic selenium prevented further losses.
Selenium administration was also effective when the condi-
tion appeared in herds that were not receiving cod liver oil
in the diet.

Michel t al. (1969) described the Characteristic

pathology of selenium and/or vitamin E deficiency in pigs

24
from 7 different Michigan swine herds. Most of these ani-
mals were on corn-soybean meal diets without added vitamin
E. The corn in most cases had been artificially dried at
harvest time. In one epizootic the feed contained 6.5 mg
g-tocopherol per kg and 0.057 ppm selenium. In another out-
break the feed was found to contain 4.0 mg a-tocopherol per
kg and 0.04 ppm selenium. Losses in all these herds ceased
when Vitamin E at the rate of 22 IU per kg was added to the
rations. Trapp at a1. (1970) reported on 97 animals from
37 swine herds in Michigan. This report included those de-
scribed by Michel §£_§l, (1969) above. Most of these pigs
weighed 20 to 40 kg and had been reared in an intensified
confinement operation on corn-soybean meal rations. Many
of these rations included arsanilic acid as a growth stimu-
lant. Most losses ceased with high levels of dietary vita-
min E supplementation or injections of selenium and Vitamin
E or both. The Clinical signs and gross as well as micro—
scopic pathology observed in the field cases in these two
reports correlated very well with that seen in the experi-
mentally-induced deficiency.

As has been observed in many of the studies previous-
ly related, Klein gt 31. (1970) found no effect of vitamin
E or selenium on rate or efficiency of gain. These obser-
vations were made in a factorial study comparing normal

and Opaque-2 corn. Mahan 35 31. (1971) reported studies

in which injections of vitamin E reduced death losses in
young pigs born to sows maintained in confinement while
injections of selenium or selenium plus vitamin E com-

pletely prevented these death losses. The sudden deaths

and lesions noted in the pigs that died were

25

typical of selenium and/or vitamin E deficiency in swine.

Pathology of selenium and/or vitamin E deficiency in swine

 

Gross and microscopic pathological changes

 

One of the earliest definitive descriptions of selenium
and/or vitamin E deficiency in swine (Obel, 1953) is also
one of the most comprehensive. The animals died suddenly
without signs of illness or after a short period of dullness.
Some exhibited dyspnea, muscle weakness, blood in the feces,
diarrhea and vomiting. Icterus was observed only in cases
with a relapsing course. The gross necropsy lesions de-
scribed included an anemic carcass, yellow fat, subcutaneous
edema, fluid accumulations in the thoracic cavity, abdominal
cavity and pericardium. There was a patchy discoloration
of the liver with varying degrees of roughening of the sur-
face of the liver. Ulcers in the fundus of the stomach
were observed in 22% of the cases. Microscopic lesions in—
cluded congestion and necrosis of liver lobules confined by
the lobular septa with normal lobules being adjacent to ones
with severe necrotic changes. Heavy mineralization of some
of these necrotic lobules was noted. Waxy degeneration of
skeletal and heart muscle was observed with a predilection

of the thigh muscles, the musculus iliopsoas, the musculus

 

subscapularis, the pectorals, the intercostals and the

 

diaphragm for this degeneration. Twenty-seven percent of
the pigs examined exhibited a fibrinoid degeneration of

arterial walls. Many also had acute nephrosis. Excellent

26
photographs of gross and microscopic lesions were included
in this article.
Orstadius et al. (1959) using a biopsy technique con-
cluded there were no real differences in the lesions of

muscular dystrOphy noted in the semitendinosus, triceps

 

brachialis or longissimus dorsi muscles. In selenium and/or

 

 

vitamin E deficiency of swine one may observe one, all, or
any combination of the pathological changes described by
Obel (1953). Lesions compatible with the deficiency were
described in most of the trials cited above under experimen-
tal or field occurrence of the deficiency (Adamstone et al.,
1949; Hove and Seibold, 1955; Eggert gt al., 1957; Grant and
Thafvelin, 1958; Lannek 33 al., 1960; Augustinsson gt al.,
1960; Hartley and Grant, 1961; Lannek 33 al., 1961;
Orstadius SE al., 1963; Reid 33 al., 1968; Wastell 35 al.,

1968; Ewan gt 1., 1969; Mahan et al., 1971; and Wastell 32

al., 1972). Michel gt _l. (1969) compared and contrasted
the lesions as they occurred intje experimentally induced
deficiency and in field cases in Michigan. Trapp SE.3£°
(1970) presented a comprehensive treatment of usual history,
gross lesions, microscopic lesions and differential diag-
nosis of selenium and/or vitamin E deficiency in swine.

In addition to the lesions already discussed, these authors
believed that edema of the mesentery of the spiral colon,
the lungs and submucosa of the stomach are significant

diagnostically. They also discussed other less well-de-

fined herd problems which may be related to a selenium

27

and/or vitamin E deficiency. Although the lesions of the
deficiency in young pigs have been described extensively,
many cases are probably still being missed due to improper
techniques (e.g., failure to perform histopathologic and
microbiologic exams), inadequate education of diagnostic or
veterinary medical personnel in the differential diagnosis
of the problem and a refusal to accept the occurrence of the

condition under field conditions.

Clinical pathology of selenium and/or vitamin E defic-

 

iency of swine

 

Various hematological and serological parameters have
been tested as possible diagnostic criteria for selenium
and/or vitamin E deficiency in swine. In general, standard
hematological examinations have been of little use (Michel

l., 1969; Trapp EE.3£°' 1970). Some parameters such as

it;
susceptibility of erythrocytes to hemolysis, which had been
the basis for biological assays of vitamin E using rats,
have proven to be of minor import in field diagnosis in
swine (Klein 25 31,, 1970) although researchers have been
able to show differences in this parameter in severely-
depleted animals of other species (Horses: Stowe, 1968;
Rats: Rotruck 35 al., 1972). Depressed albumin to globulin
ratios have been suspected, due to the severe edema and
exudation seen in many cases, but have been difficult to
consistently demonstrate. Rahmann 3E.il' (1960) found that

depressed serum albumin to globulin ratios in chicks with

exudative diathesis were prevented by 0.05 ppm selenium.

28

Serum or plasma enzyme assays have proven to be the
most effective means of monitoring the course of the de-
velopment of the deficiency and as an auxiliary tool for
diagnosis in the living animal. The most commonly employed
assays have been those for serum (SGOT) or plasma (PGOT)
glutamic-oxaloacetic transaminase, serum (SGPT) or plasma
(PGPT) glutamic-pyruvic transaminase. Others in fairly
common usage were ornithine carbamyl transferase (OCT) and
lactic dehydrogenase (LDH).

Blincoe and Dye (1958) reported that SGOT increased
markedly in calves and lambs with white muscle disease and
that the activity appeared to be proportional to the extent
of muscle damage.

The Swedish research groups have conducted extensive
studies using these enzymes. Wretlind, Orstadius and Lind-
berg (1959) reported values for normal pigs of: PGOT 28.03
: 15.39 units, PGPT 20.37 i 5.54 units, and OCT 5.93 i 2.42
units. They also examined the concentrations of these
enzymes in the organs of normal pigs. Skeletal and heart
muscle were fiaund to contain large amounts of GOT and GPT
but were very low in OCT, while liver was very high in OCT.
They suggested that this information might be used to dif-
ferentiate between muscle damage and liver damage in the
living animal. Michel 33 _l. (1969) reported increased
serum OCT levels in pigs with experimental dietary hepatic
necrosis. Orstadius e£_al, (1959) reported spontaneous

"liver dystrophy" of pigs to be associated with elevations

29
of PGOT, PGPT and POCT early in the course of the disease
with a gradual decline with time. Similarly pigs with
spontaneous muscular dystrophy exhibited elevated PGOT and
PGPT early in the course of the disease with a gradual de-
cline with time. These results are in accordance with the
tissue enzyme levels discussed previously, and the results
reported by Lannek 32 al. (1960), Augustinsson SE al.
(1960) and Ewan and Wastell (1970). They concluded that
PGOT and OCT were more specific for selenium and/or vitamin
E deficiency in swine since PGPT may be elevated in several
other diseases of swine. Several other authors have uti-
lized SGOT or PGOT activity to monitor the deficiency in
swine (Lannek 33 al., 1961; Orstadius gt 1., 1963; Ewan

t al., 1971 and Wastell gt al.,

1970) and Wastell et al. (1972)

and Wastell, 1970; Mahan e
1972). Ewan and Wastell (
found serum LDH activity to be elevated in experimental
selenium and/or vitamin E deficiency in young pigs. These
authors considered serum LDH activity to be a sensitive in-
dicator of subclinical nutritional muscular dystrophy in
sheep (based on the work of Blincoe and Marble, 1960 and
Paulson 2E._l" 1968), and suggested that it may serve like-
wise in young pigs. These researchers have also reported
work on the effect of the experimentally induced deficiency
on certain serum electrolytes. Some of these effects might

prove questionable since their diets contained very high

levels of vitamins A and D.

30

Tissue and blood selenium levels in healthy, deficient and

supplemented animals

 

Selenium is generally accepted to be present in all
plant and animal tissues although insufficient sensitivity
of analytical techniques may preclude its detection in some.
Dickson and Tomlinson (1967) using radioactivation analysis
reported human blood to contain 0.18 ug selenium/ml. Allaway
'gt gt. (1968) reported whole blood from male donors in the
United States to range from 0.10 to 0.34 ug selenium/ml
with a mean of 0.21 ug/ml. These values are very similar to
those reported by Burk gt_gt. (1967) for whole blood of
healthy adults (0.22 ug selenium/ml)and normal Guatemalan
children (0.23 ng selenium/ml).

Burk gt gt. (1968) monitored blood, liver and kidney
selenium levels during the development of liver necrosis in
rats on Torula yeast diets. Selenium levels in these
tissues declined from initial levels of 0.33 ppm, 0.75 ppm,
and 0.96 ppm, respectively, on a wet basis to 0.20 ppm, 0.07
ppm and 0.27 ppm in four weeks on the selenium and vitamin E
deficient basal diet. With supplements of 0.25 or 0.50 ppm
added selenium (as sodium selenite), whole blood selenium
levels plateaued at 0.4 ppm at two weeks. Liver selenium
levels were approximately 0.7 ppm on a wet basis on both
levels of added selenium, while kidney selenium levels were
1.5 and 1.1 ppm selenium on the 0.5 and 0.25 ppm added
selenium diets. Hurt, Cary and Visek (1971) reported that

commercially-obtained rats fed an amino acid diet with 20 IU

31

vitamin E added per kilogram had initial blood, liver and
skeletal muscle selenium levels of 0.32, 0.60 and 0.23 ppm
on a wet basis, respectively, which declined to 0.05, 0.04
and 0.02 ppm, respectively, after 20 weeks on the deficient
diet. Seventy percent of these declines occurred in the
first 6 weeks. Supplementation of these diets with 0.5 ppm
selenium as selenomethionine produced increases in blood
(0.68 ppm) and liver (1.46 ppm) selenium for 20 weeks but
skeletal muscle selenium remained unchanged.

Andrews, Hartley and Grant (1968) reported that lambs
with white muscle disease in New Zealand had 0.016, 0.048,
and 0.24 ppm selenium on a wet basis in whole blood, liver
and kidney, respectively. Calves in the same survey af-
fected with white muscle disease had 0.035 and 0.36 ppm
selenium on a wet basis in the liver and kidney, respectively.
Hidiroglou _t _l. (1968) reported selenium levels of 1.32,
0.44 and 0.76 ppm on a dry basis for liver, muscle and heart,
respectively, from sheep fed a selenium-adequate diet and
values of 0.17, 0.08 and 0.09 ppm on a dry basis for sheep
fed a selenium-deficient diet. These values for sheep are
not greatly different from those reported for normal sheep
by Pierce and Jones (1968) of 1.0, 0.3 and 1.0 ppm on a dry
basis for liver, muscle and heart, respectively.

Tissue levels of selenium in sheep are increased
slightly when selenium injections, oral drenches or dietary
supplements are used to prevent losses in deficient areas.

In New Zealand, Andrews, Grant and Stephenson (1964)

32
reported selenium levels of 0.102 and 1.42 ppm for liver
and kidney, respectively, on a wet basis for control lambs
and levels of 0.347 and 1.95 ppm on a wet basis for the
same tissues from similar lambs given 1.25 mg selenium
orally once a week for 8 months. Similar levels resulted
when heavy iron-selenium intraruminal pellets were adminis-
tered to mature ewes by Handreck and Godwin (1970) and when
Paulson _t _l. (1968) offered selenized trace mineral salt
containing up to 132 ppm selenium as sodium selenate.

Hidiroglou, Carson and Brossard (1965) reported that
calves sick or dead from white muscle disease were born to
cows with low selenium content of hair (0.06-0.23 ppm) while
no white muscle disease was observed in calves born to cows
with hair selenium levels above 0.25 ppm.

Taussky _t _l. (1963 and 1965) determined that the hen
egg contained a mean of 11 ug total selenium, most of which
was in the yolk. Essentially all this selenium was recovered
in the chick at hatching. Unfortunately no information was
given concerning dietary levels of selenium in these studies.

Scott and Thompson (1971) reported an extensive series
of studies on the effects of naturally-occurring and selen-
ite selenium in the diet on tissue selenium levels of chicks
and turkey poults. The individual data were too extensive
to relate here so only general observations and effects will
be presented. Selenium deposition in blood, muscle, liver,

kidney and skin had a direct relationship to the inorganic

selenium content ok the diet up to the dietary levels of

33
approximately 0.2 to 0.3 ppm. Further increases in dietary
inorganic selenium to 0.4, 0.6 or 0.8 ppm produced no
further increases in blood or muscle selenium in chickens
or turkeys. The addition of organic selenium in the form
of soybean meal, fish meal with solubles and wheat to raise
the dietary selenium to 0.67 ppm resulted in considerably
higher selenium levels in blood and muscle than when equi-
valent levels of selenium were provided as sodium selenite.
These studies indicated that poultry tend to excrete reason-
able excesses of selenium added to the diet once their needs
have been met without substantial increases in tissue
selenium levels.

Lindberg and Siren (1963 and 1965) reported liver
selenium levels of normal pigs to range from 0.52 to 2.12
ppm on a dry basis, those of pigs with muscular dystrophy
to range from 0.13 to 0.28 ppm dry and those with liver
dystrophy to range from 0.13 to 0.24 ppm dry. These same
authors reported mean kidney selenium levels for normal
pigs, pigs with nutritional muscular dystrophy and pigs with
dietary hepatic necrosis to be 10.98, 3.40 and 3.33 ppm on
a dry basis, respectively. Very similar values were re-
ported by Lindberg (1968). In New Zealand, Andrews gt_gt.
(1968) reported the mean selenium levels of liver and kid-

ney in pigs dying of hepatosis diaetetica to be 0.047 and

 

0.53 ppm on a wet basis, respectively.
Sharp gt gl. (1970) reported several feeding trials

with growing finishing pigs. The tissue selenium levels

34

they observed in deficient and selenium-supplemented ani-
mals were consistent with those already discussed. Several
dietary ingredients were used in these studies, most of
which are not too common in swine feeding operations in this
country. These workers noted a tendency for added vitamin
E to increase kidney selenium levels while reducing liver,
heart and muscle selenium levels. This effect was noted on
both unsupplemented diets and diets supplemented with selen-
ium in the form of selenium sulfide. They expressed agree-
ment with earlier observations that a liver selenium level
of 0.2 ppm selenium on a dry basis is the minimum level
compatible with protection against selenium responsive dis-
eases and that liver selenium levels were a more sensitive
indicator of selenium status than were kidney selenium levels.

In a study of tissue composition of young pigs fed
Torula yeast diets with or without supplements of selenium
as sodium selenite and/or vitamin E, Ewan (1971) reported
that muscle and liver selenium decreased significantly when
the deficient diet was fed for 8 weeks. Muscle selenium
was higher in pigs fed selenium than in those not fed selen-
ium, but muscle selenium levels on all diets were lower
after eight weeks on trial than they were initially. The
same situation held true for liver selenium levels in
general. The one exception was that liver selenium was
slightly higher after 8 weeks than initially in the pigs
fed both supplemental selenium and vitamin E. Kidney selen-

ium levels were higher in selenium-fed animals.

35
Unfortunately no initial kidney selenium levels were re-
ported, however, the highest levels of kidney selenium in
this study (7.50 ppm dry) were well within the normal ranges
discussed above.

Trials conducted by Lindberg and Lannek (1965) using a
commercial diet that contained 0.126 ppm selenium or this
diet supplemented with 1.2 ppm selenium as sodium selenite
indicated that feeding this supplemental selenium for 78 days
did not increase kidney selenium levels, but did increase
liver and muscle selenium levels somewhat over those ob-
served for pigs on the basal diet. After a fourteen day
withdrawal period, muscle selenium levels had returned to
control levels and liver selenium levels were nearly back to
control levels. This level of selenium supplementation was
designed to provide a daily selenium consumption equivalent
to the dosage recommended for therapeutic injection.

Ku _t gt. (1972) established a linear correlation of
0.95 between dietary selenium level and the selenium concen—

tration of longissimus muscles of pigs collected from 13

 

locations in the United States. Dietary selenium levels
ranged from deficient to adequate (0.036 to 0.493 ppm air
dry) but no potentially toxic muscle selenium concentrations
were observed (0.046 to 0.521 ppm on a wet basis).

Selenium levels in blood and tissues of animals were
closely related to the levels available from their diets.
The form in which selenium was presented in the diet had a
profound effect on the degree to which it accumulated in the

tissues of the animal consuming it.

36

Selenium balance in animals

 

Most of the early work concerning selenium turnover in
animals was directed toward elucidating the mechanisms
whereby the organism attempts to rid itself of toxic levels
of this element once they have been taken into the body.
Since such studies often resulted in death or at least
permanent impairment due to selenization, most of this work
was done with laboratory animals. After Schwarz and Foltz
(1957) established that selenium was an essential trace
element some researchers began to employ more physiologi-
cally-compatible levels of selenium in their studies. The
lack of simple analytical techniques of sufficient sensi-
tivity to reliably measure the trace element levels of
selenium found in feeds, animal tissues and excreta in
studies with physiological levels of selenium has hampered
research in this area considerably. Thus, much of the work
reported here on the routes of excretion and factors af-
fecting them were performed with levels of selenium that
represent chronic to subacute toxic levels. The extrapola-
tion of this data to animals on normal intakes of selenium
is Open to question. Details of experiments will not be pre-
sented here as they would tend to detract from the continuity

of the discussion.

Absorption and tissue distribution of selenium

 

Much of the work on the absorption of selenium has in-

volved the injection of radioactive selenium (75Se)

37
compounds into laboratory or farm animals and the monitoring
of their course in the body by measuring the relative radio-
activity of various tissues, organs and fluids at time inter-
vals after administration of the isotope. In most cases the
758e used was in an inorganic form such as selenious acid or
sodium selenite. Unfortunately many researchers incorrectly
assumed that inorganic selenium followed the same pathways
in the body as selenium serving a structural function in
organic materials. Consequently, they reached erroneous
conclusions about selenium homeostasis in the animal
organism. It has been shown by Jaffe and Mondragon (1969)
that rats adapted to different levels of selenium intake and
thus tolerated previously toxic levels. Molnar and Darby
(1971) found that female rats were more resistant to selenium
depletion than males. These and other factors may have had
an unknown bearing on the data to be discussed. There were
no marked differences in the ultimate tissue distribution of
inorganic selenium when injected 21g intravenous, intra-
muscular or intraperitoneal routes. Selenium administered
in this manner produced rapid elevations in blood selenium
levels which were lowered slowly by selective absorption by
certain tissues; notably the liver, kidney, pancreas,
ovaries and endocrine glands (Smith, Westfall and Stohlman,
1938; Blau and Manske, 1961; Orstadius and Aberg, 1961;
Ekman, Orstadius and Aberg, 1963; Lindberg and Tanhuanpaa,
1965; Wright, 1965; Caravaggl, 1969; Lopez, Preston, and

Pfander, 1969). Following this redistribution of the blood

38
pool of selenium to the tissues, the material was slowly
excreted (Smith, Westfall and Stohlman, 1937; McConnell and
Martin, 1952; Kuttler, Marble and Blincoe, 1961). Blincoe
(1960) described two rate constants for the turnover of
75Se in the rat after intraperitoneal injection of sodium
selenite. These two constants, or a diphasic depletion
curve, suggested that absorbed selenium is transferred from
a rapidly-excreted form to another more-slowly-excreted
form in the body. The uptake, tissue distribution and ex-
cretory patterns of parenterally administered inorganic
selenium were subject to effects of previous selenium in-
take or selenium status of the animal (Muth gt gl., 1967).

Orally ingested inorganic selenium followed generally
the same pattern of tissue distribution and excretion as
parenterally-administered material (Grant, Thafvelin and
Christell, 1961; Ewan, Baumann and Pope, 1963).

In transport across cell membranes and in enzymatic
conversions selenium and sulfur were in general interchange-
able (Spencer and Blau, 1962). McConnell and Cho (1965)
using everted intestinal sacs in hamsters determined that
selenomethionine was transported against a concentration
gradient while selenite and selenocystine were not. The
transport of L-selenomethionine was inhibited by its sulfur
analog L-methionine. Sulfite and cystine did not inhibit
the transport of selenite and selenocystine, respectively.
It was possible for organic selenium analogues to support

some of an organism's functions. It was even possible for

39
an organism to adapt and to thrive in the presence of a
selenium compound that was originally inhibitory (Shrift,
1961).

Wright and Bell (1966) using 75Se-tagged sodium selen-
ite found distinct differences in the sites of absorption of
this material in sheep and swine. In sheep there was no net
absorption of selenite selenium in the rumen and a very
limited absorption from the abomasum. Resecretion of selen-
ium was noted into the first section of the small intestine
followed by considerable absorption of selenium from the
rest of the small intestine with negligible flux evident in
the cecum or colon. Net absorption of selenite selenium in
the whole gut was 36%. In swine selenium was resecreted
into the first section of the small intestine and then re-
absorbed from succeeding sections of the small intestine to
a much greater degree than in sheep to result in a net ab-
sorption of selenite selenium of 86%.

There are several compounds that will alter selenium
excretory patterns and selenium balance in the animal when
administered parenterally or in the diet. The most widely
recognized effects are those of sulfate or arsenic adminis-
tration. Unfortunately most such studies have employed much
greater than physiological levels of selenium in order to
monitor the effects and the application of results from such
studies to the situation in animals on normal selenium in-
takes is at best tenuous. Dietary sulfate alleviated up to

40% of the growth depression of chronic selenosis in rats

40
(Bonhorst and Palmer, 1957;Halverson and Monty, 1960).
Sulfate additions to the diet were much more effective
against chronic toxicity of sodium selenate than they were
against the chronic toxicity of sodium selenite or seleni-
ferous grains (Halverson, Guss and Olson, 1962). This pro-
tective effect of sulfate was most pronounced at selenium
levels in the diet approaching chronic to subacute toxicity
(Franke and Moxon, 1936; Miller and Williams, 1940). Sul-
fate was more effective than sulfite in effecting such pro-
tection. This antagonistic effect led to postulations
that increased dietary sulfur might increase the incidence
of white muscle disease, but Whanger E£._£° (1969) found no
such effect in lambs. With sulfate additions to the diet
there was very little change in the net absorption of selen-
ium. There was,however, an increase in urinary selenium
excretion and a decrease in the excretion of selenium into
the gastrointestinal tract. The net result was a slight
decrease in the tissue retention of selenium (Ganther and
Baumann, 1962b). Oral sulfate had little effect on the
distribution or excretion of injected selenium. These re—
sults suggested a competition with selenium for transport in
the gastrointestinal tract. In McConnell and Cho's studies
(1965) they did not test the selenate-sulfate competition
for transmucosal movement although they reported no competi-

tion between sulfite and selenite which is in agreement with

results just discussed.

41

Inorganic and organic arsenic compounds were effective
in reducing deaths from selenium toxicity. Workers in South
Dakota have demonstrated the beneficial effects of dietary
arsenic additions in selenium toxicities produced by selen-
ium salts or seleniferous grains in swine (Wahlstrom,
Kamstra and Olson, 1955, 1956; Wahlstrom and Olson, 1959a,b)
and poultry (Carlson gt gl., 1954; Carkxny Guss and Olson,
1962; Thapar SE.3$°' 1969). The reSponse to arsenic as with
sulfate was dependent on the level of selenium administered.
Within an hour after parenteral arsenic administration, an
increased excretion of an injected dose of selenium into the
gastrointestinal tract was noted (Ganther and Baumann,
1962a; Sastri and Baumann, 1965; Levander and Baumann,
1966a). The effect of injected arsenic on the retention
of injected selenium was not evident if the injections were
more than one hour apart (Palmer and Bonhorst, 1957).
Arsenite injections increased the blood level of selenium
three-fold and produced a twenty-fold increase in biliary
excretion of selenium administered in the form of selenite
to rats (Levander and Baumann, 1966b). Arsenite injections
increased selenate selenium excretion in the bile also, but
to a lesser extent. Paradoxically, arsenic reduced the loss
of selenium in expired air on high levels of selenium
(Kamstra and Bonhorst, 1953; Olson SE.§£" 1963; Levander
and Argrett, 1969). Levander and Argrett (1969) also found
that mercury and thallium exerted a similar effect while

lead did not. The administration of arsenic retarded the

42
transfer of selenium from blood to the liver and other tis-
sues with little or no effect on long term final tissue
selenium levels (Klug, Lampson and Moxon, 1950; Palmer and
Bonhorst, 1957; Ganther and Baumann, 1962a). The mechanism
that provided protection from toxic levels of selenium
without markedly affecting total carcass or tissue selenium
levels is still tolae explained.

Cadmium and phosphate also alter net selenium retention.
Cadmium had the opposite effect of arsenic in that it in-
creased selenium concentrations in blood and tissues and
depressed all major routes of selenium excretion resulting
in a 30 to 50% increase in retention of selenium over con-
trol values (Ganther and Baumann, 1962a). In yeast cells
Mahl and Whitehead (1961) found that phOSphate ion suppressed
selenite uptake, and selenite suppressed phosphate uptake.
This effect has evidently not been tested in animal systems.
Levander and Morris (1970) found that dietary supplements of
methionine and vitamin E or synthetic antioxidants protected
rats against 10 ppm selenium from sodium selenate. They
concluded that these treatments made 32 methyl group of
methionine more available to form major expiratory (dimethyl
selenide) and urinary (trimethyl selenide) detoxified selen-
ium excretion products. Some recent studies such as those
by Ganther _t gt. (1972) indicated that selenium in the
tissues may protect the animal body against the toxic ef-
fects of several heavy metals (cadmium, mercury, and tel-

lurium).

43

Tissue selenium concentrations were considerably higher
after feeding equal quantities of selenium in an organic
form than after feeding an inorganic form (Smith gt gl.,
1938; Ehlig _t gl., 1967). The distribution of radioactive
selenium taken up by plants and then fed to mice showed a
similar pattern of tissue selectivity as discussed earlier
for inorganic forms (Jones and Godwin, 1963). It is unfor-
tunate that in most of the absorption work reported in which
75Se was used the level of naturally-occurring selenium in
the test period diet or the previous level of selenium in-
take was not reported. These factors were shown to markedly
affect selenium absorption and retention in animals whether
it is administered by oral or parenteral routes (Hopkins,
Pope and Baumann, 1966). The fact that these factors were
not controlled or even reported in most cases makes it very
difficult to draw any general conclusions concerning selen-
ium absorption from much of the radioactive selenium work.

The major portion of the research concerning the trans-
port of selenium in the animal body has been conducted by
McConnell and co-workers using subcutaneous injections of
radioactive selenium salts in dogs. Twenty-four hours after
three injections of radioactive sodium selenate at 24-hour
intervals, 2 to 4% of the total 75Se administered was in the
circulating blood with 80% of the whole blood activity in
the erythrocytes and 20% in the plasma. Radioactive selen-
ium was found in similar concentrations in albumin,

globulin, euglobulin and pseudoglobulin fractions of serum

44
proteins. 'The selenium in these serum proteins was non-
dialyzable suggesting the presence of an organo-selenium
complex or compound. In erythrocytes selenium was found in
both the hemin and globin fractions of hemoglobin with the
greater concentrations in the hemin fraction. No 75Se
activity was detected in the bone marrow of these animals
(McConnell and Cooper, 1950). McConnell, Roth and Hoch
(1968) found that the apparent life span for selenium in
the canine red blood cell approximated that of the life
span of this cell. Withrow and Bell (1969) used 75Se to
estimate the erythrocytic life span in sheep and swine.
Weswig gt gt. (1966) and Wright and Bell (1963) found in-
creased rates of 75Se uptake in red blood cells from selen-
ium deficient sheep and suggested that this test might be
useful diagnostically.

McConnell and Roth (1964) found that injected radio-
active selenium was present in both protein and non-protein
bound forms in liver fractions studied. Levander, Young and
Meeks (1970) studied the effects of various dietary and £2
vitro treatments on selenium binding capacity of liver homog-
enates. In 1957 McConnell and Wabnitz working with liver
homogenates from a dog killed 48 hours after 75Se injection
found 3 different compounds in hydrochloric acid hydrolysates
which contained radioactive selenium. When paper chromato-
graphed, two of these compounds moved as cystine and methi-
onine and the third was located near the leucines. McConnell

and Hoffman (1972) concluded that in rat liver as in E, coli

 

45
selenomethionine is incorporated into polypeptides gig the
methionine pathway. Earlier work had both supported
(Rosenfeld, 1962; Ochoa-Solano and Gitler, 1968) and dis-
claimed (Cummins and Martin, 1967; Jenkins, 1968) the incor-
poration of inorganic selenium into animal proteins. These
studies have indicated in general that radioactive selenium
is rather rapidly incorporated into seleno-amino acids and
thence into proteins.

There has not been a specific transport protein identi-
fied for selenium. However, the occurrence of radioactive
selenium in the varied fractions of the blood after dosing
does not preclude thisgxssflfllity. A portion of such selen-
ium could be in transport complexes while the remainder
could be performing a structural function in seleno-amino
acids in the serum proteins. Dickson and Tomlinson (1967)
found the Q- and S-globulins of human serum to be the major
selenium-bearing proteins. The B-lipoprotein of Cohn frac-
tion III-O was found to contain the highest level of selen-
ium of the serum fractions checked. McConnell and Levy
(1962) found that only 3.5 to 8.8% of the total protein-
bound selenium of serum was bound to the o- and E-lipopro-
teins with a greater proportion in the a-lipoproteins. Burk
and Consolazio (1972) have reported rough characterizations
of selenium-containing rat plasma proteins using 75Se in-
jections.

McConnell (1948) demonstrated the passage of radioac-

tive selenium given as sodium selenate injections into the

46
milk of the white rat. McConnell and Roth (1964) demon-
strated the transfer of 75Se into the canine mammary gland
where it was incorporated into the milk proteins and the
passage across the placental membranes into pups. Radio-
active selenium was detected in pups delivered by Caesarean
section after 75Se administration. Jacobsson, Oksanen and
Hansson (1965) found that excretion of 75Se into the milk
of sheep was markedly higher when 75Se - selenomethionine
was administered than when 758e - sodium selenite was in-
jected. Baby mice contained 758e, which had been incorpor-
ated into the protein of lucerne leaves, within four hours
after the dam ate the leaves (Jones and Godwin, 1962). The
radioselenium distribution pattern in fetal tissues is simi-
lar to that in maternal tissues. Wright and Bell (1964)
concluded that there was a definite placental barrier to
selenium transport in sheep because single fetuses contained
twice the concentration of 75Se as twin fetuses after the
pregnant ewes received oral doses of 75Se.

The transport of absorbed selenium and its relation-
ship to selenium function in the body is poorly understood
and may remain so for some time due to the varied forms and
functional roles in which selenium is encountered in the

animal organism.

Excretion and secretion of selenium

 

It is not practical to separate secretions of selenium

from excretions of selenium in most animal studies. As

47
mentioned earlier the majority of selenium metabolism
studies have involved dosing animals with radioactive
selenium and monitoring the course of 75Se in the organism.
The failure to control or at least report the levels of
selenium in the diets or the previous exposure of the test
animals to selenium makes meaningful interpretation of the
data difficult.

The predominant excretory route depended on several
factors. These include species of animal, age of animal,
level of selenium, form of selenium, route of administra-
tion, other elements in the diet and previous exposure to
selenium (Ewan, Pope and Baumann, 1967; Gortner and Lewis,
1939). Much of the work reported has involved chronic to
subacute toxicity levels which results in an inverse pattern
of excretion to that seen with more physiological levels of
selenium (Paulson, Baumann and Pope, 1966).

Selenium in levels approaching chronic toxicity is
excreted primarily ytg the urine and feces in monogastrics
and ruminants when given orally or injected in inorganic or
organic forms. At the higher levels of this range the ex-
cretion of selenium in the expired gases may approach or
even exceed that in the urine (McConnell and Roth, 1966).
Westfall and Smith (1941) reported that animals consuming
toxic levels of selenium in the form of seleniferous grains
excrete up to 70% of the ingested selenium in the urine.
Rosenfeld and Beath (1945) stated that in chronic selenosis

the urinary excretion of selenium may decrease as the

48
condition progresses due to kidney damage. There was a
much higher urinary excretion of selenium in animals chron—
ically poisoned with inorganic selenium than when similarly
poisoned with naturally-occurring selenium in foods (Smith
_t _t., 1938). The proportion of administered selenium ex—
creted gig the urine after subcutaneous administration was
very similar to that seen after feeding of inorganic selen-
ium salts at chronically toxic levels. Selenium in the urine
was primarily in an organic form and was reported by Anderson
and Moxon (1941) to be extracted with the ethereal and
neutral sulfur fractions. In chronically selenized rabbits
only 15% of the urinary selenium was in an inorganic form.
Trimethyl selenide was reported by Byard (1969) to be a
urinary metabolite of selenite. Trimethylselenonium ion
has been identified as a general major urinary excretory
product of inorganic and organic selenium (Palmer gt al.,

1969; Palmer gt _l., 1970).
In ruminant animals approximately equal amounts of in-

jected or ingested selenium at chronically toxic levels were

excreted via the feces and urine (Rosenfeld and Beath, 1945;

 

Wright and Bell, 1964; Wright, 1965; Ehlig 324-: 1967).
With more physiological levels given orally to these ani-
mals the fecal route tended to predominate, especially when
naturally occurring organic forms of selenium are utilized
(Westfall and Smith, 1941; Butler and Peterson, 1961;
Cousins and Cairney, 1961; Petersen and Spedding, 1963;

Jacobson, 1966; Ehlig t gt., 1967; Handreck and Godwin,

49
1970). The major portion of the selenium in the feces of
ruminants was determined to be in some water-insoluble in-
organic form by Cousins and Cairney (1961) and to be un-
available to plants in a 75-day test by Peterson and Sped-
ding (1963). The selenium recovered in the feces naturally
included some of that excreted in the bile. Biliary excre-
tion accounted for one half or less of the selenium found
in the feces after subcutaneous injections of inorganic
selenium which indicates that selenium must enter the di-
gestive tract by pathways other than the bile (Jacobsson,
1966). The selenium in the bile led McConnell and Martin
(1952) to postulate that biliary selenium represented that
contained in degradation products of hemoglobin. The ad-
ministration of p-bromobenzene to ruminants was found to
increase selenium excretion and lower blood and tissue
selenium levels in selenized animals by Moxon _t _l. (1940).
Westfall and Smith (1941) reported that the same compound
had no effect on selenium excretion when administered to
rabbits on a seleniferous grain diet. Moxon gt gt. (1940)
theorized that p-bromobenzene conjugates with selenium and
the conjugate was excreted as p-bromophenylmercapturic acid.
Subcutaneously injected 75Se was recovered from the mercap-
turic acid fraction of dog urine. Since tissue-bound rather
than free amino acid sulfur was believed to be the source
of sulfur for mercapturic acid sysnthesis, McConnell,
Kreamer and Roth (1959) interpreted this finding as further

evidence that selenium appears as seleno-amino acids in the

tissues of the dog.

50

The selenium excretory pathways in monogastric farm
animals do not differ appreciably from those of ruminants
at chronic toxicity levels of injected or ingested selenium.
The urinary route seems to be more important with inorganic
forms than with naturally-occurring selenium in food.
Jensen, Walter and Dunlap (1963) reported that at physio-
logical selenium levels vitamin E did not affect relative
distribution of 75Se in the chick. Buescher, Bell and Berry
(1961) found that excessive calcium levels in normal selen-
ium diets did not alter the excretion or tissue distribution
of oral 758e in swine:

As stated previously, expiratory losses of selenium
vary greatly in their contribution to total selenium excre-
tion according to several factors. The most important of
these, as reported by Ganther, Levander and Baumann (1966),
were level of selenium, form of selenium and previous ex-
posure to selenium. Most of this work has been done with
small laboratory animals due to the problems involved in
collecting expired gases from large animals. Of historical
note is the fact that the odor of garlic on the breath of
animals afflicted with "alkali disease" in seleniferous
areas of the United States was a principle clue leading to
the establishment of selenium as the toxic factor in the of-
fending plants. The garlic-like odor of some purified prep-
arations of Factor 3 upon adding alkali also prompted
Schwarz and Foltz (1957) to test the effectiveness of inor-

ganic selenium compounds against dietary hepatic necrosis in

51

rats which led to the establishment of selenium as an es-
sential trace element.

with chronic to subacute toxicity levels of selenium,
whether injected or ingested, various researchers have re-
ported that 2 to 52% of the intake was recovered in the ex-
pired gases (Shultz and Lewis, 1940; McConnell, 1942;
Peterson, Klug and Harshfield, 1951; Olson gt gl., 1963;
McConnell and Roth, 1966). This rate of excretion was
altered by many factors as discussed under tissue distri-
bution (e.g., arsenite, level of selenium used, previous
exposure to selenium). At trace or physiological levels of
selenium administration, generally less than 1% of the
selenium intake was excreted ytg the lungs (McConnell,
1941; Hopkins, 1965; Handreck and Godwin, 1970). McConnell
and Roth (1966) reported that at these lower levels the
respiratory excretion of selenium was greater when admin-
istered in an inorganic form than when administered as
selenomethionine. It has generally been accepted that
selenium was excreted ytg the lungs after methylation in
the liver in the form of dimethyl selenide. Ganther (1966)
has demonstrated the enzymatic synthesis of dimethyl selen-
ide in mouse liver homogenates $2 vitro. Arsenite inhibited
dimethyl selenide formation by this it vitro system. Mc-
Connell and Portman (1952b) found that 20% and 79% of di-
methyl selenide administered subcutaneously to rats was
eXpired in the first 10 minutes and the first 6 hours,

respectively. Using 75Se sodium selenate they were able to

52
verify that selenium was methylated in the animal organism
and appeared in the expiratory gases as dimethyl selenide.
These same workers (McConnell and Portman, 1952a) deter-
mined that selenium was 300 times less toxic in the form
of dimethyl selenide than in the form of sodium selenate or
selenocystine. A check of procedures during a study by
Heinrich and Kelsey (1955) suggested that there were signi-
ficant losses of 75Se from excreta and tissues when dried
before measuring radioactivity. This would suggest the
presence of volatile selenium compounds in the tissues as
well as the excreta.

Other routes of selenium excretion are largely unex-
plored. One 75Se study with lambs produced a very high 75Se
concentration in the pelt (Lopez _t_gl., 1969). Since these
lambs were killed at 12 or 15 days after dosing this could
have been due to selenium excretion in sebaceous secretions
or perspiration as the authors suggest or probably more
likely was due to fecal and urinary contamination of the
pelt. Some of the activity could also arise from the
deposition of seleno-amino acids in the proteins of hair
and skin.

The lack of sensitive analytical techniques for selen-
ium until rather recently has resulted in the accumulation
of considerable data in the literature on selenium balance
in animals which was difficult to evaluate due to the lack
of information concerning modifying factors in the experi-

mental conditions and materials. It would seem that

53
radioactive selenium compound studies, as useful as they
have been in studying the absorption and excretion of this
nutrient, will not provide all the answers in this area.
The ease of monitoring the course of these tagged compounds
in the body has frequently resulted in poorly-documented
studies some of which have served to confuse as much as
clarify.

It is evident that there are many factors that affect
the absorption and excretion of selenium. Lindberg and
Lannek (1965) stated the conviction that studies with
physiologically-compatible levels of selenium indicated
that animals will retain inorganic selenium in proportion
to their physiological needs. Once these physiological
stores are filled, the animal will excrete the remainder
unless detoxification and excretory mechanisms are over-
whelmed, in which case significant quantities of selenium
will be retained and selenium intoxification may result in

extreme cases.

Selenium Content of CrOps

 

Factors affecting selenium content of crOps

 

Geographical considerations

 

The selenium content of native and crop plants is a
function of the available selenium content of the soils in
which they grow. Several excellent surveys of the selenium

content of plants in the United States have been conducted.

54
Muth and Allaway (1963) correlated the incidence of white
muscle disease with the known distribution of selenium in
soils. They delineated the extensive areas around the Great
Lakes, the northern part of the Appalachian Plateau and
New England as an area where white muscle disease was a
problem and related this to the parent materials of soils
in these areas. Another area where white muscle disease was
recognized as a serious problem was in the extreme north-
western United States. In 1964, Allaway and Hodgson con-
cluded that white muscle disease is fairly common in areas
of the United States where forages contain less than 0.1 ppm
selenium. They also reported that forages obtained from the
"western part of the Cornbelt", a region where white muscle
disease was rare, were markedly higher in selenium content
than forages from areas where white muscle disease was a
problem. These levels of selenium in forages, however, were
much lower than recognized toxic levels. Kubota gt gl.
(1967) selected alfalfa as the plant to be sampled over the
United States after corn and soybeans were found to be simi-
lar in selenium content to alfalfa in the same area. They
classed the Pacific Northwest and southeastern United States
(Florida and the seaboards of Georgia, South Carolina and
North Carolina) as very low selenium areas (80% or more of
the alfalfa samples from these areas contained less than
0.05 ppm selenium). The authors recognized that white
muscle disease was not a problem in this southeastern area

and postulated that an increased toc0pherol intake due to a

55
much longer grazing season might be sufficient to protect
grazing animals in these areas. Most of Michigan, the Corn-
belt and New England were classed as low selenium areas
(80% or more of all samples contained less than 0.10 ppm
selenium; 65% contained less than 0.05 ppm selenium and 31%
contained 0.05 to 0.1 ppm selenium). Eastern Wisconsin and
the eastern thumb area of Michigan to near Lansing, were
classed as variable selenium areas (26% of the samples con-
tained less than 0.05 ppm selenium, 18% contained 0.05 to
0.1 ppm selenium, 49% contained 0.1 to 0.5 ppm selenium and
only 7% contained 0.5 to 1.0 ppm selenium). In a survey of
wheat and wheat products from seleniferous areas of the
United States, Williams, Lakin and Byers (1941) reported
that most samples contained subtoxic levels of selenium
(83% contained 1 ppm or less and 92.5% contained 4 ppm or
less). There was considerable variation in selenium content
within areas. For example in Tripp County, South Dakota
corn "grown north of town" contained 2 ppm selenium while
corn "grown south of town" contained only 0.1 ppm selenium.
Patrias and Olson (1969) found similar variations in the

selenium content of corn samples from some Midwestern states.

Species differences

 

Miller and Byers (1937) proposed three classes of
plants with regard to their ability to accumulate and tol-
erate selenium. Class I was made up of plants that absorb

selenium readily. In this group were some but not all

56

species of the Astrggalus, Stanleya, ApploEappus and Xylor-
rhiza genera. Class II consisted of plants that are able
to absorb moderate, or even large, quantities of selenium
without severe injury. In this class were the common
cereals (wheat, rye, barley, and corn) and several native
shrubs and grasses. Class III was made up of plants that
have a very limited tolerance of selenium and are able to
absorb only small quantities even when grown on selenifer-

ous soils. Examples of this group are buffalo grass and

 

the grama grasses, Bouteloua gracilis and B. curtipendula.

 

Virupaksha and Shrift (1965) reported that in selenium
accumulator plants 75Se in selenite was incorporated pri-
marily into Se-methyl (75Se) selenocysteine, while Se-
methyl (75Se) selenomethionine could only be found in traces.
The opposite situation was found to be true in non-accumu-

lator species of the Astragalus genus. These findings were

 

borne out by the studies of Olson _t gt. (1970) with wheat.
Beeson (1961) states that only "organic" forms of selenium
were detected in alfalfa, young wheat plants and yellow
sweetclover or in barley, corn and oat grains. In contrast
to this, high levels of inorganic water-soluble selenium
were found in accumulator and indicator plants. He also
concluded that grasses in general were poor accumulators of
selenium. Peterson and Butler (1962) reached similar con-
clusions in a greenhouse radioisotOpe study utilizing

plants from each of Miller and Byers' classes mentioned

above. These same authors (1966), using the insoluble

57
inorganic selenium present in sheep feces, found that

browntop (Agrostis tenius) absorbed the most selenium,

 

ryegrass absorbed an intermediate quantity and red clover
absorbed the least selenium of the three Species examined.
Davies and Watkinson (1966) reached similar conclusions
using similar plant types. As mentioned earlier, Kubota

_t _t. (1967) concluded that corn, soybeans and alfalfa

in a given area had similar selenium contents. In an
excellent study Ehlig gt_gt. (1968) monitored species
differences in selenium uptake on soils low in avail—

able selenium under both field and greenhouse condi-

tions. Such soils are associated with enzootic areas of
selenium deficiency. Alfalfa (and dicotyledonous plants

in general) increased selenium accumulation in proportion

to dry matter production while timothy (and other mono-
cotyledonous plants) did not. Therefore the selenium con-
centration in grasses decreased with increases in the rate
of dry matter production, whereas the selenium concentration
in dicotyledon species did not decrease with increases in
the rate of dry matter production. The relative differences
observed in selenium accumulation by the different species
were much less than the species differences observed on
soils high in available selenium. Scott and Thompson (1971)
reported that within a given geographical area, higher pro-
tein feedstuffs contained proportionately more selenium than
lower protein materials. The high protein material tested

most commonly was soybean meal, thus lending some additional

58
credence to the findings just discussed. Some of the dif-
ferences between grasses and clovers might be due to the
high levels and the inorganic forms of selenium added in

most of these greenhouse studies.

 

Soil and climatic effects

Selenium is generally present in soils in selenides,
sulfide ores, iron pyrites, pyritic concretions, selenates,
organic seleno-compounds, selenites and small amounts of
elemental selenium (Rosenfeld and Beath, 1964). Cary and
Allaway (1969) reported that in some acid-to-neutral soils,
water—soluble selenite salts reacted rapidly with the solid
phase to form a relatively insoluble absorption complex.

The selenium in this complex was less available to alfalfa
than the selenium added to the same soil as Fe(OH)3 - HSeoa.
These authors found that in this particular soil the oxida-
tion of elemental selenium was not pH dependent. They sug-
gested that soluble selenite might be a practical form of
selenium to add to low-selenium soils in order to produce
selenium-adequate crops. Geering gt gt. (1968) reported

the solubility of selenium in seven soils indicated that
selenium concentration in the soil solution was governed
primarily by a ferric oxide-selenite adsorption complex in
which selenium was in an oxidation state of +4. Their data
indicated that over a pH range of 4 to 8 a one-fold increase
in pH doubled the concentration of Se+4 in the soil solution

and that beyond a pH of 8 the adsorption complex completely

59
broke down. While working in seleniferous areas, Miller
and Byers (1937) concluded that soil pH did not affect
selenium absorption by plants. All soils they studied,
however, as is characteristic of most seleniferous areas,
were quite alkaline (pH 7.5 to 8.4).

The forms of selenium added to greenhouse cultures of
plants had less marked effects on selenium accumulation by
plants than they did on the retention of selenium by ani-
mals (Hurd-Karrer, 1935; Hamilton and Beath, 1963a, b, 1964).
This lack of differences between the forms added may have
been the result of a conversion of added soil selenium to a
common intermediate by chemical and microbiological proces-
ses taking place in the soil. Studies concerning the feasi-
bility of fertilizing pastures with selenium have indicated
that this could be a solution to the production of selenium
deficient crops; but many problems can arise due to varia—
tions in soil and plant types and modes of application
(Grant, 1965; Allaway _t gt., 1966; Cary, Wieczorek and
Allaway, 1967).

In a survey of selenium deficient soils in Western
Australia, Gardiner and Gorman (1963) noted a tendency for
plant selenium levels to be higher in areas of heavy soil

and lower in areas of high rainfall.

l. (1966) and Allaway, Cary and Ehlig

Allaway gt
(1967) have done an excellent job of discussing rationally
the cycling of physiological levels of selenium from soils,

through plants and animals and back to the soil again.

60

They also discussed the merits of a natural selenium cycle

proposed by Shrift in 1964.

Selenium levels of common feedstuffs

 

Morris and Levander (1970) have conducted a rather ex-
tensive survey of the selenium content of human foodstuffs.
They concluded that a well-balanced human diet was likely
to be adequate in selenium. Higgs _t _t. (1972) reported
variable effects of cooking on the selenium content of
human foods. Some information from these studies is per-
tinent to our consideration of animal feeds. Grain products
varied widely in their selenium content (from 0.025 ppm for
corn flakes to 0.66 ppm for barley cereal). Whole wheat
flour and bread contained two to four times more selenium
than did white flour or bread. Seafoods were generally
higher in selenium content than other meats.

Patrias and Olson (1969) reported selenium levels of
135 samples of corn from several midwestern states. These
ranged from a low of 0.01 ppm for a sample from Indiana to
a high of 2.03 ppm for a sample from South Dakota with an
average of 0.11 ppm selenium. Sixty percent of the samples
contained 0.05 ppm selenium or less. Five samples col-
lected from the southwestern section of Michigan ranged
from 0.03 to 0.04 ppm selenium with an average of 0.03 ppm
selenium. Corn samples from Indiana and Illinois had mean
selenium levels of 0.04 and 0.05 ppm selenium, respectively.

Arthur (1970) reported values for a general survey of

61

Canadian animal feedstuffs. Of the grains analyzed, corn
was the lowest in selenium content (average of 0.04 ppm)
and Durum wheat was the highest (average of 1.47 ppm).
Bran, shorts and middlings contained more selenium than
the wheat itself. The western-grown grains were consider-
ably higher in selenium content than those grown in central
Canada. Soybean meal averaged 0.13 ppm selenium. In pro-
ducts of animal origin, milk products were lowest (0.04 to
0.17 ppm) in selenium and fish by-products were highest
(1.65 to 2.86 ppm). Products of the meat packing and
poultry industry contained intermediate selenium levels.
These values for fish meal agreed well with those reported
by Kifer and Payne (1968) and Kifer, Payne and Ambrose (1969).
Scott and Thompson (1971) reported the selenium content of
several common poultry feedstuffs. They concluded that the
selenium content of these feedstuffs depended largely upon
the selenium content of the soils on which they were grown.
Within a given geographical area they noted that the higher
protein feedstuffs contained proportionately more selenium
than lower protein materials. Dent corn from Lafayette,
Indiana and New York State contained 0.025 ppm while Opaque-
2 corn from Lafayette, Indiana contained 0.0?8 ppm selenium.
Other corn samples from the midwestern United States and the
Nebraska-South Dakota area contained 0.05 and 0.38 ppm
selenium, respectively.

The selenium content of plants is influenced by many

factors; but it is quite evident that the most important of

62
these is the level of available selenium in the soil in
which these plants grow. The level of available selenium
in the soil is likewise altered by many factors. The most
relevant determinant of soil selenium levels is evidently

the geological derivation of the soil in question.

EXPERIMENTAL PROCEDURE

Introduction

 

Three feeding trials with growing-finishing swine and
six selenium balance studies with young pigs were conducted
over a two year period. A survey of the selenium levels of
17 samples of Michigan 275-2X hybrid corn grown in 13 dif—
ferent counties in Michigan and 20 varieties of hybrid corn
grown at one location in Clinton County, Michigan was cone
ducted. These samples were collected in the fall of 1969.
Our intent in these latter studies was to determine if
there were significant location, soil pH, varietal or yield
effects on the selenium content of corn in Michigan.

A brief description of the ten experiments conducted
follows:

Feeding trials

1. Selenium or vitamin E supplementation of practical
diets for finishing swine. A preliminary study.

2. Selenium and/or vitamin E supplementation of practical
diets for growing-finishing swine. Included effects of
sow pretreatment on pigs.

3. Selenium and/or vitamin E supplementation of practical
diets for growing-finishing swine. Further definition
of levels of supplementation and effects of selenium

withdrawal.
63

 

64
Selenium Balance Trials

4. Selenium balance in the young pig. A preliminary study.

5. Selenium balance in the young pig. Further definition
of levels of supplementation. No added vitamin E.

6. Selenium balance in the young pig. Repeat of Experi-
ment 5 with added vitamin E.

7. Selenium balance in the young pig. Effect of inorganic
or natural selenium sources with or without added
vitamin E.

8. Selenium balance in the young pig. Fresh Xi’ un-
stable sodium selenite premix and effect of added
vitamin E.

9. Selenium balance in the young pig. Repeat of Experi-
ment 8 after pigs had received respective diets in
self-feeders for 17 days.

Corn Study
10. Survey of location, soil pH, variety and yield effects

on selenium content of Michigan—grown corn.

General Conduct of Experiments

 

The experimental animals used in these studies were
Yorkshire, Hampshire or crossbred pigs obtained from the
Michigan State University Swine Research herd. In most
cases the treatment of the dams with regard to dietary
vitamin E supplementation was considered to be of possible
pertinence to the outcome of experiments with their off-

spring. This history will be noted in the discussion of

65
individual experiments. Good animal husbandry practices
were applied prior to placing the young pigs on experiment.
Needle teeth were clipped and each pig was individually ear
notched at one day of age. The pigs received an injection

of 100 mg of iron as iron dextran1

at three days of age.
All males were castrated and well-healed before being
placed on experiment with the exception of two balance
trials (experiments 5 and 6) in which the males were in-
tact. These pigs were routinely weaned at 5 to 6 weeks of
age and had been offered creep feed prior to weaning.
Feeding trials 1 and 2 were conducted in an open-
fronted building with concrete floors. The back portions
of these pens were bedded lightly in severe weather. Feed-
ing trial 3 was conducted in a completely-enclosed, slotted-
floor, environmentally-controlled building. In most cases
the floor space allotted per pig was somewhat excessive in
the early part of the trials and adequate in the latter
parts of the feeding trials. Feed and tap water (less than
1 ppb selenium) were provided gg libitum. These animals
were weighed initially and biweekly thereafter. Feed con-
sumption was determined for each weigh period. They were
observed daily. All dead animals were necropsied as soon as
was feasible following their discovery. Routine gross,
histOpathological and microbiological examinations were

performed on all dead animals by the staff of the Veterinary

Diagnostic Laboratory at Michigan State University.

 

1Armidexan. Bradley Products Company, Division Armour
Pharmaceutical Company, Omaha, Nebraska 68103.

66

The balance trials were conducted in a well-ventilated
metabolism area with thermostatically controlled (29 C) hot
water heat. The pigs were housed in stainless steel-lined
metabolism cages. Three times daily they were removed to
stainless steel-lined feeding cages where they were offered
a near gg libitum portion of finely ground diet as a gruel
with deionized water. The basal diet in all the selenium
balance trials had the formulation of the basal grower diet
in experiment 3 (table 1). Immediately after thay had
finished eating, their snouts were wiped clean, and they
were returned to the metabolism cages. After a short train-
ing period these young pigs would clean up the diet offered
in a 3 to 5 minute period. The feed intake data were ad-
justed for any refused feed by drying this material,
weighing it, adjusting this weight to air-dryness and sub-
tracting this quantity from the total offered. Urine was
collected in 6 N hydrochloric acid separate from the feces.
The total volume voided was measured and an aliquot frozen
for subsequent analysis. The feces were spread on trays to
air dry before being ground for analysis. The metabolism
cages and accessories were scrubbed and rinsed with deion-
ized water before the 3-day collections began. The feeding
cages and troughs were rinsed with deionized water after
each feeding.

Diets for the feeding trials were mixed in a two-ton
horizontal batch mixer. They were stored in 50 pound kraft

polywall bags until fed. Diets for the balance trials were

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68
mixed in 10 kg batches in a stainless steel institutional-
type mixer2 and stored in large plastic bags at room temper-
ature. Sodium selenite3 was the source of supplemental
selenium in all trials unless specified otherwise. Selenite
premixes were made up in cerelose (glucose monohydrate)4
and finely ground corn and substituted for corn in the
formula. The source of added vitamin E in all trials was
d-o-tocopheryl acetate.5 All diets were analyzed for selen-
ium and some were analyzed for a-tocopherol.

Animals from the feeding trials were slaughtered at ei-
ther the Michigan State University Meats LaboratoryCX'atPeet
Packing Company in Chesaning, Michigan, In the case of those
slaughtered at the former locajon,aithorough examination of
the vhwra and carcass was possible while at the latter loca-
tion only a cursory examination was possible. These feeding
trials were conducted under agreement with and inspection
by the Food and Drug Administration of the U.S. Department

of Health, Education and Welfare (FDA) who specified a 60-day

 

2Reco 22 quart four-speed mixer, Reynolds Electric Company,
River Grove, Illinois.

3Alfa Inorganics, Inc., Beverly, Massachusetts.
4Corn Products Company, Argo, Illinois.

5Myvamix-Type 125. 275.6 IU vitamin E per gram as d-a-toco-
pheryl acetate with dextrin. Distillation Products Indus—
tries, Rochester, New York. Note: 22 IU vitamin E per
kilogram of diet is equivalent to 20,000 IU vitamin E per
ton .

69
minimum withdrawal period before selenium—supplemented swine

could be utilized for human consumption. Therefore, several
of the carcasses from these trials had to be burned or
tanked per their directions.

The survey of corn selenium levels was made possible
through the unselfish cooperation of Dr. E. C. Rossman of
the Crop and Soil Sciences Department on campus. The sam-
ples were collected in an annual outstate corn hybrid com-
parison test. The details of handling these will be deline-

ated later.

Experimental Details

 

Experiment 1. Eighteen purebred and crossbred pigs

 

weighing approximately 45 kg were allotted to three dietary
treatments, housed six to a pen in the open-fronted building
and managed as described above. The dams of these pigs had
not received added vitamin E in their gestation or lactation
rations. The following diets were fed for 8 weeks: (1)
basal (table 1) (0.040 ppm selenium as fed), (2) basal + 22
IU vitamin E per kilogram of diet and (3) basal + 1.0 ppm

selenium. The basal diet was a standard 12.5% crude protein
fortified corn soybean meal diet with arsanilic acid added
in an effort to accentuate the selenium needs of these pigs.

This experiment was conducted during May and June, 1969.

Blood samples were collected from the anterior vena
cava initially and at the end of the experiment. Hemo-
globin, hematocrit and serum glutamic-oxaloacetic trans-

aminase (SGOT) determinations were made initially and at

70
the end of the experiment. Total serum protein determina-
tions and electrOphoretic separations of serum proteins
were also conducted on the final serum samples. Albumin to
globulin (A/G) ratios were calculated from the electro-
phoresis data. Serum samples were frozen for subsequent
selenium analyses. Three pigs from each of the basal and
the basal + 1.0 ppm selenium groups were slaughtered after

8 weeks on experiment. They were examined at slaughter for

 

lesions, and tissue samples were obtained for subsequent
histopathological examination and selenium analysis. Sam-
ples of liver, myocardium, skeletal muscle (in most cases
iliopsoas muscle), kidney and pancreas were examined histo-
logically. Samples of liver (left or right central lobe),
kidney (cross-section excluding renal pelvis), heart (left
ventricle), three skeletal muscles (ham, loin and shoulder)

and serum were analyzed for selenium.

Experiment 2. One hundred eight purebred and cross-

 

bred pigs averaging 8.9 kg were assigned to nine dietary
treatments in a 2 x 2 x 3 x 3 factorial arrangement. They
were housed 12 to a pen in the open-fronted building and
managed as described above. The pigs' dams had been on a
previous experiment concerning the supplementation of gesta-
tion and lactation diets with d-a-tocopheryl acetate. Six
male and six female pigs were assigned to each dietary
treatment. One-half of the pigs in each group were off-

spring of sows maintained in concrete drylots with no

71
supplemental vitamin E in the diet during gestation and
lactation. The other half were offspring of sows which re-
ceived 22 IU of supplemental vitamin E per kilogram of diet
in concrete drylots during gestation and lactation. The
dietary treatments are presented in table 2. The basal diet
(table 1) was a standard 16% crude protein fortified corn
soybean meal diet with the exception that the corn was pur-
chased from a local farm with a history of pig losses con-
firmed by necropsy to be due to selenium and/or vitamin E
deficiency. Corn from the same supply was utilized in the
13% crude protein "withdrawal" diet (table 1) and assayed
0.016 ppm selenium as fed and 0.018 ppm on a dry basis.

Blood samples were taken from four pigs per treatment
as in experiment 1 for hemoglobin, hematocrit, total serum
protein and SGOT determinations, as well as for electro-
phoretic separation of serum proteins initially and after
10 weeks on experiment. Blood samples were also taken the
day prior to slaughter from the 27 pigs slaughtered to ob-
tain tissues for histological examination and selenium
analyses and were used for the same hematological determina-
tions..

The animals were observed closely for clinical signs
of the deficiency or other health problems. Samples of the
following tissues were taken at necropsy or at slaughter
for histological examination: liver, myocardium, skeletal
muscle, kidney, adrenal gland and pancreas. Only longissi-

mus muscles were analyzed for selenium in this study,

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73
although in addition to these, samples of myocardium, liver,
lung, kidney and spleen were collected and frozen at
slaughter. Initial and 10-week serum selenium levels were
determined for the four pigs per treatment that were sampled.
Final serum selenium levels were also measured in the 27
pigs slaughtered to obtain information on tissue selenium
levels.

All 108 pigs received their respective dietary treat-
ments for a period of 14 weeks. After this time, all re-
maining animals were fed the 13% crude protein "withdrawal"
diet (table 1) in order that we might salvage most of these
animals for human consumption by feeding them non-selenium-
supplemented diets for 60 days prior to slaughter as re-
quired by the FDA. Three animals from each of treatments 1,
2 and 3 were slaughtered after 0, 30 and 65 days of selenium
withdrawal to obtain tissues needed for histOpathological
examination and tissue selenium analyses. These pigs con-
stitute the group of 27 mentioned earlier. The remaining
animals were either retained for breeding purposes or
slaughtered at Peet Packing Company after 65 days of selen-
ium withdrawal.

Weight gain and feed consumption data reported in this
experiment represent these parameters over the first 14
weeks of the trial only. When selenium withdrawal was
instituted, some pens were consolidated (within reSpective

previously-added selenium levels) in order to conserve

 

1:.

74
space in the finishing pens. This trial was conducted from

July to December, 1969.

Experiment 3. Seventy-two purebred and crossbred pigs

 

weighing an average of approximately 14.4 kg were assigned
to eight dietary treatments. They were housed nine per pen
in the completely-enclosed, slotted-floor, environmentally-
controlled building and managed as outlined above. Since
the pen partitions in this building were not solid, the pens
were arranged in such a manner that pens receiving like
levels of added selenium were adjacent, with another pen of
pigs (not involved in this trial) serving as a buffer be-
tween the pairs of pens on different levels of added dietary
selenium to minimize fecal contamination problems. These
pigs were all offSpring of sows maintained in concrete dry-
lots with no supplemental vitamin E during gestation or
lactation. The dietary treatments are outlined in table 2.
The basal grower diet (table 1) was a standard 16% crude
protein fortified corn-soybean meal diet which assayed 0.052
ppm selenium as fed and contained no supplemental vitamin E.
The corn used in this diet contained 0.026 ppm selenium and
the soybean meal 0.087 ppm selenium, both as fed. This
trial was conducted from May to September, 1970.

After 11 weeks on trial (average weight of approxi-
mately 62 kg) all pigs were switched to the 13% crude pro-
tein finisher diet in table 1. Three pigs from each selen-

ium-supplemented group (a total of 18 pigs) were moved into

75
a commercially-built portable swine nursery. The smaller
pens in this house-trailer-sized unit had completely-slot-
ted floors of aluminum slats. These 18 pigs received the
13% crude protein finisher diet plus their originally as-
signed levels of selenium and vitamin E supplementation
until slaughtered 60 days later. These eighteen pigs will
be referred to as the non-withdrawn pigs (added selenium
fed until slaughter). The remaining pigs were housed in
their original pens and fed the 13% crude protein finisher
diet either with or without supplementary vitamin E, as
they had received in their respective original diets, until
slaughtered 60 days later. These pigs will be referred to
as withdrawn pigs (added selenium withdrawn 60 days prior
to slaughter) in later discussions.

All animals were observed closely for clinical signs
of deficiency or other health problems. Blood samples were
collected from the anterior vena cava of the 18 non-with-
drawn pigs and three pigs from each of the eight withdrawn
groups (a total of 42 pigs) the day before slaughter.
Heparinized whole blood and serum samples were retained for
hematocrit and selenium determinations. After hematocrit
determinations were made, both the whole blood and the
serum samples were frozen in acid-washed vials at -20 C
until needed for selenium analyses. Samples of the follow-
ing tissues were collected at slaughter from these 42 pigs

placed in plastic bags,6 and frozen at -20 C until selenium

 

6Whirlpak bags, Nasco, Inc., Fort Atkinson, Wisconsin.

76

analyses COUld be conducted: longissimus muscle, myo-

 

cardium, liver and kidney. Samples of the following
tissues were taken from the 42 sampled pigs described
above for histOpathological examination: liver, myo-

cardium, skeletal muscle, kidney and pancreas.

Experiment 4. Three male and three female crossbred

 

littermate pigs weighing approximately 12 kg were used in

a preliminary study of the influence of added inorganic
dietary selenium upon selenium excretion and retention.

The pigs were housed and managed as previously discussed
for a 1-week adjustment period and a subsequent 3-day col-
lection period while consuming a constant daily intake

(450 g) of diet. The dietary treatments were: (1) basal
(table 1) (0.042 ppm selenium as fed), (2) basal + 0.1 ppm
selenium and (3) basal + 0.5 ppm selenium. The dam of this
litter had received 22 IU of supplemental vitamin E per

kilogram of diet during gestation and lactation.

EXperiment 5. In this trial a litter of 12 Yorkshire

 

pigs weighing an average of 9 kg was selected to study the
effects of more moderate levels of dietary selenium supple-
mentation than those employed in experiment 4. One pig in
this litter never adapted to the meal-feeding regime and
had to be excluded from the trial. Thus three pigs were
assigned to each of diets 1, 2 and 3 while there were only
two pigs on diet 4. The dietary treatments were: (1)

basal (table 1) (0.048 ppm selenium as fed), (2) basal +

77
0.05 ppm selenium, (3) basal + 0.1 ppm selenium and (4)
basal + 0.2 ppm selenium. The pigs' dam had not received
supplemental vitamin E during gestation and lactation.

The pigs were housed and managed as previously de-
scribed for a 10-day adjustment period and a subsequent 3—
day collection period while consuming a constant daily in-
take (375 or 450 g) of the test diets. Blood samples were
collected from the anterior vena cava of each pig initially
and at the end of the collection period. Serum samples
were stored in acid-washed vials at -20 C for subsequent

selenium analyses.

Experiment 6. This experiment was a continuation of
experiment 5 and involved the same experimental animals and
diets with the exception that all were supplemented with 22
IU vitamin E per kilogram of diet. The basal in this ex-
periment assayed 0.053 ppm selenium. These vitamin E-
fortified diets were fed to the pigs for a 4-day adjustment
period and a subsequent 3-day collection period. During
this trial all pigs consumed 450 g of diet daily. Blood
samples were collected again at the end of the second col-

lection period. Serum samples were stored as above.

Experiment 7. Twelve crossbred littermate pigs from a

 

sow that had received no supplemental vitamin E during ges-
tation or lactation, with an average initial weight of 5.4
kg were assigned to one of four dietary treatments in a

study of the effect of inorganic XE' natural selenium on

78
selenium balance in the young pig. The pigs were housed
and managed as previously discussed for a 6-day adjustment
period and a subsequent 3-day collection period while being
offered 210 g of diet daily.

The dietary treatments were: (1) basal (table 1)
(0.041 ppm selenium as fed) + 0.2 ppm selenium as sodium
selenite, (2) basal + 0.2 ppm selenium as sodium selenite
+ 22 IU vitamin E per kilogram of diet, (3) basal + 0.2 ppm
selenium as seleniferous corn and (4) basal + 0.2 ppm
selenium as seleniferous corn + 22 IU vitamin E per kilo-
gram of diet. The seleniferous corn, graciously provided
by Dr. Oscar Olson of South Dakota State University, had
been harvested 10 years before in a seleniferous area of
that state. Our analysis of this material indicated it con-
tained 25.1 ppm selenium which is in good agreement with
the value of 24.5 ppm obtained in Dr. Olson's laboratory.
This corn was finely ground and substituted for an equal
quantity of whole diet in diets 3 and 4 (80 g per 10 kg
batch). Blood samples were collected initially and at the
conclusion of the excreta collection. Samples of heparin-
ized whole blood were used for hematocrit determinations,
then frozen in acid-washed vials at -20 C for subsequent
selenium analyses. Serum samples were also stored in a

like manner for future selenium assay.

EXperiment 8. Nine crossbred littermate pigs, from a

 

sow that had received no supplemental vitamin E during

79
gestation and lactation with an average initial weight of
6.4 kg were assigned to one of three dietary treatments, in
a study to determine if the feeding of a seemingly unstable
sodium selenite premix would affect the excretion or reten-
tion of added dietary inorganic selenium by the young pig.

In November of 1969 a selenium premix was prepared
from anhydrous sodium selenite and cerelose. The selenite
was ground in a mortar and pestle with a small portion of
cerelose. The pestle was then "rinsed" by grinding several
small portions of fresh cerelose in it. This material was
all transferred to a 4-liter brown glass jar where it was
mixed by shaking with the remainder of 3 kg of cerelose to
produce a premix containing 1500 ppm selenium. This materi-
al was stored in the same jar in a laboratory cabinet at
room temperature. A 15 ppm selenium premix was prepared by
dilution of the original premix with cerelose. This materi-
al was stored in a 3-quart freezer container in a walk-in
cooler.

In August of 1970, approximately 9 months after the
premixes were prepared, the more concentrated one in the
brown jar was noted to have a pink tinge and pink to dark
red lumps of material in the preparation when some of it
was being weighed out for use. One could also detect a
musty, sweetish odor from this material. The 15 ppm selen-
ium premix in the cooler was checked at this time and found
to have a weaker, but similar musty odor and scattered

lumps of pinkish material were evident in it also. The

80
1500 ppm premix just described is the “unstable" sodium
selenite premix utilized in experiments 8 and 9.

The dietary treatments were: (1) basal (table 1)
(0.044 ppm selenium as fed) + 0.2 ppm selenium as a fresh
sodium selenite in cerelose premix, (2) basal + 0.2 ppm
selenium as a fresh sodium selenite in cerelose premix +
22 IU vitamin E per kilogram of diet and (3) basal + 0.2
ppm selenium as the old unstable sodium selenite in cerelose
premix.

The pigs were housed and managed as previously de-
scribed for a 7-day adjustment period and a subsequent 3-
day collection period while being offered 300 g of diet a
day. Blood samples were collected initially and at the con-
clusion of the collection period for serum and whole blood

selenium determinations as well as hematocrit determinations.

Experiment 9. The nine pigs of experiment 8 were pen-

 

ned in groups of three in large stainless steel-lined feed-
ing cages with their respective diets available in self
feeders for 17 days. At the end of this self-fed period
they were returned to the metabolism cages for a 5-day ad-
justment period followed by a 3-day collection using the
same management procedures and diets as in experiment 8.

At the initiation of this collection period the pigs had an
average weight of 13.3 kg. The basal diet contained 0.047
ppm selenium as fed. Blood samples were collected at the

end of the collection period for the determination of

81

hematocrit, whole blood selenium and serum selenium.

Expgriment 10. The samples of Michigan 275-2X (a
single cross hybrid corn) were collected in an outstate com-
parison of corn hybrids conducted in 1969. The collection
of these samples was supervised by Dr. E. C. Rossman of the
Crop and Soil Sciences Department. Information concerning
cultural practices and growing season data were compiled by
him or his assistants. This hybrid was selected because it
is a popular hybrid in the state and was planted at all loca-
tions. This is an early maturing hybrid. Corn cannot be
successfully grown for grain in all portions of Michigan,
therefore, most of the trials reported here were conducted
in the southern two-thirds of the lower peninsula.

All plots were planted between May 1 and May 28, 1969.
Wet, cold weather delayed field work during May and June
resulting in some delayed plantings and water damage. Ex-
cessive to adequate soil moisture existed until late July.
Temperatures were below normal during most of the season.
August rainfall was very light with at least five consecu-
tive weeks with little or no rainfall. September was dry
and warm. Absence of a general killing frost in September
and early October favored development of late-maturing corn.
Light to moderate frost occurred in some areas on October 9,
with a hard killing frost in all areas on October 23. Wet
weather during the latter half of October and November
slowed harvest and reduced the rate of grain moisture dry-

down in the field. Prolonged dry weather in August and

82
blight resulted in an appearance of early maturity and con-
tributed to excessive stalk breakage for late harvests.

An equal number of seeds were planted in each plot
(except for high plant population plots) with standard two-
or four-row corn planters adapted for use in small plots.

A pre-emergence herbicide7 was used on all plots. Soil pH
was determined on soil samples taken in the spring prior to
planting and fertilizers were applied to attain maximum
yields according to the results of soil tests. The soil
types will be presented in a later table. Two plots were
irrigated. Plots were harvested with a one—row picker—
sheller. Moisture content of the grain at harvest ranged
from 19.6% to 34.3% for the outstate plots and 22.3% to

33% for the Clinton County varieties. The outstate samples
were placed in plastic bags without drying and frozen at
-20 C until ground for selenium analyses. The Clinton
County variety samples were dried to a safe storage mois-
ture in a forced air oven (41 C) before being frozen at -20
C until ground for selenium analyses. These 20 varieties
were all commercially-available hybrids. Moisture content
was determined on an aliquot to adjust all analytical values
to a dry basis. Yields in the tables are eXpressed as
bushels per acre of 15.5% moisture shelled corn (equivalent

to U.S. No. 2 market grade).

 

7Atrazine 80W, Geigy Agricultural Chemicals, Division of
Geigy Chemical Corporation, Ardsley, New York.

83

Laboratorngrocedures

 

Hemoglobin

 

Hemoglobin was determined by the cyanmethemoglobin
method of Crosby, Munn and Furth (1954). A Coleman Junior
II SpectrOphotometer8 was used for the optical density

determinations.

Hematocrit

 

Hematocrit was determined by the micro method (Mc-
Govern, Jones and Steinberg 1955). Blood samples in
sealed capillary tubes were centrifuged for five minutes at

10,000 RPM in an International "Hemacrit" centrifuge.9

Total serum protein

 

Total serum protein was determined by Miller's (1959)
modification of the Lowry method. Ten d1 of serum were
added to 5 ml of deionized distilled water, and 1 ml of the
resulting dilution was used in the determination. Optical
densities were measured on a Coleman Junior II spectrophoto-

meter.

Serum glutamic-oxaloacetic transaminase (SGOT)_determinations

 

The serum activity of this enzyme was determined on

fresh serum samples using the reagents and procedure

 

8Coleman Instruments Corporation, 42 Madison Street, May-
wood, Illinois 60154.

9International Equipment Company, Boston, Massachusetts.

84
provided by the Sigma Chemical Company (Sigma Technical
Bulletin, 1964). Optical densities were measured on a
Beckman DU Spectrophotometer.10 Activity is expressed in

Sigma-Frankel units per milliliter of serum.

Electrophoresis of serum proteins

 

Electrophoretic separations of serum proteins (Cawley
and Eberhardt, 1962) were conducted and serum albumin to
globulin ratios were calculated after quantitation of the
serum protein fractions with a Beckman Spinco Analytrol
densitometer.11 Approximately 10 ul of serum were streaked

on the agar strips for separation.

Histology

 

Tissues taken for histopathological examination in the
feeding trials were fixed in either 10% formalin in physio-
logical saline or 10% buffered neutral formalin solution.
After fixation, they were imbedded in paraffin, cut and
stained with hematoxylin and eosin using standard techniques

(Armed Forces Institute of PathOIOgy, 1957).

 

1°Beckman Division, Beckman Instruments, Inc., Fullerton,
California.

11Spinco Division, Beckman Instruments, Inc., Palo Alto,
California.

85

Sample preparation for selenium analyses

Feed and grain samples were ground twice through a
screen with 2 mm diameter openings in a Wiley mill.12 Prior
to and after grinding they were stored in plastic bags at

-20 Co

Myocardium and longissimus muscle samples were homog-

 

enized in either a Virtis13 or a Polytron14 tissue grinding
mill with twice their weight of deionized distilled water
to facilitate uniform sampling and pipetting. Liver and
kidney samples were homogenized as above with three and
five times their weight of deionized distilled water, re-
spectively. Homogenates were then frozen at -20 C in either
acid-washed vials or plastic bags. Acid washed pipettes
and disposable Pasteur pipettes were used to transfer the
homogenates and serum or blood samples, respectively, into
tared digestion flasks for selenium analysis.

Feces were ground with a hand grist mill15 after air
drying on trays. This mill was used because it did not
pulverize the hair contaminating the fecal collection as a

high-speed electric mill would. This allowed one to watch

 

12Thomas-Wiley Mill, Model ED-5, Arthur H. Thomas Company,
Philadelphia, Pennsylvania 19105.

13Virtis No. 45, Virtis Company, Gardiner, New York.

14Willems Polytron R, Brinkman Instruments, Westbury, New
York.

15Quaker City Mill, Philadelphia, Pennsylvania.

86
for these hairs when weighing out samples for selenium
analyses and discard them, since hair was found to be rela-
tively high in selenium content even in unsupplemented pigs.
After grinding, fecal samples were stored in plastic bags

at -20 C until needed for analysis.

Dry matter determination

 

Dry matter determinations were conducted by drying
samples overnight in a vacuum oven at 60 C. Tissue homog-
enates were pre-dried overnight in a 37 C forced air warm
room to prevent overloading of the moisture trap on the

vacuum oven 0

Selenium analyses

 

Selenium analyses of feed, grains, tissues, serum,
whole blood, urine and feces were conducted fluorometri-
cally according to the method of Hoffman, Westerby and
Hidiroglou (1968) with modifications to utilize the equip-
ment available in our laboratory. The glassware used for
selenium analyses was segregated and used only for this
procedure. After each set of analyses, the glassware was
rinsed several times in flowing distilled water, rinsed
with a 1:1 mixture of concentrated nitric and sulfuric
acids, rinsed 3 or 4 times with flowing deionized distilled
water and then inverted for drying in a 37 C forced air
warm room. The digestion of up to 1 gm dry matter (sample

size was adjusted according to estimated selenium content)

87
was carried out in a 100 ml semimicro Kjeldahl flask with
an expansion bulb,16 according to Hoffman gt gl.(1968) after
a 12 to 14 hour predigestion in the complete digestion acid
mixture as suggested by Olson (1969). This predigestion
period eliminated most charring problems during subsequent
digestion. The sulfuric acid used for digestion and prepar-
ation of reagents was treated with hydrobromic acid as sug-
gested by Morris and Levander (1970) to lower selenium con-
tamination from this source. Olson's (1969) suggestion of
working in dim or yellow light with the 2,3 diaminonaphtha-
lene solution, and all succeeding preparations containing it,
was observed and seemed to give a more stable preparation.
In the complexing step the various reagents were placed in
a 250 ml Phillips beaker and swirled by hand. After the
incubation period, the reaction mixture was extracted with
6 ml of cyclohexane in a 125 m1 separatory funnel for 15
minutes. The fluorescence was read using an Aminco-Bowman
Spectrophotofluorometer17 with an excitation monochromator
setting of 372 ma and an emission monochromator setting of
516 millimicrons. The fluorometer was zeroed against a
reagent blank carried through the entire procedure. The
method gave good precision and good linearity up to at
least 0.5 ug selenium and the standard curve consistently

passed through the origin. This is a very brief treatment

 

16Aminco No. 4-1874, American Instrument Company, Silver
Springs, Maryland.

17American Instrument Company, Inc., Silver Springs, Mary-
land.

88
of a fairly complicated and demanding analytical procedure.
The reader is referred to a more thorough treatment of the
procedure (appendix 1) if he is contemplating instituting
this assay procedure in his own laboratory. Standards were
prepared from selenium metal18 dissolved in a minimal
quantity of redistilled concentrated nitric acid and made
up to volume with 0.1 N sulfuric acid. The concentrated
stock solution was stored in a walk-in cooler while the
working standard solution (0.1 ug selenium per milliliter)
was stored at room temperature. These standards have re-

mained stable for at least 3 years.

Statistical Analyses

 

Appropriate statistical analyses were applied to the
data from these trials as outlined by Steel and Torrie
(1960). The tests applied included Student's t-test, one-
and two-way analysis of variance and least squares analysis
of variance. Duncan's multiple range test was used to
determine which means in a group of 3 or more treatments
were significantly different. It is recognized that for
the selenium level comparisons, the assumption of non-
ordered response was not met; however, the Duncan's multiple
range test was used as an approximate test of differences
among means for the tissue, serum and blood selenium levels

analyses. Correlation coefficients were calculated for the

 

18Alfa Inorganics, Inc., Beverly, Massachusetts.

89
selenium content of the corn samples with soil pH and yield.
A considerable portion of the data from the feeding and
balance trials was punched onto IBM cards and the statisti-

cal analyses performed with the aid of a computer.

RESULTS AND DISCUSSION

Experiment 1. The performance data and hematological

 

parameters of the finishing pigs in experiment 1 are pre-
sented in table 3. There were no significant differences
among treatments in average daily gain or feed conversion.
Initial hematocrit, hemoglobin or SGOT values were not dif-
ferent among treatments. Likewise there were no differences
among treatments for these same three hematological param-
eters, total serum protein levels, or serum albumin to
globulin ratios at the conclusion of the experiment. Treat-
ment mean serum selenium levels were all significantly
(P < 0.01) different from each other with the level being
the highest (0.167 ppm) for pigs receiving added selenium.
The addition of vitamin E to this basal diet increased the
serum selenium levels almost two-fold (0.025 X53 0.042 ppm).
The tissue selenium levels at slaughter for the basal
and the selenium supplemented groups are presented in table
4. Skeletal muscle selenium levels were similar among the
three muscles analyzed on a wet tissue or dry basis although
a significant pig effect was noted in the basal group. The
basal + 1.0 ppm selenium group had significantly (P < 0.001)
higher selenium levels in skeletal muscle, liver and heart
at the time of slaughter on both a wet tissue basis and a
dry basis. In the case of kidney selenium, the added

90

91

 

 

 

TABLE 3 . PERFORMANCE AND HEMATOLOGICAL PARAMETERS
(EXPERIMENT 1)
Dietary treatments
Basal + Basal +
Item Basal Vit. E 1.0 ppm Se

No. of pigs 6 6 6
Initial weight, kg 45.5 45.0 45.0
Avg daily gain, kg 0.75 0.80 0.75
Gain/feed 0.30 0.32 0.31
Initial blood values

Hematocrit, % 34.1 33.5 34.1

Hemoglobin, g/100 ml 11.7 11.5 11.8

SGOT, S-F units/ml 42.4 77.4 51.6
Final blood values

Hematocrit, % 33.7 35.2 35.2

Hemoglobin, g/100 ml 11.8 12.6 12.3

SGOT, S-F units/ml 43.1 47.1 43.2
Total serum protein, g/100 ml 8.0 7.8 8.1
A/G ratio 0.70 0.65 0.77
Serum Se, ppma 0.025 0.042 0.167

 

aAll treatment means (serum selenium) significantly
(P < 0.01) different from each other.

92

TABLE 4. FINAL TISSUE SELENIUM LEVELS (EXPERIMENT 1)

 

Dietary treatments

 

 

Item Basal légsgém+8e
Selenium, ppm (wet tissue basis)a
Ham 0.054 0.121
Loin 0.052 0.126
Shoulder 0.051 0.133
Skeletal muscle mean 0.052 0.127
Heartb 0.086 0.327
Liver 0.100 0.797
KidneyC 1.134 2.102
Selenium, ppm (dry tissue basis)a
Ham 0.213 0.477
Loin 0.192 0.423
Shoulder 0.195 0.525
Skeletal muscle mean 0.200 0.475
Heartb 0.384 1.432
Liver 0.343 2.669
Kidneyb 5.332 10.148

 

aMean of three pigs per treatment.

b1.0 ppm added selenium group significantly (P < 0.001)

higher than basal.

c1.0 ppm added selenium group significantly (P < 0.01)
higher than basal.

93
selenium group was also significantly (P < 0.01 for wet
tissue basis and P < 0.001 for dry basis) higher than the
basal group. There were no significant differences between
treatments in dry matter content of the tissues analyzed.
A general discussion of tissue selenium levels in all three
feeding trials will be presented with the discussion of
experiment 3.

None of the pigs in this study exhibited clinical
signs attributable to the deficiency and none of the six
pigs examined at slaughter showed gross or histological
lesions of the deficiency. This was not unexpected, since
these pigs were past what seems to be the most critical age
for overt signs of injury due to the deficiency (Obel, 1953;

Trapp gt 1., 1970).

Experiment 2. No significant differences in gain or

 

feed efficiency were noted in the first 14 weeks of the
trial. These parameters were both within normal ranges for
pigs of this age on all treatments. As noted earlier,
treatment feed consumption and weight records were not main-
tained after selenium withdrawal was instituted at 14 weeks.
The effects of sex and sow pretreatment on initial
hematological parameters are presented in table 5. Sex or
sow pretreatment had no main effect on initial hemoglobin,
hematocrit, total serum protein or serum albumin to globulin
ratio. A significant (P < 0.01) interaction with regard to

serum A/G ratios did occur. Added vitamin E in the sow's

 

94

TABLE 5. INITIAL HEMATOLOGICAL PARAMETERS. EFFECT OF
SEX AND sow PRETREATMENT (EXPERIMENT 2)

 

Pigs from

 

 

Item No B sows Males Females
E sows

Hemoglobin, g/100 ml 11.1a 10.9 10.8a 11.2
Hematocrit, % 31.9 31.1 30.7 32.2
Total serum protein, g/100 ml 6.0 6.0 6.2 5.8
A/G ratiob 0.75 0.69 0.72 0.71
SGOT, S-F units/m1C 55.9 45.9 46.5 55.3
Serum selenium, ppm 0.031 0.030 0.027 0.034

 

aMean values for 18 pigs in each cell.

bSignificant (P < 0.01) interaction of sex and sow pretreat-
ment noted with regard to A/G ratios. Added vitamin E in
sow's ration increased A/G ratios in male offspring (0.65
XE' 0.79) and decreased them in female offspring (0.84 35'
0.59).

CPigs from sows which received no supplemental vitamin E

in gestation and lactation had significantly (P < 0.01)
higher SGOT levels than pigs from sows receiving vitamin

E. Female pigs had significantly (P < 0.05) higher initial
SGOT levels than male pigs.

95

ration increased serum A/G ratios in male offspring and de—
creased them in female offspring. Pigs from sows which
received no supplemental vitamin E during gestation and
lactation had significantly (P < 0.01) higher initial SGOT
levels than pigs from sows receiving supplemental vitamin
E (55.9 Xi' 45.9 S-F units per milliliter of serum). These
SGOT values were all in the upper range of the normal
values reported by Wretlind gt gt. (1959). These SGOT
levels were much lower than those previously reported for
pigs showing overt signs of the deficiency (Orstadius SE.3£"
1959). One pig which had chronic liver fibrosis at slaughter
had normal SGOT activity the day prior to slaughter. This
was probably due to the apparent chronicity of this pig's
condition and indicates that active necrosis must be pres-
ent to be detected by increased SGOT activity.

At the 10-week bleeding the hemoglobin, hematocrit,
total serum protein levels, SGOT levels and serum albumin
to globulin ratios were similar for all treatments. At this
bleeding the male pigs had significantly (P < 0.05) higher
total serum protein levels than the female pigs (8.0 25' 7.3
g per 100 ml serum). The effect of dietary treatments on
serum selenium concentration is presented in table 6.
Over all vitamin E levels, the means of the added selenium
levels were all significantly (P < 0.01) different from
each other. A significant (P < 0.001) interaction was
noted between the levels of dietary selenium and vitamin E.

On the basal level of dietary selenium the addition of

TABLE 6. EFFECT OF SELENIUM AND/OR VITAMIN E SUPPLEMENTATION
0N 10-WEEK SERUM SELENIUM LEVELS (EXPERIMENT 2)

 

Added vitamin E

 

 

Added Se 0 11 IU/kg 22 IU/kg Meanc
0 0.0698'b 0.018 0.016 0.034
0.1 ppm 0.082 0.096 0.128 0.102
0.5 ppm 0.145 0.173 0.153 0.157
Mean 0.099 0.096 0.099

 

aMean of four pigs per treatment (ppm).

bSignificant (P < 0.001) interaction of selenium and vita—
min E levels. On the basal level of selenium the addition
of vitamin E decreased serum selenium levels, while on
both levels of added selenium, the addition of vitamin E
tended to increase serum selenium levels.

CAll treatment (selenium levels) means significantly
(P < 0.01) different from each other.

97
vitamin E, in contrast to results in experiment 1, decreased
serum selenium levels, while on both levels of added selen-
ium the addition of vitamin E tended to increase serum
selenium levels.

The final hematological parameters and longissimus

 

muscle selenium level data for the 27 pigs sampled from
lots 1, 2 and 3 were combined for the three slaughter dates
(0, 30 and 65 days of selenium withdrawal) and analyzed

by least squares analysis of variance. These data are pre-
sented in table 7. No significant differences were noted
in final hemoglobin, hematocrit or SGOT levels due to sex,
selenium treatment or slaughter date. Total serum protein
levels increased significantly (P < 0.01) between the first
and third slaughter dates. This is quite possibly due to
the increasing age of the pigs at slaughter (Miller SE.3£"
1961). Neither sex nor treatment modified total serum
protein levels. The serum A/G ratios of pigs fed the basal
diet were significantly (P < 0.05) lower than those of pigs
fed the basal + 0.1 ppm added selenium (0.45 2g. 0.60).
Both of these values, however, were not different from the
intermediate serum A/G ratio (0.49) of the basal + 0.5 ppm
added selenium group. No differences in serum A/G ratios
attributable to sex or slaughter date were observed.

Serum selenium levels were significantly (P < 0.05) lower
on the basal than on the added selenium diets with no dif-
ference between this parameter on the latter two diets.

Serum selenium levels had dropped significantly (P < 0.05)

 

98

 

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99

by 30 days of selenium withdrawal, with no significant dif-
ference between the 30 and 65 day withdrawal serum selenium
levels.

All three dietary treatments differed significantly (P

< 0.01) from each other with regard to longissimus muscle

 

selenium levels on both a wet tissue and a dry basis. Across
treatments, selenium levels were not significantly lower in

longissimus muscle after 30 days of selenium withdrawal, but

 

were significantly (P < 0.01) lower 65 days after selenium
withdrawal. No differences in muscle selenium levels attri-
butable to sex were noted.

In experiment 2, 29 pigs were examined for gross and
microscopic lesions of selenium and/or vitamin E deficiency.
Twenty-seven of these were the animals of lots 1, 2 and 3
slaughtered to obtain muscle samples for selenium analyses.
The other two were male pigs which died suddenly after 4
weeks on experiment. The lesions observed in these two pigs
will be discussed in some detail. A numerical scoring sys-
tem was used in all microscopic examinations to compare any

histopathological changes observed. The system was as fol—
lows: 0 = no tissue taken or lost in processing; 1 = nor-
mal; 2 = very slight pathological changes; 3 = slight patho—
logical changes; 4 = moderate pathological changes; and 5 =
severe pathological changes.

The first pig which died was from a vitamin E-supple-
mented sow and weighed 26 kg at death while the second pig
was from a non-supplemented sow and weighed 24 kg at death.
The first pig displayed no overt signs of illness the even-

ing before he was found dead in the early morning. The

100
second pig was found dead approximately 24 hours after the
first death loss. Paleness, listlessness and signs of
reSpiratory distress were noted in the second animal the
afternoon prior to his death. No particular environmental
or social stresses were evident immediately prior to or
during this two-day period that might explain why both
losses occurred in such a short period. The gross lesions
observed upon necropsy of the first pig were a mottled myo-

cardium with dilatation of the right ventricle and hemor-

 

rhage in the gastric submucosa. The second pig had pale

streaks in the myocardium, the semitendinosus and semimem-

 

branosus muscles. Diffuse edema of the lungs, a hard
roughened liver and a large ulcer in the fundus of the
stomach were also noted in the second pig. Histopathologic
examination revealed fragmentation, hyaline degeneration,
swelling and loss of striations of the skeletal muscle

fibers of both pigs with focal areas of mineral deposition.
Microscopic examination of the myocardium of both animals
revealed pyknosis, fragmentation, loss of striations and
focal areas of necrosis or mineral deposition. The suffusion
hemorrhages in the lamina propia and submucosa of the stom—
ach wall of the first pig as well as the fundic ulcer of

the second pig were confirmed by histopathologic examination.
The liver tissue of the first pig was normal. In the second
pig centrolobular necrosis occurring in individual or small
groups of hepatic lobules with replacement of necrotic cells

by blood and/or mineral deposits plus a marked increase of

101

interlobular fibrous connective tissue were noted. Inter—
lobular and intra-alveolar edema was observed in sections
of lung tissue from the second pig. The histopathologic

scores on tissues of the first and second pigs, respectively,

 

were: skeletal muscle 5, 5; myocardium 4, 4; liver 1, 5;
adrenal gland 2, 2; kidney 0, 0; pancreas 0, 0. The adrenal
glands were scored 2 because of extensive congestion of the
cortical region.

These same tissues were examined from the 27 pigs

slaughtered to obtain longissimus muscle selenium level

 

data. The skeletal muscles, adrenal glands and pancreas
examined from all 27 pigs were judged to be normal. All
myocardial sections examined were normal except two which
had small foci of mononuclear inflammatory cells which were
thought to be due to a chronic respiratory infection rather
than a consequence of any of the imposed dietary treatments.
These same two animals also had similar foci of inflammatory
cells in their renal tissue while the renal tissue sections
of the other 25 pigs appeared to be normal. One pig fed

the basal diet was found to have severe chronic fibrosis of
the liver at slaughter. Upon histological examination this
liver was scored 4. The changes noted in the liver sections
of the remainder of the pigs (mostly mild eosinophilic
hepatitis with some increase in interlobular fibrous con-
nective tissue) were not those usually seen in selenium
and/or vitamin E deficiency and were attributed to allergic

reactions (e.g., to parasites) or to general debilitation.

102
Pneumonia was a problem in all lots after a cold rain blew
into the pens on September 1, 1969 (11 weeks into the experi-
ment). This condition was treated with sulfa drugs in the
water and injections of oxytetracycline hydrochloride. It
seemed that the condition was about evenly distributed
through the experimental lots. The animals' rate of gain
recovered quite quickly after treatment, but residual lung
damage was evident in many of them at slaughter. The major
pathological findings on the two pigs that died and the 27
examined at slaughter are summarized in table 8. No sudden
death losses occurred in pigs fed diets containing supple—

mental selenium and/or vitamin E.

TABLE 8. SUMMARY OF PATHOLOGICAL FINDINGS (EXPERIMENT 2)

 

Dietary treatments
Basal + Basal +

 

 

Item Basal 0.1 ppm Se 0.5 ppm Se
Pigs started 12 12 12
Pigs examined 11 9 9
Sudden deaths 2 0 0

Pigs showing moderate to severe
pathological changes in:

Skeletal muscle 2 0 0
Myocardium 2 0 0
Liver 2 0 0

 

103

Experiment 3. As in the two previous trials, no dif-

 

ferences in average daily gain or feed efficiency could be
attributed to the dietary additions of selenium and/or
vitamin E. The pigs in this trial grew well and the gains
and feed conversions were within normal ranges.

In the statistical analysis of the final data from 42
pigs from experiment 3, the main effects of added vitamin E
Zi' no added vitamin E and selenium withdrawal (60 days)
23' no withdrawal were of prime interest. In the other
statistical treatment applied to the data, comparisons were
made of the results on the combined basal diets (basal and
basal + vitamin E) Xi' the six diets which contained added
selenium. A separate comparison was then made within the
selenium treatments to determine if differences attributable
to the levels of added selenium were evident.

The effects of added dietary vitamin E and selenium
withdrawal on blood parameters are presented in table 9.
Red blood cell selenium concentrations were calculated from
whole blood selenium levels, serum selenium levels and
hematocrit. Red blood cell selenium levels were signifi-
cantly (P < 0.05) lower in pigs receiving supplemental
vitamin E (11 IU per kilogram of diet) than in pigs re-
ceiving no supplemental vitamin E. Serum, whole blood and
erythrocyte selenium levels were all significantly (P S 0.01)
lower after 60 days of selenium withdrawal. Selenium with-
drawal had no effect on hematocrit. Added vitamin E had

no effect on hematocrit, serum selenium level or whole blood

selenium level.

104

TABLE 9. EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM
WITHDRAWAL ON BLOOD PARAMETERS (EXPERIMENT 3)

 

 

 

E level Se withdrawal
Item 0 11 IU/kg 0 60
No. of pigs 21 21 18 18
Hematocrit (%) 40.1 39.6 41.0 38.7
Serum Se (ppm) 0.102 0.110 0.150b 0.071
Whole blood Se (ppm) 0.137 0.132 0.190b 0.102
RBC Se (ppm) 0.191a 0.167 0.233b 0.155

 

aRed blood cell selenium significantly lower (P < 0.05) on

vitamin E-supplemented diets.

bSerum, whole blood and red blood cell selenium levels were
all significantly lower (P < 0.01) after 60 days of selen-

ium withdrawal.

TABLE 10. EFFECT OF SUPPLEMENTARY DIETARY SELENIUM ON
BLOOD PARAMETERS (EXPERIMENT 3)

 

Supplementary selenium (ppm)

 

 

Item Basal 0.05 0.10 0.20
No. of pigs 6 12 12 12
Hematocrit (%) 39.5 40.8 39.1 39.7
Serum Se (ppm) 0.046a 0.110 0.115 0.121
Whole blood Se (ppm) 0.062b 0.139 0.145 0.154
RBC Se (ppm) 0.088b 0.181 0.193 0.207

 

aSerum selenium levels on the basal diets were not signifi-
cantly different from the selenium withdrawn diets; but
serum levels were significantly (P < 0.01) higher on the

non-selenium withdrawn diets.

b

the selenium-supplemented diets.

Significantly (P < 0.05) lower on the basal diet than on

105

The effects of the different levels of added dietary
selenium on blood parameters are presented in table 10.

The level of added dietary selenium had no effect on hemato-
crit. Serum selenium levels of pigs on the basal diet were
not different from those of the withdrawn pigs, however,
serum selenium levels were significantly (P < 0.01) higher
in the non-withdrawn pigs than in either the basal or with-
drawn groups. Whole blood and erythrocyte selenium levels
were significantly (P < 0.05) lower in pigs on the basal
diet than in pigs on selenium-supplemented diets.

Tissue selenium levels that resulted from selenium
supplementation in this trial were very similar to those
noted in experiments 1 and 2. The tissue selenium levels
and how they were affected by added dietary vitamin E and
selenium withdrawal are presented in table 11. The addition
of supplementary vitamin E produced significantly (P < 0.05)
higher kidney selenium levels on both a wet and dry basis.
Higher kidney selenium levels with the addition of vitamin
E were noted by Sharp _t gl.(1970) who also reported a con-
comitant reduction in liver and muscle selenium levels. No
effect of vitamin E on the selenium concentration of any
of the other tissues analyzed was noted in experiment 3.

As might be eXpected, from the results of experiment 2, the

selenium levels in longissimus muscle, myocardium, liver

 

and kidney were all significantly (P < 0.01) lower after
60 days of selenium withdrawal on both a wet and dry basis.
The effect of the level of added dietary selenium on

tissue selenium concentrations is presented in table 12.

106

TABLE 11. EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM
WITHDRAWAL 0N TISSUE SELENIUM (EXPERIMENT 3)

 

 

 

 

 

E level Se withdrawal

Item 0 11 IU/kg 0 60 days
No. of pigs 21 21 18 18

Longissimus muscle wet 0.082 0.081 0.096b 0.076

dry 0.303 0.297 0.347 0.286

Myocardium wet 0.169 0.174 0.218 0.142

dry 0.802 0.816 1.034 0.664

Liver wet 0.353 0.357 0.552 0.221

dry 1.224 1.214 1.898 0.757

Kidney wet 1.731: 1.843 2.026 1.643

dry 8.755 9.301 10.286 8.212

 

aKidney selenium levels on both a wet and dry basis were
significantly (P < 0.05) higher on vitamin E—suplemented
diets.

bSelenium levels for all tissues analyzed were significantly
(P < 0.01) lower after 60 days of selenium withdrawal on
both wet and dry bases.

 

 

 

 

TABLE 12. EFFECT OF SUPPLEMENTARY DIETARY SELENIUM ON
TISSUE SELENIUM (EXPERIMENT 3)
Supplementary selenium (ppm)
Item Basal 0.05 0.1 .
No. of pigs 6 12 12 12
. . a b c d d
LongISSImus muscle wet 0.055b 0.075C 0.091d 0.093d
dry 0.199 0.277 0.333 0.341
Myocardium wet 0.122: 0.178: 3 0.1743 0.1893
dry 0.571 0.841 ' 0.815 0.891
Liver wet 0.1662 0.358: 0.3963 0.4053
dry 0.569 1.219 1.362 1.400
Kidney wet 1.5052 1.8853 1.819: 1.798:
dry 7.705 9.567 9.033 9.146

 

aMeans in the same row with different superscripts are
significantly different (P < 0.05).

107
The effect of the level of added dietary selenium on
tissue selenium concentrations is presented in table 12.

Selenium concentrations in longissimus muscle, myocardium,

 

liver and kidney were all lower for pigs on the basal diets
than for pigs on selenium-supplemented diets. Within the
selenium-supplemented diets, the tissues varied with re-
spect to selenium levels at slaughter in response to added
dietary selenium. Kidney selenium levels were not dif—
ferent for the three levels of added selenium on a wet or

dry basis. Longissimus muscle and liver selenium levels

 

were significantly (P < 0.05) lower on the 0.05 ppm added
selenium level than on either the 0.1 or 0.2 ppm added
selenium diets. On a wet basis, myocardial selenium con-
centrations were significantly (P < 0.05) lower on the

0.05 and 0.1 ppm added selenium diets than on the 0.2 ppm
added selenium diet. On a dry basis, the 0.1 ppm added
selenium treatment had significantly lower myocardial
selenium levels than the 0.2 ppm added selenium treatment
with the group receiving 0.05 ppm selenium having an inter-
mediate level of myocardial selenium.

Two interesting effects on tissue dry matter content
are presented in table 13. Liver dry matter content was
found to be significantly (P < 0.01) higher on vitamin E-
supplemented diets. Feeding supplemental selenium until
slaughter Significantly (P < 0.01) increased longissimus
muscle dry matter content. The import of these Observa-

tions is still uncertain. These two tissues are recognized

TABLE

108

EFFECT OF ADDED DIETARY VITAMIN E AND SELENIUM

WITHDRAWAL ON TISSUE DRY MATTER CONTENT (EXPERIMENT 3)

 

 

 

 

 

E level Se withdrawal
Item 0 11 IU/kg 0 65 days
No. of pigs 21 21 18 18
Tissue dry matter (% of fresh)
Longissimus muscle 27.18 27.35 27.89b 26.56
Myocardium 21.12 21.41 21.11 21.36
Liver 28.74a 29.59 29.13 29.20
Kidney 19.81 19.84 19.71 20.05

 

aLiver dry matter significantly (P < 0.01) higher on vitamin

E-supplemented diets.

bLongissimus muscle dry matter significantly (P < 0.01)

 

higher when supplemental selenium was fed until slaughter.

109
as being major target organs of selenium and/or vitamin E
deficiency in swine (Obel, 1953; Trapp gt_gt., 1970). Dis-
orders of membrane permeability are also recognized as
prominent lesions of the deficiency by the same authors.
One could Speculate that the differences in dry matter con-
tent may be due to minor abnormalities of fluid balance in
a sub-clinical deficiency state. The final conclusion on
this matter cannot be drawn from available data.

A survey of the data on tissue selenium concentrations
in tables 4, 7, 11 and 12 indicates that in muscle and the
other organs analyzed the increase in selenium concentra-
tion tends to level off and is not proportional to further
increases in added dietary selenium in the form of sodium
selenite. This is apparently the case with the levels
tested (0.05 to 1.0 ppm selenium), all of which were within
physiologically-compatible ranges. The muscle selenium
levels noted after selenium supplementation, even without
a withdrawal period, were much lower than those reported
for porcine muscle in selenium-adequate areas of the United
States (Morris and Levander, 1970; Ku gt gt., 1972).
Lindberg and Lannek (1965) fed a commercial swine diet
(0.126 ppm selenium) or this diet + 1.2 ppm selenium as
sodium selenite to pigs of 19.6 kg initial weight for 78
days. Muscle from control and selenium-supplemented ani—
mals contained 0.52 and 0.76 ppm selenium, reSpectively, on
a dry basis. They found that the muscle selenium levels of

the selenium-supplemented pigs returned to normal in 14

 

110

days. These authors expressed the opinion that this rapid
decline in muscle selenium level may have been due to the
relatively high natural selenium level of their basal diet.
Grant, Thafvelin and Christell (1961) reported a series of
studies concerning retention of selenium by pig tissues.
Their method of analysis (neutron activation) was relative—
ly insensitive but trends could be noted. They reported
that the selenium content of muscle and liver tissue in
non-selenium—supplemented pigs fluctuated around the same
lower limits as in pigs fed 0.2 mg added sodium selenite
per kilogram of diet. In their experiments the distribu~
tion patterns and absolute amounts of retained selenium
were not consistently affected by the duration of selenite
supplementation (13 to 367 days) or the length of the
selenium withdrawal period before slaughter (0 to 47 days).

The muscle selenium levels, in the trials reported
here, on selenium-supplemented rations are of similar mag-
nitude to those reported by Lindberg and Lannek (1965) for
pigs fed a diet containing 0.126 ppm natural selenium. The
tendency for muscle selenium levels to remain fairly stable
after supplemental selenium is withdrawn from the selenium—
deficient ration could be seen in the results of the serial
slaughter dates of experiment 2.

The relative tissue selenium concentrations noted in
these studies were similar to those reported in the litera—
ture (Lindberg and Siren, 1963, 1965; Lindberg, 1968;

Sharp gt 1., 1970) with kidney being the highest in

 

111

selenium content followed by liver, heart and skeletal
muscle, respectively. In general the tissue selenium
levels of the pigs on the basal diet, even the pen-mates
of the two death losses in experiment 2, were not quite as
low as those reported for pigs dying of the deficiency in
enzootic selenium-responsive disease areas (Andrews gt gt.,
1968; Sharp gt gt., 1970; Lindberg, 1968). Most of these
authors have considered liver selenium levels to be the
best indicator of selenium status of the animal and that a
level of at least 0.2 ppm on a dry basis is required for
survival. Even animals on the basal diets in the trials
reported here had about twice this level of liver selenium.
It is unfortunate that the tissue samples from the two
death losses in experiment 2 were lost in storage before
selenium analyses could be conducted. Some of the devia-
tion of these values from earlier reported values may arise
from differences in analytical techniques and age of the
pigs at sampling. Most Of the pigs that died of selenium
and/or vitamin E deficiency, for which tissue selenium
levels have been reported, were near weaning age, whereas
in these studies all the tissues analyzed were from animals
that had survived this critical period and were near
slaughter weights when sampled.

No death losses recognized as being caused by selenium
and/or vitamin E deficiency occurred during the course of
eXperiment 3 although four animals did die. One pig that

died was found to have a fibrinohemorrhagic enteritis;

112
another had a gastric ulcer with fatal hemorrhage and
another died of undetermined causes. These three pigs
died rather early in the course of the experiment. Another
pig died in transit to slaughter and had necropsy lesions
suggestive of the so-called porcine stress syndrome.

No gross lesions that could be attributed to the
dietary treatments were noted in necrOpsy of the death
losses or in posbhortem examination of the 42 pigs slaugh—
tered at the Michigan State University Meats Laboratory in
experiment 3. One male pig on the basal + 0.2 ppm added
selenium (non-withdrawn) had a severe fundic ulcer at
slaughter. One female pig on the basal f 11 IU vitamin E
per kilogram diet slaughtered at Peet Packing Company had
a hard roughened liver which on histopathological examina-
tion was scored 4 due to extensive interlobular fibrosis
with eosinophils. Two other pigs (a female on the basal
and a male on the basal + 0.1 ppm selenium withdrawn diet)
had large gastric ulcers when slaughtered at Peet Packing
Company. Histological examinations of the tissues from the
42 pigs sampled in this study also failed to reveal any
definite lesions of the deficiency. Some liver damage was
noted but the universal presence of eosinophils in these
sections indicated that these changes were probably due to
allergic reactions rather than to a nutritional deficiency.
One female pig on the basal + 11 IU vitamin E per kilogram
diet exhibited a slight diffuse myositis with necrosis of

occasional skeletal fibers and infiltration of mononuclear

113
cells in the region. These changes could have been consis—
tent with resolving nutritional muscular dystrophy. The
only changes noted in the sections of myocardium were very
mild focal inflammatory changes in three of the 42 pigs ex-
amined. Foci of a mixture of lymphocytes and plasma cells
were noted in kidney sections of approximately one-half of
the 42 pigs examined. This finding is quite common in

swine kidneys and was considered to be of no significance.

EXperiment 4. The results of this preliminary selenium

 

balance study are presented in table 14. The fecal excre-
tion of selenium indicated that approximately equal per—
centages of both levels of supplemental selenium were ap—
parently absorbed from the diet. Fecal and urinary excre-
tion of selenium increased significantly (P < 0.01) as the
level of dietary selenium was increased. Urinary selenium
excretion increased 20-fold and a three-fold increase occur-
red in fecal selenium excretion with the diet containing

0.5 ppm added selenium over that observed with the diet
containing 0.1 ppm added selenium. The absolute daily
retention of selenium was significantly (P < 0.01) higher
with the selenium—supplemented diets. It must be recognized
in all these selenium balance trials that "retention" in-
cludes any eXpiratory losses of selenium. The results of
earlier studies (McConnell, 1941; Hopkins, 1965; Handreck
and Godwin, 1970) indicate that such losses would not be

great (probably less than 1% of the ingested selenium) at

 

114

TABLE 14. DAILY SELENIUM BALANCE (EXPERIMENT 4)

 

Dietary treatments

 

 

 

Item Basald Basal + Basal +
0.1 ppm Se 0.5 ppm Se
ppm Se (as fed) 0.042 0.147 0.543
Se intake, ug 18.9 66.2 244.4
Total Se excretion, ugb 10.3 25.3 207.3
Fecal Se, ugb 8.4 18.4 57.7
Urinary Se, ugb 1.9 6.9 149.6
Se retention, ugc 8.6 40.9 37.1
Percent retentiond 45.5 61.7 15.2
aTable 1.

bAll treatment means were significantly (P < 0.01) differ—

ent from each other.

CSignificantly (P < 0.01) lower selenium retention on basal
diet.

dSignificantly (P < 0.01) lower percentage of dietary selen-
ium retained on the 0.5 ppm added selenium diet.

115
the physiologically-compatible levels of selenium fed in
these studies. A significantly (P < 0.01) lower percentage
of dietary selenium was retained with the higher level of
selenium supplementation than with the basal or the 0.1 ppm
added selenium diets. The prOportion of dietary selenium
retained was not different between the basal and the lower

level of selenium supplementation.

Experiment 5. The selenium balance and serum selenium

 

data for experiments 5 and 6 are presented in table 15. In
eXperiment 5 there were no differences in percent retention
or percent of the selenium intake excreted in the feces.
Absolute selenium retention increased up to 0.10 ppm added
selenium. This absolute daily selenium retention of 40.0
pg observed on the 0.10 ppm added selenium diet is almost
identical to the daily retention (40.9 ug) observed on a
Similar diet in experiment 4. The plateauing effect of
absolute selenium retention at levels of added dietary
selenium of 0.1 ppm or above is evident in this study as

it was in experiment 4. The percent of the selenium intake
excreted ytg the urine was significantly higher on the
highest level of selenium supplementation. Serum selenium
levels of pigs on the different diets were all significant-
ly (P < 0.05) different from each other at the end of the

collection period in experiment 5.

Experiment 6. The selenium balance and serum selenium

data are presented in table 15. As in experiment 5 there

116

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117

no differences in the proportion of the selenium intake
excreted in the feces. In contrast to the results of the
balance in eXperiment 5 the percent retention of selenium
on the 0.2 ppm added selenium diet was significantly (P <
0.05) lower than on the other three diets. This decreased
retention was due to marked increase in urinary selenium
excretion on the highest level of supplemental selenium.
The urinary excretion of selenium on the 0.1 ppm added
selenium diet was also increased in relation to the basal
and basal + 0.05 ppm added selenium diets. The absolute
daily retention of selenium on the two higher levels of
added selenium was significantly (P < 0.01) greater than on
the two lower levels of dietary selenium with no differ-
ences between the members of these respective pairs. In
parallel with urinary excretion and retention data for the
collection of experiment 6, added dietary selenium increased
serum selenium levels up to an added dietary selenium level
of 0.1 ppm with no further increase in serum selenium
levels being evident at 0.2 ppm added dietary selenium.

When we consider the data from experiments 5 and 6 in
tandem it is noted that the addition of dietary vitamin E
seems to have increased the urinary excretion of added
dietary inorganic selenium and thus lowered both the pro-
portionate and the absolute retentions of this nutrient.
Urinary excretion increased from 26.0 to 45.4% of intake
and percent retention drOpped from 41.5 to 24.9% on the

highest level of selenium supplementation. These trends

118
may also be noted on the 0.1 ppm level of added dietary
selenium. The absolute retention of selenium on the two
higher levels of added selenium was reduced from the first
collection period (40.0 to 35.6 and 48.2 to 29.8 ug per
day for the 0.1 and 0.2 ppm added selenium levels, respec-
tively). Since this comparison of no added vitamin E with
added vitamin E data involves a time span, the validity of
such a comparison is rightfully Open to question. The in-
creased excretion and lowered retention on the higher levels
might be indicative of a selenium deficit in the tissues of
these pigs that was filled more quickly from the diets with
the higher levels of selenium supplementation while having
insufficient time during these trials to be filled on the

lower levels of dietary selenium.

Experiment 7. This experiment was designed to consider

 

the effect of added vitamin E on selenium balance in the
young pig as well as to compare the retention of natural
and selenite selenium. The main effect comparisons of the
selenium balance data and blood selenium levels of experi-
ment 7 are presented in table 16. A significantly (P <
0.05) higher percentage of the selenium intake was ex—
creted X23 the feces on the seleniferous corn diets. A
significantly (P < 0.01) lower percentage of the selenium
intake was excreted gig the urine on these same diets. The
addition of vitamin E lowered fecal selenium excretion.
There were no differences in absolute selenium retention

or in percent of selenium intake retained on these diets.

 

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120
No simple main effect influences on absolute selenium re-
tention or percent of selenium intake retained were evi-
dent, however, a significant (P < 0.01) interaction was
noted in which added vitamin E reduced the absolute reten-
tion of selenite selenium (27.4 gg. 25.0 ug per day) (as in
experiment 6) and increased the absolute retention of
aflenium from seleniferous corn (22.5 gg. 31.7 ug per day).
The increase in selenium retention from the seleniferous
corn was due primarily to a decrease in fecal selenium ex-
cretion with the addition of vitamin E to this diet. In
the main effect comparisons of blood selenium levels the
final whole blood selenium levels were significantly (P <
0.01) lower on the seleniferous corn diets than on the
selenite diets. This difference was attributable to the
significant (P < 0.01) differences in final serum selenium
levels since final erythrocyte selenium levels were very
similar for both selenium sources.

Since there were no significant differences in selen-
ium retention between natural and inorganic selenium in
these trials, the marked difference in urinary selenium
losses, when considered with fecal selenium losses, would
seem to indicate that selenite selenium was more readily
absorbed by the young pig and then excreted gig the urine
to a greater extent than naturally occurring selenium.

The differences in serum selenium levels would also be
consistent with this assumption. The lack of differences

in overall retention from the two sources was unexpected

 

121
and can possibly be partially explained by the fact that
this was a short-term trial with selenium-deficient, rapid-
ly-growing animals. Tissues from animals on natural diets
in selenium-adequate areas of this country consistently
have had much higher selenium levels than pigs from earlier
discussed feeding trials where similar levels of selenium
have been added as sodium selenite (Ku gt gi., 1972).

The action of vitamin E in lowering the retention of
selenite selenium was evident in this trial as in experi-
ment 6. No explanation was apparent from the balance data
for this effect. If there were some effect, such as ele-
vated serum selenium levels, one might postulate that this
might lead to increased urinary selenium losses. Such a
trend in serum levels was not evident in these short-term
balance trials. In one feeding trial (experiment 3) it was
found that added vitamin E did increase kidney selenium
levels in growing-finishing pigs at slaughter. In this
same feeding trial vitamin E additions significantly (P <
0.05) lowered erythrocyte selenium concentrations but had
no effect on serum or whole blood selenium levels. Canad-
ian workers have reported a similar increase in kidney
selenium accompanied by a decrease in liver selenium levels
when corn-soy diets were supplemented with 5.5 IU of vita-
min E per kilogram (Sharp gt_gi3, 1970). At the 10-week
bleeding of experiment 2, supplemental vitamin E had
drastically reduced serum selenium levels (0.069 gg. 0.016

ppm) of pigs on diets containing only natural selenium

 

122

(0.041 ppm) while it tended to increase serum selenium
levels of pigs fed this diet plus added selenite selenium.
Unfortunately, whole blood was not preserved for selenium
analyses in this study. From these results one might postu-
late that supplemental vitamin E lowers the tissue reten-
tion of selenium, eSpecially selenium of inorganic origin,
resulting in higher serum levels Of selenium which in turn
leads to greater urinary excretion of inorganic selenium.
The increased concentrations of selenium in kidney tissue
may simply reflect the greater quantity of selenium being
handled by the renal apparatus on the vitamin E-supple-
mented diets in an effort to maintain some homeostatic
level of selenium in the body. The serum selenium levels
tended to plateau within a reasonable range after levels of
added inorganic selenium reached about 0.1 ppm with the
basal diets used. This would further suggest the existence
of a threshold for selenium of inorganic origin in the serum
and tissues. The facts just outlined, along with the reali—
zation that resultant tissue selenium levels are much lower
on selenite-supplemented diets than on diets containing
equal levels of natural selenium, lead one to believe that
inorganic selenium and selenium occurring naturally in
plants are metabolized in a different manner by the animal
organism.

The effect of vitamin E in experiment 7 whereby it in-
creased the retention of natural selenium but decreased the

retention of selenite selenium lends further credence to

123
this conviction. The manner in which vitamin E exerts this
effect on natural selenium likewise can only be surmised
from the available data. Selenium retention was increased
on the seleniferous corn diets primarily due to a decrease
in fecal excretion of selenium. Selenium excretion in the
feces represents the net result of both the absorption of
selenium from the gastrointestinal tract and its resecre-
tion into the same. Wright and Bell (1966) reported that
selenium was secreted in fairly large quantities into the
first section of the small intestine of the pig and then
reabsorbed to a considerable extent from succeeding sec-
tions of the small intestine. Resecretion of selenium in
this first section of the small intestine probably is a
result of biliary excretion in this area. The bile pig-
ments are derived from breakdown products of hemoglobin
from defunct erythrocytes. McConnell and COOper (1950)
reported that 75Se from selenite was incorporated into both
the hemin and globin fractions of hemoglobin with the con-
centration being greatest in hemin. In experiment 3, added
vitamin E did lower erythrocyte selenium levels. It is
doubtful, however, that this change would be enough to
lower fecal selenium levels as drastically as was observed
on such a short-term study. It is clear that more defini-
tive work needs to be done on the interrelationship of
vitamin E and selenium with regard to their reSpective in-

fluences on selenium balance in animals.

124

Experiment 8. Selenium balance and blood selenium,

 

data of this experiment are presented in table 17. Signifi-
cantly (P < 0.01) less selenium was retained on an absolute
basis on the old unstable premix than on the fresh premix.
This was due to a tendency for increased selenium excretion
gig the feces and urine with significant (P < 0.05) differ-
ences in urinary selenium excretion. There was a tendency
for added vitamin E to lower the retention of selenite
selenium as in experiments 6 and 7 but this difference was
not significant. There were no differences among treat-
ments for the three blood selenium parameters measured.

The expected rise in blood selenium levels in these margin-

ally selenium-deficient pigs was observed.

Experiment 9. In table 18 is presented the data from

 

the second collection on the animals of experiment 8,
after they had access to their respective diets in self-
feeders for 17 days. Absolute selenium retention for

the unstable selenium premix was not significantly lower
than that on the fresh premix. These values may have
been influenced by significant (P < 0.01) differences

in selenium intake during the collection. Significant

(P < 0.05) differences in the percent of intake excreted
gig the feces also influenced the absolute retention
values. The addition of vitamin E significantly (P <
0.05) reduced the absolute retention of selenite selenium.
Again there were no differences in blood parameters among

the treatments. The increases in serum and whole blood

selenium levels were again evident as was a marked

125

TABLE 17. EFFECT OF UNSTABLE 0R FRESH SODIUM SELENITE
PREMIXES AND VITAMIN E 0N SELENIUM BALANCE IN THE
YOUNG PIG ( EXPERIMENT 8)

 

 

Fresh Se premix Ugiggiie
Item 0.2 ppm 0.2 ppm + Ea 0.2 ppm
Selenium balance:
Se intake, ug/day 52.0 50.0 53.2
Excretion, % of intake
Fecal 41.0 46.0 51.7
Urinary 12.7d 17.4d'e 29.8e
Se retention, ug/day 23.7b 12.3b’C 9.9C
Se retention, % 46.3 26.1 18.6
Selenium levels in blood:
Initial serum Se, ppm 0.043 0.045 0.036
Initial whole blood Se, ppm 0.068 0.068 0.060
Initial RBC Se, ppm 0.102 0.102 0.094
Final serum Se, ppm 0.113 0.100 0.102
Final whole blood Se, ppm 0.102 0.095 0.106
Final RBC Se, ppm 0.086 0.085 0.113

 

aVitamin E added at a level of 22 IU per kilogram of diet.

b

d

’CMeans on the same line with different superscripts are
significantly (P < 0.01) different.

’eMeans on the same line with different superscripts are
significantly (P < 0.05) different.

TABLE 18. EFFECT OF UNSTABLE OR FRESH SODIUM SELENITE
PREMIXES AND VITAMIN E 0N SELENIUM BALANCE IN THE
YOUNG PIG (EXPERIMENT 9)

 

 

Fresh Se premix Ugigggie
Item 0.2 ppm 0.2 ppm +Ea 0.2 ppm
Selenium balance:
Se intake, ug/day 129.5b 113.3C 132.2d
Excretion, % of intake
Fecal 22.5e 29.2f 25.9e'f
Urinary 41.7 54.5 47.7
Se retention, ug/day 46.5e 18.2f 34.9e’f
Se retention, % 35.9 16.4 26.4
Selenium levels in blood:
Initial serum Se, ppm 0.113 0.100 0.102
Initial whole blood Se, ppm 0.102 0.095 0.106
Initial RBC Se, ppm ’ 0.086 0.085 0.113
Final serum Se, ppm 0.133 0.141 0.124
Final whole blood Se, ppm 0.182 0.188 0.178
Final RBC Se, ppm 0.267 0.263 0.271

 

aVitamin E added at a level of 22 IU per kilogram of diet.

b’C'dMeans on the same line with different superscripts are

significantly (P < 0.01) different.

e'fMeans on the same line with different superscripts are

significantly (P < 0.05) different.

127

increase in erythrocyte selenium levels. Serum selenium
levels did not change proportionately as much as red blood
cell selenium levels did with time during the course of
these two experiments. This finding is suggestive of a
threshold level for selenium of inorganic origin in the
serum of young pigs. The failure to achieve consistent
results in these two collections left the question of the
availability of selenium in the unstable selenite premix
unanswered; but the results did lend further support to
the validity of the effect of vitamin E on selenite selen-

ium retention by the young pig.

Conclusions from Swine Studies

 

In feeding trials it was determined that the supple-
mentation of selenium-deficient practical corn-soybean meal
diets with 0.1 ppm selenium from sodium selenite or 11 IU
vitamin E per kilogram of diet prevented death losses,
gross pathology and histopathological lesions of nutritional
muscular dystrophy or dietary hepatic necrosis in growing-
finishing swine. The period when death losses were observed
was in the early post-weaning and early feeding segment of
the life cycle. Pretreatment of the dams with added die—
tary vitamin E did not seem to affect the susceptibility
of their offSpring to the deficiency although it did cause
minor alterations in SGOT activity of the pigs at weaning.
After 10 weeks of selenium and/or vitamin E supplementation,

added vitamin E had depressed serum selenium levels on the

128
basal diet while increasing them on the diets with added
selenite selenium. In a second study supplemental vitamin
E depressed erythrocyte selenium levels without showing
any effect on serum or whole blood selenium concentrations.

Selenium concentrations in skeletal muscle, myocardium,
liver and kidney were increased over levels observed in the
animals fed the deficient basals by supplementation of the
basal corn-soybean meal diets with sodium selenite. The
degree of increase in tissue selenium noted with graded
levels of added selenite was not directly proportional to
the increments of added selenium. Even the highest levels
of tissue selenium that resulted from feeding 0.05 to 1.0
ppm added selenium as sodium selenite until slaughter were
well below those reported for the same tissues from swine
receiving selenium-adequate diets containing only naturally-
occurring selenium. The levels of tissue selenium in
selenite-supplemented swine declined significantly 60 days
after supplemental selenite selenium was removed from the
diet. Supplemental vitamin E increased kidney selenium
levels in one study, but had no apparent effect on the se-
lenium levels of the other tissues analyzed.

The data from the balance trials indicated a parallel
to the observed tissue selenium levels in the feeding
trials. Supplemental selenite selenium was absorbed and
retained quite well when added to selenium-deficient prac-
tical corn—soybean meal diets at levels up to 0.1 ppm added

selenium. Levels of added selenium above 0.1 ppm (0.2 and

129
0.5 ppm) were readily absorbed and then excreted in greater
proportions in the urine to give absolute selenium reten-
tions very similar to those observed on the 0.1 ppm added
selenite selenium diets. Serum selenium levels showed a
plateauing at about the same level of supplemental dietary
selenite selenium indicating the possible existence of
tissue and serum thresholds for selenium of inorganic ori-
gin in the young pig. Erythrocyte selenium tended to in-
crease proportionately more than serum selenium. This
could possibly be due to the incorporation of seleno—amino
acids into new red cells as there was a definite lag fol-
lowed by a marked increase in erythrocyte selenium concen—
tration in the one 35 day balance study in which this
effect was observed.

Over short-term balances, added dietary vitamin E de-
creased the retention Of selenite selenium by increasing
its urinary excretion, an effect that may have been related
to its effect on serum selenium levels as discussed for the
feeding trial above. Additions of dietary vitamin E in-
creased the retention of selenium from seleniferous corn
primarily by reducing the fecal excretion of selenium.
Urinary excretion of selenium was higher for selenite
selenium than for seleniferous corn. The higher serum
selenium levels in pigs fed selenite selenium correlate
well with this finding. Fecal selenium losses were greater
on seleniferous corn than on selenite indicating poorer

absorption or greater resecretion of selenium into the

 

130
gastrointestinal tract. The changes occurring in the un-
stable selenite premix had no consistent effect on selenium
balance in the young pig.

As implied by Lindberg and Lannek (1965), it seems
that the pig retains and stores physiological levels of
dietary inorganic selenium in proportion to its require-
ments. Once the physiological stores are filled, the body
tends to excrete any excesses as long as excretory mechan-
isms are not overwhelmed by toxic levels of this nutrient.
It is fairly obvious that the resultant tissue levels are
affected by the form in which dietary selenium is provided
to the animal. This probably is due to selenium serving
both metabolic and structural roles in the tissues of the
body.

A schematic flow sheet depicting the postulated meta-
bolic pathways for selenium in monogastrics is presented in
figure 1. The information utilized in the preparation of
this figure was drawn primarily from that available in the
literature, but also includes data obtained from the ex—

periments reported in this manuscript.

Experiment 10. The selenium levels of Michigan 275-2X

 

corn from 17 plots, along with the soil type, soil pH and
yield data for each plot, are presented in table 19. The
mean level of selenium in these corn samples (0.033 ppm
dry) is in close agreement with the mean of 5 Michigan corn

samples (0.03 ppm air dry) reported by Patrias and Olson

131

Figure 1. Metabolic pathways of selenium in monogastrics.
Inorganic Se includes sodium selenite and sodium selen-
ate. Natural Se refers to the selenium naturally present in
common forages and grains. E refers to vitamin E or more
specifically in our work, d—Q—tOCOpheryl acetate. The rela-
tive widths of the arrows indicates the relative importance
of the pathway for the form of selenium represented by those
arrows. Modifying factors placed between major pathway
arrows signify equal effect on both major Se forms; while
factors placed outside signify a unilateral effect or repre-
sent reports of effects observed in studies in which only

one Se form was evaluated.

 

132

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134
(1969). The selenium concentration in corn was signifi-
cantly (P a 0.01) correlated with soil pH (r = 0.73).

Geering S£._£° (1968) reported that pH had a marked effect
upon the concentration of selenium in the soil solution
through its effect on the formation of an insoluble ferric
oxide-selenite adsorption complex. Selenium in solution
increased with increasing pH. The correlation of selenium
concentration with yield approached significance (r =
—0.475; for P < 0.05, n of 17 requires r = 0.482). Ehlig
gt _i, (1968) reported that in monocotyledonous plants the
selenium concentration decreased as dry matter production
increased while the selenium concentration in dicotyledon-
ous plants did not decrease with increasing dry matter pro-
duction. From this limited number of samples representing
several soil types it is difficult to draw any conclusions
about the effect of soil type on selenium levels in corn.
The clay loams seemed to produce higher selenium levels in
corn grain. Gardiner and Gorman (1963) noted a tendency
for higher plant selenium levels in heavy soils in Western
Australia.

In the three cases where two plots were grown on the
same type of soil within the same county there was some
indication that a stress situation led to higher selenium
levels in the grain. In Monroe and Saginaw Counties, selen—
ium levels were higher with high plant populations while

plant population had very little effect on yield per acre.

This effect of plant population could also possibly be

135
attributed to a greater proportion of root absorptive sur-
face area to grain produced in the high plant population
plots since there were no real differences in grain yield
per acre between the two plant populations. In Montcalm
County, irrigation increased yield and decreased selenium
levels in the grain produced. In this county the observed
differences may reflect both stress and dry matter produc-
tion effects as discussed earlier. Three samples are too
few on which to base general conclusions, but these were
interesting observations.

The effect of location is readily apparent from the
selenium levels of these corn samples with a six—fold dif-
ference between the highest and lowest values (0.013 to
0.089 ppm). In the earlier national surveys reported, the
conclusion was generally that the level of available selen-
ium in the soil, which is a reflection of the geological
derivation of the soil, is the major factor in determining
the selenium content of plants grown on these soils (Muth
and Allaway, 1963; Allaway and Hodgson, 1964; Kubota gt_gi3,
1967). In the survey of Kubota gt _i. (1967) they classed
most of Michigan as a low selenium area. In their survey
65% of the plants analyzed contained less than 0.05 ppm
selenium, 31% contained 0.05 to 0.10 ppm selenium and 4%
contained 0.1 to 0.5 ppm selenium. They classed the thumb
area and the southeastern quadrant of the lower peninsula
as variable selenium areas in which 26% of the samples con—

tained less than 0.05 ppm selenium and only 7% contained

136
0.5 to 1.0 ppm selenium. The only samples in our survey
containing over 0.05 ppm selenium were grown in these south-
eastern and thumb areas (figure 2). As in the survey above,
there was a considerable variation in selenium content of
corn grown in this area. Previous workers (Williams, Lakin
and Byers, 1941; Patrias and Olson, 1969) have reported
similar variations within rather small areas.

The data on the varietal effects on selenium content
of corn grown at one location in Clinton County, Michigan
are presented in table 20. All these grain samples were
very low in selenium content. Most of them approached the
practical lower limits of detection in our selenium analyti-
cal procedure. For this reason, precision was not as high
as one would prefer. The mean selenium content of these
samples is only about half of the mean reported for samples
collected over the state in table 19. The existence of
differences due to variety is apparent since the range from
lowest to highest levels of selenium (0.007 to 0.024 ppm)
represents a three-fold difference. The correlation of
grain yield with selenium level on these samples was non-
significant (r - -0.14) although it was a negative correla—

tion as in the statewide survey.

Conclusions from Corn Study

 

It is apparent from these data that marked location
and/or soil pH effects are manifested in the selenium con-

tent of corn. Soil type and stress factors may play a role

 

 

137

Figure 2. Selenium content (ppm dry) of Michigan 275-2X

corn.

H = high plant population
I = irrigated

M = muck soil

U - upland soil

   

 

 

138

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

aunt. 73:60 urm
arm 1.
an 0 03am atom
pascal. occur mm
m m: moan an: mm
M
mm"I
MY
cam mm Mtcosu mum .0 34
mm we
at. momma wmr am
.014 I .089 H .030
um
.02: .056 um W"
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139

TABLE 20. SELENIUM CONTENT OF 20 VARIETIES OF CORN GROWN
IN CLINTON COUNTY, MICHIGAN (EXPERIMENT 10)a

 

 

 

b YieldC Se,
Variety bu/A ppm dry

1 116.3 0.021

2 136.1 0.024

3 143.6 0.011

4 168.9 0.011

5 145.0 0.008

6 154.8 0.014

7 154.5 0.016

8 140.1 0.023

9 156.2 0.015

10 149.5 0.020

11 152.8 0.015

12 148.1 0.016

13 161.4 0.008

14 132.7 0.007

15 144.7 0.013

16 143.1 0.007

17 125.5 0.015

18 132.9 0.008

19 130.7 0.008

20 141.0 0.010

Mean 143.9 0.014
Standard error 2.83 0.0012

“Soil type - Miami loam 4505.
Soil pH - 6.0.
Planted - May 23, 1969.
Harvested - November 5, 1969.

bAppendix II, Table 1.

CYield is expressed as bushels per acre of 15.5% moisture,
shelled corn (equivalent to U.S. No. 2 market grade).

140
also. The possibility of a varietal effect on corn selenium
content cannot be ignored. The data in this trial do not
definitely prove the existence of a varietal effect but
they strongly suggest that such an effect may be real.

If corn-soybean meal rations for swine were compounded,
using soybean meal from the midwestern or eastern United
States and any of the Michigan corns analyzed in this sur-
vey, rations with very deficient to marginal selenium con—

tent would result.

 

B IB LIOGRAPHY

 

I

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.._.4

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APPENDICES

APPENDIX I

Fluorometric Selenium Analysis

 

Foreword

If one is to have reasonable success with this method
he has to accept the "rules" dictated by the procedure.
This is a demanding procedure: in time, care, and a certain
amount of technique. The entire analysis, from the start
of digestion to completion of reading the fluorescence of
the samples requires a minimum of 5 1/2 to 6 1/2 hours de-
pending on the agility and adeptness attained by the
technician. There is a 1-1 1/2 hour incubation period in
the procedure; otherwise the procedure requires constant
attention.

If this is your first experience with fluorometric
analysis as it was ours, you may tend to take lightly some
of the precautions herein. You will have muct less trouble
if you take them seriously. Assume that nothing is clean
unless you cleaned it. Reagents should be standardized as
much as possible and each new batch purchased used with

caution until it has proven to be acceptable.

159

160

Materials

 

Apparatus:

 

(a) Eberbach shaker —
Fitted with an improvised rack for holding 12
125-ml separatory funnels in an upright position (made from
a sulfuric acid box which fits perfectly on the platform —
wooden rack was too heavy). A cardboard "spacer" is used
over the funnel necks to keep the funnel stems from touch-

ing and breaking. Shaker is set at a maximum or nearly so.

(b) Fluorometer -

Aminco-Bowman Spectrophotofluorometer - This
machine is used "manually", i.e., the scanner is not used
and the percent transmittance is read directly off the
meter -

READ THE INSTRUCTION MANUAL BEFORE OPERATING FLUOROMETER.
Settings:
Excitation monochromator 372 mu
Emission monochromator 516 mu

Slit Widths:

Excitation 3 mm
Emission 3 mm
Photomultiplier 3 mm
Meter multiplier range .01
Meter sensitivity 50

Use the small round cuvettes. These are cleaned with

alcohol and dried with acetone between samples.

161
Periodically they should be cleaned with the cleaning solu-
tion. Never, repeat never touch any part of the cuvette
except the extreme lips with the bare fingers. Don't spare
the kimwipes when handling and drying cuvettes. They cost
$25.00 each and cannot be used if etched or scratched.

For stable operation, the instrument and lamp should
be allowed to warm up for 30-45 minutes before making read-
ings. When finished, turn off the lamp and photometer; but
do not turn off the blower until the lamp is completely
cooled. Remove the slot holders from the machine since

they will rust in tight if left in position.

(c) Glassware -

12 microneldahl digestion flasks (100 ml with
expansion bulb, Aminco 4-1874)

12 Phillips beakers with watchglasses

12 125 ml separatory funnels with Teflon stopcocks

1 250 ml separatory funnel with Teflon stopcock
2 100 ml beakers

1 100 ml graduated cylinder

1 10 ml graduated cylinder

1 5 ml volumetric pipette for DAN solution

1 1 ml volumetric pipette for standards

1 2 ml volumetric pipette for standards

1 25 ml burette with Teflon stopcock

2 50 ml burettes with Teflon stopcock

1 250 ml burette with Teflon stopcock

1 10 ml repipette to greatly speed the adding
of the water and EDTA solution.

162

The ground glass stOppers of the separatory funnels
m3§£_be replaced with No. 4 soft rubber stoppers (wrapped
with a fresh piece of Saran wrap for each run). Use only
Saran wrap. We lost a whole week trying to figure out the
source of interference when it was only a new batch of
brand X plastic wrap we had just begun using.

The above listed glassware should be segregated from
circulation with the general lab glassware to avoid prob-
lems of contamination. Once this glassware has been initi-
ally cleaned with Alconox and acid washed: one should not
use soap on it again. Don't worry about the outside of
glassware, it's the inside that counts. The washing pro-
cedure is as follows: (1) Rinse at least 3 times in flowing
distilled water (use a segregated nylon brush only if neces-
sary -'Qg soap). (2) Pour 250 ml of cleaning solution
into a Phillips beaker, use this to acid wash all glassware
once and then discard. (3) Rinse all acid washed items
3 to 4 times in flowing deionized distilled water. (4) In—
vert and dry in a reserved section of the drying room or a
forced air oven. Whenever possible wash the items in this
order; digestion flasks, Phillips beakers, separatory fun-

nels.

Reagents:

 

(1) Nitric Acid — sp. gr. 1.42 — Redistilled from
glass-use lots of acid-washed boiling beads, dis-

card first portion of distillate and do not boil

163
down too low in distillation flask. One can use
Baker nitric acid without redistilling. This
material is satisfactory for all except very low
Selenium samples (< 0.01 ug in a 1 gram sample).
Sulfuric Acid Solution - Add 140 ml selenium—free
H2804 - sp. gr. 1.85 - to deionized distilled
water and dilute to 1 liter.
Ammonium Hydroxide Solution - dilute 400 ml NH4OH
- sp. gr. 0.90 - to 1 liter with deionized dis—
tilled water.
Disodium Ethylenedinitrilotetraacetate Solution
(EDTA) — 0.01M; dissolve 7.4450 g dihydrate in
water and dilute to 1 liter.
2,3-Diaminonaphthalene (DAN) Solution. Reagent
from Aldrich Chemical Company, Milwaukee, Wiscon—
sin or G. Frederick Smith Chemical Co. Grind
finely with mortar and pestle, store in brown
bottle in the dark. We store excess DAN in 1
gram vials in the freezer. Weigh in reduced light.
Preparation: Insert a glass wool plug in stem
of 250 ml separatory funnel. At point indicated
in Procedure transfer 0.0650 g DAN reagent to
separatory funnel. Wash in with 65 ml of H2804
solution. Place on shaker for 15 min.; then add
65 ml of cyclohexane and shake for another 15
min.; let phases separate for 5 min. and draw off

lower phase for immediate use.

164
Note: The shaker should be kept in the darkroom,
and all operations with the DAN solution performed
in dim light. The samples after extraction into
cyclohexane should be protected from light also.
Cyclohexane-Eastman Kodak-Reagent grade.
Digestion Acids:
Concentrated Nitric Acid-Reagent grade redistilled
over glass or J. T. Baker nitric acid may be used
without redistilling.
Perchloric Acid (70% Reagent grade) (G. Frederick
Smith)
Selenium-free Sulfuric Acid - Dilute concentrated
sulfuric acid (J. T. Baker) with an equal volume
of deionized distilled water. Add 15 ml of re-
agent grade 48% hydrobromic acid per 200 ml of
the mixture. Heat strongly until volume is the
same as that of the original concentrated sulfuric
acid. This process requires several hours on a
large hotplate.
Cleaning Solution — 1:1 mixture of concentrated
nitric and concentrated sulfuric acids. Add
H2804 to HNO3. Do not mix in a soft glass bottle

as considerable heat is generated. This is bad

 

stuff - use under a hood; wear heavy rubber gloves
and keep bicarbonate solution handy. Wash it off
your gloves with deionized distilled water as it

will slowly eat through them too.

165

(9) Standard Selenium Solution -
Stock solution - Dissolve 100 mg selenium metal
(Alfa Inorganics - 99.999% pure) in 10 ml concen-
trated redistilled nitric acid. Add 28.0 ml con-
centrated sulfuric acid and dilute to 1 liter to
give 100 ug Se/ml in 1 N sulfuric acid. Store in
cold room. To prepare a working solution dilute
1.0 ml of stock solution to 1 liter with 0.1 N
H2504 to give 0.1 ug Se/ml in 0.1 N H2804.

(10) Hydrogen Peroxide (30%) - Reagent grade (Store in

cold room or refrigerator).

Procedure

 

Sample Preparation:

 

Grain, feed, feces and other dry samples should be
finely ground to insure good mixing and efficient prediges—
tion and digestion. Tissues are homogenized with deionized
water on either the Polytron or the Virtis homogenizer to
facilitate pipetting. In general, the following dilutions
have worked well for us: Skeletal muscle and heart are
homogenized with twice their weight of deionized water.
Liver and kidney are homogenized with three and five times
their weight of deionized water respectively. Clean all
glassware, including storage vials, with cleaning solution
and rinse them well with deionized water. The disposable
plastic Whirl—paks seem to work well for storing. Freeze
homogenates until you are ready to analyze them. Tissue
homogenates keep better if they are thawed rapidly in a

container of warm water than they do it allowed to thaw

166
slowly at room temperature. This rapid-thaw procedure works
especially well with the Whirl-paks.
Sampling error seems to be the greatest source of repli—
cation deviation with this method, so due care in sample

preparation is warranted.

Predigestion:

 

All samples (feeds, grains, tissues homogenates, etc.)
except strictly liquid samples (e.g. urine and standards)

should be predigested for at least 12 hours in the complete

 

digestion acid mixture.

 

Place accurately weighed sample (containing up to 0.4
ug selenium and preferably not greater than 1.0 g dry mat-
ter) along with 3 glass beads in a 100 ml Kjeldahl flask.
Add in_seguence 6.0 ml redistilled nitric acid, 2.0 ml 70%
perchloric acid, and 5.0 ml H2804 (sp. gr. 1.84). Burettes
will greatly speed the addition of these many reagents.

Use Teflon stopcocks only. Cover the flasks with a Nalgene
centrifuge tube to prevent contamination during the pre—

digestion period.

Digestion:

 

CAUTION: Since this is a modified perchloric acid digestion;
there is a potential danger for explosive perchlorates
to be formed. One should be especially careful with
samples containing fat, always swirl them to mix before

putting on heat. Keep the hood glass pulled down level

167
with the top edge of the heaters. This protects you
and makes a draft over the flasks giving more efficient
condensation action. Turn on showers in the perchloric

hood during digestion.

Always work in an efficient hood. Use the Aminco
digestor with individual rheostats for each pair of heating
elements. Heat carefully in initial stages to avoid loss
by foaming. Increase heat as digestion proceeds. Turn the
flasks 1800 occasionally to "wash" down all sides with con—
densate. Starting with cold heaters, set rheostat at 5, for
20 minutes, increase to 7 for 20 minutes, then to 9 to fin—
ish. Take back to 7 for H202 addition. Add small amounts
of nitric acid with a Pasteur pipette at first signs of
darkening due to charring. As digestion proceeds, the dark
brown nitric acid fumes will dissipate and eventually be re-
placed by white fumes. The liquid will become bluish-green

and then slowly yellow. This is the stage that samples

 

will char if they are going to; watch them_glg§gly. The
yellow color will slowly disappear and the liquid become
water clear in most cases. With feces and mixed feeds or
high ash samples, precipitation will occur before the solu-
tion becomes water clear. These samples are cooked until
turbid rather than water clear. Once they are water clear
(or turbid) remove flask from heat and swirl contents to
wet entire bulb area and lower neck of flask. Some yellow
coloring will reappear. This will be a "healthier" yellow

in the case of standards and tissue homogenates than it will

168
be for feeds and grains. Replace flask on heater and con-
tinue heating until solution becomes water clear and white

fumes appear. Make a check list, with flask number and

 

columns for "yellow" and "washed" to check off when you
note yellowing or wash down the flask, otherwise you will
not know if a flask has yellowed and cleared or just cleared.
When flasks become water clear and white fumes appear
remove them from heat and place in rack until all are
cleared. Turn the heaters down to 7 and replace flasks on
heater. Remove second flask from heater and let cool in
rack while you are working with first flask. This allows
the flasks to cool just enough, they need to be fairly warm
to react with the H202. Take the partially-cooled flask,
swirl contents, and add 1.0 ml of 30% hydrogen peroxide
slowly down walls of flask, swirl until all action ceases,
and resume heating until contents are boiling briskly and
white fumes appear. By cooling one and working with another
this works well to go around all 12 flasks and start over
again. Add 1.0 ml hydrogen peroxide twice more and continue
final heating for 5 minutes after appearance of white fumes.
Set flasks aside to cool while you are getting materials
ready for the next steps in the procedure. Keep them near
the hood to keep fumes out of the lab.

NOTE: Turn on hot plate NOWl

Use a repipette to add 20 ml of deionized distilled

water to the digested material, carefully. Mix and quanti~

 

tatively transfer the contents of the flask to a 250 ml

169
Phillips beaker using two 10 ml washings and cover with a
watchglass. After you have transferred all 12 samples to

the Phillips beakers add in turn, with mixing, 10 ml di-

 

sodium EDTA solution, 25.0 ml ammonium hydroxide solution
and 5.0 ml DAN solution. The time one begins the DAN prep—
aration will vary with how long it takes him to transfer
the flask contents to the beakers and add the other two
reagents. You should time it so that the DAN is ready for
use when you are ready to add it to the samples. In other
words, do not let it sit around too long after preparation.

NOTE: The shaker should be in the darkroom, and all
operations with the DAN solution or the complete reaction
mixture should be performed in dim light. The samples
after extraction should be protected from light also. Give
the beaker and contents several seconds of healthy swirling
after the addition of eagh reagent. Boil the flask contents
for 2 minutes on the hotplate. Place on cart and roll into
darkroom and let them sit undisturbed ifl_£he_dark for 1 hour.
You have about 20-30 minutes leeway here, i.e. the samples
may be incubated up to 1 1/2 hours if you don't mess around
in the extraction of such samples.

After incubation quantitatively transfer the flask con-
tents to a 125 ml separatory funnel and add 6.0 ml cyclo—
hexane. Stopper with a Saran-wrapped rubber stopper and
shake for 15 minutes. Let stand for 5 minutes to allow
phases to separate. Discard lower aqueous phase and drain

cyclohexane solution into 15 ml centrifuge tube. Centrifuge

170
for 5 minutes, place tubes inEilight tight container for
trip to fluorometer.

Read fluorescence against deionized water blank taken
through entire procedure. Do not leave the blank in the
cuvette holder for any longer than necessary, as exposure
to the excitation light causes fluorescence of the solutions
to change. Plot standard graph from two standards. Plot is
linear up to at least 0.5 ug selenium and passes through

the origin consistently.

Additional notes:

 

The clean work bench is used in our lab to store re-
agents and some glassware. It is essential that all items
be kept rust-free and dust-free. Metal contamination as
well as grease contamination must be avoided. Any fluor—
escing material is a potential problem also.

It has been our experience that when fluorescent con-
tamination has been a problem (e.g. with the Brand X plastic
film) the samples have a definite blue fluorescence when
placed in the cuvette well rather than the normal bright
green fluorescence.

Once the digested material has cooled enought to work
with, it should be carried through the rest of the procedure.
We have found that we cannot get consistent results by di—
gesting samples one day and working them up the next.

In conclusion, I would say that this may seem like a

very lengthy treatment of a relatively simple procedure.

171

You were forewarned: This is not a routine or simple pro—

cedure. You will spare yourself much agony and frustration

if someone familiar with the analysis walks you through the

first several times.

APPENDIX II

TABLE 1. CLINTON COUNTY CORN VARIETIES

 

 

Number Variety Breeding type
1 Michigan 280 2X
2 Michigan 568-3X 3X
3 Michigan 402-2X 2X
4 Michigan 555-3X 3X
5 Garno S95 2X
6 Northrup King PX519 Sp
7 Blaney B601 2X
8 Pfister P.A.G. 8X52 2X
9 Blaney 6606A 2X

10 Pioneer 3773 2X
11 Michigan 500-2X 2X
12 DeKalb XL45 2X
13 Cowbell SX112 2X
14 Pioneer 3582 2X
15 Michigan 275—2X 2X
16 Michigan Exp. 67-142 3X
17 DeKalb XL 306 3X
18 Funk Bros. G17A 4X
19 Funk Bros. G4287 3X
20 Northrup King PX525 Sp

 

172

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