ABSTRACT

THE INFLUENCE OF SUPPLEMENTAL VITAMIN E AND ARSANILIC
ACID ON DIETARY SELENIUM UTILIZATION BY SWINE

BY
John Paul Hitchcock

Two areas of investigation were undertaken in this
research. The first area of research was designed to
investigate the effects of supplemental arsanilic acid,
selenium (Se) and vitamin E on performance, deficiency
signs, hematology, tissue selenium and selenium balance
in growing-finishing pigs fed diets low in vitamin E and
selenium. One feeding trial with growing-finishing pigs
and two selenium balance experiments with young pigs were
conducted to evaluate these effects. Yorkshire, Hampshire
and crossbred pigs were maintained in a modern confinement
management system in the feeding trial. Young pigs (5
weeks of age) for the balance experiments were housed,
shortly after weaning, in stainless steel metabolism cages
and received a constant, near ad Zibitum intake of experi-
mental diet in three meal feedings daily.

In the feeding trial, classic Se and/or vitamin E
deficiency lesions were observed in four animals fed diets
not supplemented with Se or vitamin E. Selenium supplemen-

tation (0.1 ppm Se, as sodium selenite) increased (P<.01)

John Paul Hitchcock
liver, kidney and diaphragm muscle selenium concentrations.
Arsanilic acid supplementation (99 ppm) increased (P<.OS)
diaphragm muscle selenium concentrations. Vitamin E (22
IU/kg) supplements had no effect on tissue selenium
levels.

The two balance studies were concerned with Se absorp-
tion and retention on diets supplemented with Se as sodium
selenite or seleniferous corn and the effects of arsanilic
acid (99 ppm) and vitamin E (22 IU/kg) on Se balance.
Supplemental selenium (0.1 ppm) from seleniferous corn
increased percent fecal Se excretion (as a percent of
intake) and decreased percent urinary Se excretion as
compared to Se from sodium selenite. Vitamin E supplemen-
tation increased Se retention from seleniferous corn by
decreasing urinary and fecal Se excretion. Arsanilic
acid decreased Se excretion and increased Se retention in
these experiments.

The second area of research was designed to study
the effects of 0.1 ppm supplemental selenium as sodium
selenite and four dietary levels of vitamin E (d-a-
tocopheryl acetate) supplementation on performance,
hematology, tissue selenium and selenium balance in
growing-finishing pigs fed diets low in vitamin E and
selenium. Pigs were housed and maintained during one
feeding trial and one selenium balance study as described

for the previous experiments. There were no lesions

John Paul Hitchcock
indicative of vitamin E-selenium deficiency and no deaths
due to the deficiency were observed in the feeding trial.
Dietary supplements of Se or vitamin E had no significant
effects on overall performance of pigs during the feeding
trial. Selenium supplementation increased (P<.01) serum
selenium concentrations, while vitamin E supplementation
had no effect on serum selenium concentrations. Selenium
supplementation increased (P<.01) rectus abdominus muscle,
liver-and kidney Se concentrations on both a wet and dry
basis. There was a significant (P<.05) interaction
between selenium and vitamin E on muscle selenium concen-
trations (wet basis). The addition of increasing levels
of vitamin E to unsupplemented Se diets decreased muscle
Se concentrations while on Se supplemented diets, muscle
Se concentrations increased with increasing levels of
vitamin E. Dietary level of vitamin B supplementation
had no significant effect on tissue Se concentrations.

The balance study was conducted to study Se absorp-
tion and retention on a selenium supplemented basal diet
(0.172 ppm Se) with dietary vitamin E added at levels of
0, S, 10 or 15 IU E/kg of diet. Supplemental levels of
vitamin E had no significant effects on selenium excretion
or retention, serum selenium or tissue selenium concentra-
tions on either a wet or dry basis in this short-term

selenium balance study.

THE INFLUENCE OF SUPPLEMENTAL VITAMIN E AND ARSANILIC
ACID ON DIETARY SELENIUM UTILIZATION BY SWINE

BY
John Paul Hitchcock

A DISSERTATION
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

1975

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to Dr. E. R. Miller and Dr. D. E. Ullrey for their guidance
during the conduction of this research and for their con-
structive review of this manuscript. The author wishes
to express his grateful appreciation for the example pro-
vided by his major professor, Dr. B. R. Miller, whose
insights into research and competence in swine nutrition
research have provided the author a constant inspiration
for achievement. His encouragement and the associations
during the course of this research will always be cherished.

Appreciation is also expressed for the helpful
guidance and interest of my other graduate committee
members, Dr. K. K. Keahey, Dr. R. W.Luecke and Dr. W. T.
Magee. The assistance of Dr. W. T. Magee with the statis-
tical analysis of the data is gratefully appreciated. A
special note of thanks is extended to Dr. A. L. Trapp for
his participation on my guidance committee, contributions
during my graduate program and for constructive review of
this manuscript.

The assistance of Dr. K. K. Keahey, Dr. A. L. Trapp,

Dr. D. L. Whitenack and the staff of the Veterinary

ii

Diagnostic Laboratory during this research is gratefully
acknowledged.

The cooperation and support of Mr. Roger Hale and
his associates at the Swine Research Center with regard
to animal care and the mixing of experimental rations is
sincerely appreciated.

Sincere appreciation is expressed to Dr. R. H.
Nelson and the Animal Husbandry Department for the use
of facilities and animals and for financial support in
the form of a graduate assistantship. The author also
expresses sincere appreciation to Dr. J. W. Thomas and
the N.I.H. Predoctoral Trainee program for financial
support and research funds.

Appreciation is extended to fellow graduate students,
laboratory personnel and departmental secretaries for
their assistance during the course of my research program.
The assistance of Mrs. Rosemary Covert with laboratory
procedures and the secretarial assistance of Mrs. Delores
Calcatera are gratefully acknowledged.

The author wishes to express sincere appreciation to
Dr. A. Wayne Groce for his encouragement, instruction and
training in the procedural steps of Se analysis. This
research would not have been possible without his quanti-
tative, scientific instruction and willingness to train

the author for this difficult quantitative procedure.

iii

Special thanks are due Ms. Kim Miller, Ms. Janice
Fuller, and Ms. Shirley Goodwin for their assistance in
typing, organizing and printing this manuscript.

The author is very grateful to his parents for their
continual encouragement, support and interest during these
ten years of college work.

The author wishes to express sincere appreciation
and gratefulness to Ms. Brenda Buzzell for her assistance,
encouragement and sacrifices during the conduction of the

research and preparation of this manuscript.

iv

John Paul Hitchcock
Candidate for the degree of
Doctor of Philosophy

DISSERTATION:

The Influence of Supplemental Vitamin E and
Arsanilic Acid on Dietary Selenium Utiliza—
tion by Swine

OUTLINE OF STUDIES:

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

Minor Areas: Biochemistry and Physiology

BIOGRAPHICAL ITEMS:
Born: April 13, 1945, Muscatine, Iowa

Undergraduate Studies: Iowa State University
1964-1968

Graduate Studies: Pennsylvania State University
1968-1970

Michigan State University
1970-1975

Experience: Graduate Research Assistant
Pennsylvania State University, 1968-1970

Graduate Research Assistant and National
Institute of Health Predoctoral trainee
Michigan State University, 1970-1975

MEMBER: American Society of Animal Science
Alpha Zeta
Phi Kappa Phi
The Society of Sigma Xi

AWARD: Recipient of the 1974, Michigan State University

Sigma Xi, Graduate Student Award for Meritorious
Research

V

I.
II.

III.

TABLE OF CONTENTS

INTRODUCTION .
REVIEW OF LITERATURE .

Selenium and/or Vitamin E Deficiency in
Farm Animals . .
Experimental diet- induced deficien-
cies. . .
Poultry. .
Cattle and sheep
Swine.
Occurrence of deficiencies under
practical conditions.
Cattle and sheep .
Swine.
Pathology of selenium and/or vitamin
E deficiency in swine .
Gross and microscopic changes.
Clinical pathology of selenium
and/or vitaminE Edeficiency
of swine . .
Selenium levels in swine blood and
tissues .
Selenium Balance in Swine. . .
Interrelationships of Selenium with Sulfur,
Vitamin E and Other Dietary Factors.
Vitamin E . . . . . .
Sulfur amino acids.
Polyunsaturated fat
Synthetic antioxidants.

Iron . . . .
Arsenic. . . . . . . . .
Vitamin E Studies in Swine . . . . . . . .

Page

13
16

22
22
23

27
27

EFFECT OF ARSANILIC ACID, SELENIUM AND VITAMIN

E ON PRODUCTIVE PERFORMANCE, TISSUE SELENIUM
CONCENTRATIONS AND SELENIUM BALANCE IN
GROWING- FINISHING SWINE. . .

Introduction . .

Experimental Procedure
Experiment 1..
Experiment 2.

vi

IV.

VI.

VII.

Page

Experiment 3. . . . . . . . . . . . . 61
Results and Discussion . . . . . . . . . . . 63
Experiment 1. . . . . . . . . . . . . 63
Experiment 2. . . . . . . . . . . . . 7]
Experiment 3. . . . . . . . . . . . . 75
Summary. . . . . . . . . . . . . . . . . . . 81

EFFECT OF DIETARY VITAMIN E LEVEL ON PERFORM-
ANCE, TISSUE SELENIUM AND SELENIUM BALANCE
IN GROWING- FINISHING SWINE . . . . . . . . 84

Introduction . . . . . . . . . . . . . 84
Experimental Procedure . . . . . . . . . . . 85
Experiment 1.. . . . . . . . . . . . 86
Experiment 2. . . . . . . . . . . . . 88
Results and Discussion . . . . . . . . . . . 89
Experiment 1. . . . . . . . . . . . . 89
Experiment 2. . . . . . . . . . . . . 98
Summary. . . . . . . . . . . . . . . . . . . 104

BIBLIOGRAPHY . . . . . . . . . . . . . . . . 106
APPENDIX A. FLUOROMETRIC SELENIUM ANALYSIS. 119
APPENDIX B. EFFECT OF ADDED DIETARY SELENIUM,

VITAMIN E AND ARSANILIC ACID ON INITIAL AND
INTERMEDIATE BLOOD PARAMETERS (EXPERIMENT 1) 133

vii

Table

10

11

LIST OF TABLES

Composition of basal diets (Experiment 1)

Effect of dietary selenium, vitamin E and
arsanilic acid supplementation upon pig
performance and liver weight (Experiment
1). O O O 0 O O O O O 0

Effect of added dietary selenium, vitamin
E and arsanilic acid on final blood
parameters (Experiment 1) . . .

Effect of added dietary selenium, vitamin
E and arsanilic acid on tissue selenium,
ppm (Experiment 1).

Effect of added dietary selenium, vitamin
E and arsanilic acid on selenium balance
in the young pig (Experiment 2)

Effect of added dietary selenium (selen-
iferous corn), vitamin E and arsanilic
acid on selenium balance in the young pig
(Experiment 3). . . . .

Composition of basal diet (Experiment 1).
Effect of dietary selenium and vitamin E
supplementation upon pig performance
(Experiment 1). . . . . . .

Effect of added dietary selenium and
vitamin E on 10 week and final hemoglobin
concentration and hematocrit (Experiment 1)

Effect of added dietary selenium and
vitamin E on serum Se levels (Experiment 1)

Effect of added dietary selenium and
vitamin E on tissue Se levels (Experiment 1)

viii

Page
56

64

67

69

72

76
87

91

92

94

95

Table

12 Effect of dietary vitamin E levels of
supplementation on selenium balance
(Experiment 2). . . . . . . . . . .

13 Effect of dietary vitamin E levels of
supplementation on blood parameters
(Experiment 2). . . . . . .

14 Effect of dietary supplemental level of
vitamin E on serum enzyme activity
(Experiment 2). . . . . . . .

15 Effect on dietary supplemental level of
vitamin E on tissue Se concentrations
(Experiment 2). . . . . . . . . .

APPENDIX
1 Effect of added dietary selenium, vitamin

E and arsanilic acid on initial and inter-
mediate blood parameters (Experiment 1)

ix

Page

99

100

101

103

133

INTRODUCTION

The role of selenium in nutrition during the past
150 years has changed dramatically. Selenium was first
recognized for its toxicity characteristics and was iden-
tified as the toxic principle in "alkali disease in
.horses." The concept of selenium as a toxic element was
generally accepted as its only role in nutrition until,
in 1957, Schwarz and Foltz (1957) showed that sodium
selenite would prevent liver necrosis in rats fed torula
yeast diets. Eggert et a2. (1957) and Grant and Thafvelin
(1958) demonstrated a relationship between selenium
deficiency and hepatosis dietetica in swine.

Thompson and Scott (1970) demonstrated that 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. This research established that selenium is
indeed an essential trace element. The discovery of a
specific function of selenium as a component of gluta-
thione peroxidase in animals has been described by Rotruck
at al. (1973) and Hoekstra (1974). These researchers

indicated that decreases in glutathione peroxidase appear

2
to explain partially the degenerative diseases induced by
selenium deficiency.

Naturally occurring selenium and/or vitamin E
deficiency in ruminant farm animals has been recognized
in certain areas of the United States for many years.
Field cases of selenium and/or vitamin E deficiency were
first observed in swine in this country in the late 1960's.
Michel et a1. (1969) were the first to describe the occur—
rence of the disease in commercial swine herds in Michigan
on practical corn-soybean meal type diets. Trapp et al.
~(1970) suggested that the condition probably existed
prior to the first observed cases in 1967. They observed
that most herds in which selenium-vitamin E deficiency
occurred were being fed corn-soybean meal diets supple-
mented with arsanilic acid as a feed additive. Arsanilic
acid has been demonstrated to counteract selenium toxicity
in swine (Wahlstrom et al., 1955). Therefore, Trapp et
a1. (1970) suggested that arsanilic acid may have been
antagonistic to selenium and thus enhanced the occurrence
of a natural selenium-vitamin E deficiency.

Preliminary estimates of the dietary selenium require-
ment for the pig have been reported by Groce et al.
(1973b). These studies indicated that the dietary
selenium requirement for the pig when supplemental
vitamin E does not exceed 11 IU/kg is 0.15 ppm. These

studies also indicated a need for further research on

3
the interrelationship of Se with vitamin E. The National
Research Council (1973) suggests that 11 IU of vitamin E
be added per kg of diet until more specific information
is obtained on vitamin E and selenium requirements of
swine.

The studies presented in this dissertation were
undertaken to define more clearly the effects of arsanilic
acid, vitamin E and selenium supplementation in corn-
soybean meal diets for swine. The studies on tissue
selenium levels and selenium balance studies were under-
taken to study the effects of arsanilic acid and vitamin
E on the utilization of natural and supplemental selenium.
Vitamin E supplementation of swine rations is relatively
expensive and determination of an optimal dietary level
of vitamin E in diets supplying required levels of

selenium was deemed an important area for investigation.

REVIEW OF LITERATURE

Selenium and/or Vitamin E Deficiency in Farm Animals

Experimental diet-induced deficiencies

The early investigations with selenium were achieved
with diets containing some selenium and usually substan-
tial quantities of unsaturated fat. These studies
revealed a relationship between vitamin E and selenium.
To induce dietary selenium deficiency, semi-purified
diets containing torula yeast as the major source of
protein were often used. These diets contained a very
low level of vitamin E and were deficient in sulfur con-
taining amino acids but very high in unsaturated fatty

acids.

Poultry. In independent discoveries in 1957,
selenium was identified as an integral part of Factor 3
active in preventing liver necrosis in rats by Schwarz
and Foltz (1957) and was shown to prevent exudative
diathesis in chicks fed a torula yeast diet low in
vitamin E (Patterson et al., 1957). The latter authors
reported that exudative diathesis in chicks fed torula

yeast diets could be prevented by either selenium or

5
vitamin E additions to the diet. In these studies
selenium levels of 0.1 ppm in the form of seleno-
cystathionine or sodium selenite prevented exudative
diathesis. Rahman et al. (1960) reported that vitamin
E, dried brewers' yeast or selenium (0.05 or 0.1 ppm)
prevented exudative diathesis in chicks fed a torula
yeast diet. Corn distillers' dried solubles and con-
densed fish solubles also prevented signs of exudative
diathesis. In the absence of vitamin E, selenium pro-
moted growth when fed at 0.05 or 0.1 ppm in the diet.
In the presence of vitamin E no growth response was
observed from selenium supplementation or dried brewers'
yeast in the diet. Selenium, vitamin E, or the use of
starch as a carbohydrate source completely prevented the
development of exudative diathesis in turkey poults fed
a torula yeast diet. Mathias and Hogue (1971) observed
that selenium, vitamin E and ethoxyquin were effective
in preventing exudative diathesis, and a significant
interaction between selenium and vitamin E was observed
in preventing mortality. This result was interpreted as
support for a biological synergism between the two
nutrients. They concluded that at relatively low levels
of dietary and body stores of selenium and vitamin E,
synthetic antioxidants are partially capable of prevent-
ing or curing exudative diathesis but not eventual death

of chicks fed torula yeast basal diets. Nesheim and

6
Scott (1961) stated that the most marked nutritional
effect of dietary selenium in chickens and turkeys is
its effectiveness in preventing exudative diathesis.
Selenium is not completely effective in preventing muscu-
lar dystrophy in chicks, and levels of selenium that
partially prevent muscular dystrophy (1-5 ppm in the
diet) are much higher than the levels effective against
exudative diathesis. Selenium appeared to have growth
promoting properties for chicks receiving diets contain-
ing torula yeast and adequate in vitamin E.

The major muscle lesions in chicks were muscular
dystrophy of the pectoral and leg muscles while myopathy
of the gizzard and myocardium were observed in turkey
poults fed selenium and/or vitamin E deficient diets.
Machlin and Shalkop (1956) observed that chickens fed
casein-gelatin diets low in vitamin E and sulfur amino
acids to four weeks of age developed a muscular degenera-
tion manifested grossly as white striations of the breast
and leg muscles and microscopically as a hyaline type
degeneration. The addition of a-tocopheryl acetate,
methionine, cystine or a high level of an antioxidant
to the diets completely prevented muscular degeneration.

Walter and Jensen (1963) reported that adding 1 ppm
selenium (as selenious acid) or 20 IU/kg of a-tocopheryl
acetate to torula yeast diets calculated to be slightly

deficient in sulfur amino acids completely protected

7
turkey poults against gizzard and skeletal muscular
dystrophy. Skeletal muscles were affected to a lesser
extent than the gizzard musculature. Supplementation
of the ration with cystine (0.15%), methionine (0.4%),
low levels of ethoxyquin (0.025%) or selenium (0.01%
or 0.1 ppm) proved ineffective in preventing the skeletal
and gizzard muscular dystrophy. A high level of ethoxy-
quin (0.3%) reduced the incidence but did not completely
protect against the skeletal and gizzard muscular
dystrophy. Selenium, vitamin E and the high level of
ethoxyquin prevented anemia and reduced albumin-globulin
ratios which is generally observed in cases of muscular
dystrophy. Hintz and Hogue (1964b) added raw kidney
beans to vitamin E supplemented torula yeast diets and
increased the incidence of nutritional muscular dystrophy
from 5-6% to 45-100%. This indicated to the authors that
the beans contained an anti-vitamin E Factor. Two vitamin
E antagonists were isolated and the authors indicated
that an alcohol soluble antagonist was due to unsaturated
fats present in the raw kidney bean. Hathcock and Scott
(1966) fed chicks a casein-soy-torula yeast diet to study
the role of cysteine as the functional compound in pre-
vention of muscular dystrophy in chicks. Sodium selenite
and ethoxyquin were included in the diet to prevent
exudative diathesis and encephalomalacia while allowing

production of muscular dystrophy. Their results

8
indicated that guanidoacetic acid accelerates the conver-
sion of methionine to cysteine and reduces the severity
of muscular dystrophy while creatinine, choline and
betaine inhibit the conversion and accentuate the
dystrophy. They concluded that cysteine, not methionine,
was the metabolically active sulfur amino acid in the
prevention of nutritional muscular dystrophy in vitamin
E deficient chicks. Hathcock et al. (1968a) studied the
effects of cysteine and several sulfhydryl compounds on
nutritional muscular dystrophy in chicks. They concluded
that cysteine is the functional sulfur compound in one
of the pathways involved in the prevention of muscular
dystrophy in the chick while vitamin B may be involved
in another pathway. Hathcock (1968b) studied the role
of cysteine in prevention of muscular dystrophy in the
chicken with recognition that one of the normal metabolic
fates of cysteine is oxidation and decarboxylation to
taurine, and taurocholate is the only bile salt produced
by the chicken. The rate of cysteine to taurine conver-
sion and the incidence of muscular dystrophy were
increased when cholic acid was added to the diet. Tauro-
cholic acid and taurine additions decreased the rate of
cysteine to taurine conversion and reduced the occurrence
of muscular dystrophy. Vitamin E additions to the basal
diet reduced the taurine excretion rate. Scott et al.
(1967) formulated a practical type corn-soybean meal diet

utilizing feedstuffs obtained from areas with known low

9
selenium soils and without vitamin or methionine additions
to the diet. They concluded that selenium is the primary
nutritional factor required to prevent myopathies in turkey
poults while vitamin E was of less importance and sulfur
amino acids were ineffective. They suggested that the selen-
ium requirement of the poult on a practical type diet ranged
from 0.18 ppm in the presence of vitamin E to 0.28 ppm
of selenium in the absence of added vitamin E. The order
of prominence of the "selenium-responsive" myopathies of
the young poult appear to be first of the gizzard, second
of the myocardium and third of the skeletal muscle.
Thompson and Scott (1969), in an attempt to establish
selenium as an essential element, reported that chicks
fed semi-purified diets were protected against exudative
diathesis by supplements of either 10 ppm d-a-tocopheryl
acetate or 0.04 ppm selenium. Chicks given crystalline
amino acid diets containing less than 0.005 ppm selenium
had poor growth and high mortality even when the diet
contained up to 200 ppm d-a-tocpheryl acetate. Higher
levels of vitamin E prevented mortality, but, even with
1000 ppm, growth was inferior to that obtained with sup-
plements of selenium and no added vitamin E. When diets
contained 100 ppm vitamin E (d-a-tocopheryl acetate),
the selenium requirement was less than 0.01 ppm, whereas
with 10 ppm vitamin B it was 0.02 ppm, and with no added

vitamin E it was approximately 0.05 ppm. Thompson and

10

Scott (1970) conducted further studies with crystalline
amino acid diets, containing high levels of vitamin E

but less than 0.02 ppm selenium. Selenium deficiency
resulted in poor growth, poor feathering and atrophy of
the pancreas which resulted in impaired fat hydrolysis

and poor absorption of lipids, including vitamin E. They
stated that selenium is, thus, an essential trace nutrient
and one of its roles is in maintenance of the pancreas.
The indirect effect of selenium deficiency on vitamin E
absorption does not explain the ability of both selenium
and vitamin E to prevent exudative diathesis since a

more direct interrelationship is probably responsible

for this effect. Noguchi et al. (1973a) studied selenium
deficiency uncomplicated by vitamin E deficiency in chicks
using a crystalline amino acid diet complete in all known
nutrients except selenium. Gross signs of deficiency
included growth retardation marked by particularly poor
growth of muscle, liver, and pancreas. \Histochemical and
biochemical studies throughout the entire experimental
period showed no sign of lysosomal disruption in the
acinar cells or in the invading fibroblasts and macro-
phages, demonstrating that the function of selenium in
protecting the pancreas from atrophy and fibrosis does
not result from its protection of the lysosomal membranes
Of the pancreatic cells. Noguchi et al. (1973b) continued

their studies using a crystalline amino acid diet very

11

low in selenium and vitamin E and demonstrated that the
glutathione peroxidase level of chick plasma is directly
related to selenium level in the diet and to the effective-
ness of selenium in prevention of exudative diathesis.
They suggested that the plasma glutathione peroxidase
present, when the diet contains adequate selenium, acts
to prevent exudative diathesis by destroying peroxides
that may form in the plasma and/or cytosol of the capil~
lary cell. Vitamin E appears to prevent exudative
diathesis by acting within the lipid membrane where it
neutralizes free radicals, thereby preventing a chain
reaction autoxidation of the membrane lipids. Combs and
Scott (1974a) determined the dietary requirements of
chicks for vitamin E and selenium utilizing a semi-
purified basal diet low in selenium and vitamin E. The
results of this study showed that both selenium and
vitamin B were required to completely protect hepatic
microsome membranes from in vitro ascorbic acid-stimulated
peroxidation. Vitamin E was required at 30 to 50 IU per
kilogram diet for optimal growth and utilization of
dietary vitamin E as plasma tocopherols in selenium-
adequate chicks. Selenium was required at 0.06 ppm (as
sodium selenite) for inhibition of peroxidation and for
Optimal growth in vitamin E-adequate chicks. Combs and
Scott (1974b) determined the effects of high-level anti-

cxxidant feeding on selenium and vitamin E function in

12
vitamin E depleted chicks fed a semi-purified basal diet
low in selenium and toc0pherols. Ethoxyquin, ascorbic
acid and vitamin A reduced selenium requirements for
growth and prevention of exudative diathesis and mortality
in vitamin E deficient chicks. Ethoxyquin and ascorbic
acid reduced the dietary vitamin E requirement for pre-
vention of in vitro ascorbic acid stimulated peroxidation
in hepatic microsomes in selenium adequate chicks.

Vitamin A severely depressed the plasma concentra-
tion of tocopherols and increased microsomal peroxidation
without response to dietary vitamin E. The results of
this study indicated that antioxidants increased the
utilization of dietary selenium without severely affecting
vitamin E antioxidant function, but vitamin A appeared
to antagonize vitamin E absorption directly.

Cantor et al. (1975a) studied the biological availa-
bility of selenium in feedstuffs and selenium compounds
for prevention of exudative diathesis in chicks using a
casein-soy-torula yeast basal diet deficient in vitamin
E and selenium. Protection against exudative diathesis
was highly correlated with plasma glutathione peroxidase
activity in chicks fed sodium selenite or selenomethionine,
indicating that biological availability was determined
by the ability of the chick to utilize the various forms
of selenium for enzyme activity. Cantor et al. (1975b)

evaluated the efficacy of dietary supplements of selenium

13
for prevention of pancreatic fibrosis in chicks fed a
crystalline amino acid basal diet containing .012 ppm of
naturally occurring selenium and 15 IU vitamin E/kg diet.
Selenomethionine was four times as effective as either
selenite or selenocystine in preventing pancreatic degen-
eration and increasing the relative weight and selenium
concentration of the pancreas. Studies on plasma and
pancreatic glutathione peroxidase activities did not
show any relationship between enzyme activity and preven-

tion of pancreatic fibrosis.

Cattle and sheep. Many experiments have been con-

 

ducted to study methods of controlling or preventing
'muscular dystrophy or white muscle disease of young
ruminant farm animals in many areas where soil selenium
levels are low. A reason for the prevalence of-white
muscle disease among cattle and sheep may be that, more
than any other domestic species, they are likely to
consume forages grown on low selenium soils as virtually
their entire diet.

Proctor et a2. (1958) fed a known dystrophogenic
diet of raw cull kidney beans and mixed hay to ewes
during gestation. Muscular dystrophy in the lambs born
to these ewes was prevented by addition of 1 ppm selenium
as sodium selenite to the diet or by feeding 100 IU a-
tocopheryl acetate per head per day or 0.25 pounds linseed

meal per head per day. The protection afforded by linseed

l4
meal was possibly due to its high selenium content (1.18
ppm). Hogue et a1. (1959) utilized a similar hay and
cull kidney bean basal diet and observed that providing
lambs every other day with 100 mg a-tocopheryl acetate
or 1 mg selenium would control muscular dystrophy.
Vitamin E at the rate of 100 IU/ewe/day was adequate but
not as effective as 1 ppm selenium in preventing muscular
dystrophy of lambs. Welch et al. (1960) produced muscular
dystrophy in lambs by feeding fish liver oil to ewes
during pregnancy and lactation. The fish liver oil
lowered ewe and lamb blood plasma vitamin E concentrations
and increased the incidence of muscular dystrophy.
Vitamin E administration to ewes was effective in prevent-
ing and curing muscular dystrophy in lambs. Selenium
decreased but did not eliminate the occurrence of muscular
dystrophy in lambs. Hintz and Hogue (1964a) compared the
addition of combinations of selenium and sulfur to the
diet of the ewe and cystine and methionine when given to
the lamb on the incidence of muscular dystrophy in the
lambs. Selenium (0.17 ppm) given during lactation had
no significant effect on clinical incidence of muscular
dystrophy but did reduce the number of lambs with elevated
SGOT values. Dietary sulfur (0.33%) increased the clinical
incidence of muscular dystrophy and, when given in combi-
nation with selenium, prevented any beneficial effect of

Selenium. Administration of cystine or methionine to

15
lambs was not effective in preventing the nutritional
muscular dystrophy.

Ewan at al. (1964) fed lambs an artificial milk diet
based on stripped lard and torula yeast. Selenium sig-
nificantly improved total weight gains, rate of gain and
survival time. The improvement in total weight gains and
survival time due to vitamin E supplementation approached
significance (P<0.l) but little effect was noted on rate
of gain. No interaction between vitamin E and selenium
was observed.

Schubert et a1. (1961) produced white muscle disease
in lambs and calves by feeding the dams during gestation
on native hays from low selenium areas. The character-
istic clinical symptoms of erratic locomotion, cardiac
failure and gross and histopathological lesions of
skeletal and cardiac muscle degeneration and calcifica-
tion were observed in preweaning offspring. Supplementa-
tion of the dam's ration during gestation through weaning
with 0.1 ppm selenium as sodium selenite provided complete
protection against the disease while vitamin E did not.
Selenium and vitamin E therapy of lambs was effective in
preventing grossly observable postmortem lesions but
not those detectable microscopically. They suggested
that sulfur antagonism could influence the biological
availability of trace amounts of selenium which are

required. Oldfield et al. (1963) reported that white

16

muscle disease produced in lambs by feeding their dams
prenatally and through lactation on an alfalfa hay-oats
diet containing less than .02 ppm selenium was prevented

by raising the dietary selenium level to 0.06 ppm.

Selenium provided in a single parenteral dose from a

slow absorption carrier was effective in protecting against
deficiency throughout pregnancy, but was less effective
when a similar amount was given orally, as sodium selenite,
in aqueous solution. Whole blood levels of 0.11 ppm
selenium in the ewes and 0.12 ppm selenium in the lambs
were compatible with prevention of white muscle disease

in this study.

Buchanan-Smith et a1. (1969) prevented selenium
and/or vitamin E deficiency in sheep fed a purified diet
containing urea as the sole nitrogen source by giving
weekly injections of 5 mg selenium (sodium selenate)
and/or a-tocopheryl acetate (700 IU vitamin E). Selenium
delayed, but did not prevent, death. Growth was improved
by selenium, but satisfactory reproductive performance
was obtained only in ewes treated with a combination of

vitamin E and selenium.

Swing. Experimental diet-induced deficiencies of
selenium and/or vitamin E in swine have generally been
produced by feeding diets containing torula yeast as the
PTOtein source or diets containing large quantities of

unsaturated or rancid fat.

l7

Adamstone et a1. (1949) first reported a study based
on a wheat flour-casein diet with 10% added rancid lard
or 50 mg d,l-a-tocopherol/head/day. Gilts fed the basal
diet or the basal diet plus rancid lard had severely
affected reproductive performance, and pigs born to sows
on these diets exhibited wobbly gaits and incoordination
of the hind legs. The livers of pigs and their dams
contained large amounts of fat and exhibited fibrosis
and accumulation of blood in some of the lobules. Vitamin
E additions to the basal diet resulted in superior repro-
ductive performance, and no clinical signs or histological
lesions were observed in the pigs. Hove and Seibold
(1955) utilized a 6% crude protein sucrose-soybean meal
diet containing 6% lard and 2% cod liver oil or this
diet supplemented with 0.01% d,l-a-toc0phery1 acetate to
study vitamin E deficiency in pigs. They observed acute
hemorrhagic necrosis of the liver in three pigs that died
during the study on the vitamin E deficient diet. At
slaughter two pigs on this diet exhibited post-necrotic
cirrhosis. However, no muscle lesions were observed in
any of the pigs. Reid et al. (1968) studied a combined
protein-vitamin E deficiency syndrome, causing severe
liver damage in the pig, by manipulating dietary levels
of methionine, choline and selenium. Animals were fed
a basal diet containing 3% isolated soy protein and 25%

corn oil during the eight week trial. Liver damage was

18

completely preventable by supplementation with a-
tocopherol, or selenium, or both. Choline supplementa-
tion aggravated the liver damage while methionine gave
partial or complete protection against necrosis and
scarring but did not prevent the appearance of hyaline
bodies in the hepatocytes. The methionine effect was
not related to contamination of the supplemental methionine
with selenium.

Eggert et a1. (1957) utilized a torula yeast basal
diet for two to three week old pigs and reported that 67%
of the pigs on the basal diet died suddenly within 53
days, and typical lesions of liver necrosis and yellow
fat were observed. Supplementation of the basal diet
with 1.0 ppm selenium as sodium selenite or 40 ppm a-
tocopheryl acetate prevented sudden deaths and gross
lesions in animals when sacrificed. Wastell et al.
(1968) and Ewan et al. (1969) studied the effects of
selenium and/or vitamin E deficiency in young pigs fed
torula yeast or isolated soy protein diets containing 5%
fortified cod liver oil. Supplements of selenium or
tocopherol or both to the semi-purified diets had no
effect on growth rate but did reduce mortality from 54%
to 7% and prevented lesions of selenium and/or vitamin E
deficiency of pigs. Wastell et al. (1972) continued
these studies by feeding growing finishing pigs the diets

Previously described. Pigs fed a torula yeast diet

19

supplemented with both vitamin E and selenium gained sig-
nificantly faster than with either vitamin E or selenium
alone. Isolated soy protein diets supplemented with
vitamin E or selenium alone or in combination had no
effect on gain. Pigs fed the torula yeast diets sup-
plemented with selenium alone had edema, hyalinization
of skeletal muscles and a yellowish-brown discoloration
of body fat and tissues. Pigs fed the torula yeast diets
supplemented with both vitamin E and selenium showed
liver fibrosis, hyalinized skeletal muscle fibers and
degenerative heart myofibrils. No tissue changes were
observed in pigs fed a torula yeast diet supplemented
with vitamin E alone or in pigs fed the isolated soybean
protein diets supplemented with vitamin E and selenium
alone or in combination.

Michel et al. (1969) fed young pigs a 6% crude
protein torula yeast diet containing stripped lard.
Dietary hepatic necrosis occurred consistently on the
basal diet or this diet supplemented with methionine or
low levels of vitamin E. Selenium, high levels of vitamin
E or additional protein prevented dietary hepatic necrosis.
However, microscopic lesions of nutritional myopathy were
observed in some experimental pigs of all singly supple-
rnented groups except those fed vitamin E or ethoxyquin.
Selenium did not prevent the myopathy. Sharp et al.

(1970) reported that torula yeast and high moisture corn

20

increased the occurrence of muscular dystrophy in young
growing pigs. Vitamin E alone or in combination with
selenium prevented death losses on deficient diets.
Mahan et a1. (1974) fed a torula yeast semi-purified
diet devoid of supplemental vitamin E and selenium to
gilts. Sows fed the semi-purified diet exhibited very
poor reproduction and had small litters. All piglets
were fed a similar semi-purified diet from 21 to 56 days
of age. Piglets from selenium supplemented sows did not
exhibit clinical or histopathological deficiency signs
by 56 days of age. Their results suggested that selenium
fed to sows at the supplementary rate of 0.1 ppm effec-
tively delays the onset of vitamin E-selenium deficiency
lesions in their progeny.

Numerous studies have reported an induced selenium
and/or vitamin E deficiency in swine fed large quantities
of fish oils, fat or damaged grains. Obel (1953)
observed that up to 14.5% of all diagnoses in her labora-
tory from 1947 to 1951 were accounted for by a naturally
occurring condition she designated hepatosis diaetetica
in swine submitted for necropsy at the State Veterinary
Medical Institute of Sweden. The condition was produced
experimentally by feeding a starch-brewers' yeast diet
containing 6% cod liver oil. She stated that vitamin E
deficiency was the major problem even though Schwarz's

Factor 3 might also be involved. Lannek et al. (1961)

21

fed 20-25 kg pigs a sucrose-casein-yeast basal diet with
added fat. Nutritional muscular dystrophy in pigs was

not observed on the vitamin E~stripped lard diet but did
occur on a diet supplemented with cod liver oil which was
rich in a-tocopherol. Further supplementation with a-
tocopherol prevented nutritional muscular dystrophy and
sodium selenite injections lowered plasma transaminase
levels in deficient pigs. Orstadius et a2. (1963) indi-
cated that intramuscular injections of sodium selenite

or vitamin E reduced the occurrence of nutritional muscular
dystrophy on grain diets containing heated cottonseed oil.
They indicated that vitamin E and selenium were synergistic
in curing the disease. Grant and Thafvelin (1958) fed
weaned pigs 3 hepatonecrogenic soybean meal diet and
observed the effect of supplementing this diet with

sodium selenite. All pigs fed the basal diet died between
the twenty-second and forty-fifth days of the experiment.
The pigs had liver necrosis, massive transudations, degen-
eration of skeletal and heart muscle, and deposits of
ceroid pigment in the adipose tissue. Those supplemented
with sodium selenite survived and had normal livers, but
degeneration of skeletal muscle and deposits of ceroid in
the fat did occur. Thafvelin (1960) and Swahn and
Thafvelin (1962) stated that when hepatosis diaetetica

and muscular degeneration were seen in pigs fed grain,

the occurrence of the lesions was associated with the

properties of the cereal fat. Heating the grains

22

increased the peroxide value and reduced the iodine number
and vitamin E level in cereal fats. The heated grains or
heated maize oil produced nutritional muscular dystrophy
and hepatosis diaetetica with increased serum transaminase
levels. They theorized that heating disrupted the

natural antioxidative system of the cereal fat and per-
mitted oxidation of the fatty acids. These changes occur
gradually in ground grains stored under usual conditions.
Sodium selenite and vitamin E prevented transudation and
hepatosis diaetetica while selenite decreased muscular
degeneration but did not completely prevent it. Lannek

et al. (1960) studied the effect of moldy grain on
selenium-vitamin E deficiency in pigs. Supplementation

of the moldy grain diet with d,l-a-tocopheryl acetate

(50 ppm) or 0.2 ppm selenium as sodium selenite prevented
the occurrence of muscular dystrophy.

Occurrence of deficiencies under
practical conditions

 

 

Cattle and sheep. A naturally occurring deficiency

 

of selenium and/or vitamin E deficiency in young lambs
and calves designated as white muscle disease has been
recognized for many years. Muth (1955) described the
occurrence of white muscle disease in Oregon and the
etiological and pathological changes observed. Hartley
and Grant (1961) described a congenital and a delayed

white muscle disease in lambs and white muscle disease in

23

yearling sheep. Selenium treatments improved fertility
and cured ill thrift of sheep and cattle. White muscle
disease was also diagnosed in calves and foals in New
Zealand.

Blaxter (1963) reported that selenium deficiency was
less severe in Scotland soils than in New Zealand or
deficient areas of the United States. Selenium drenches
or injections resulted in small improvements in weight
gain in several Scottish flocks. Shirley et al. (1966)
demonstrated that intramuscular injections of sodium
selenite were ineffective in improving weaning weights
of calves or rate of gain of lambs in Florida. No
clinical signs of white muscle disease were observed in
any of the cattle or lambs even though the selenium

content of the pastures was low.

Swing, Obel (1953) first observed and reported
sudden death in pigs without evidence of illness. She
referred to this condition as hepatosis diaetetica and
a large proportion of the pig deaths in Sweden resulted
from this condition. The incidence of the condition
occurred most frequently in 6 week old pigs during the
fall of the year. Thomke et al. (1965) reported that
vitamin E and selenium supplementation of practical skim
milk and barley diets for pigs from 20 to 90 kg had no
effect on growth rate, feed conversion or carcass quality.

Vitamin E additions did improve the stability of

24

subcutaneous fat depots. Lannek et al. (1960) and
Lindberg and Siren (1965) diagnosed the nutritional
muscular dystrophy condition on several farms.

Hartley and Grant (1961) stated that hepatosis
diaetetica was responsible for pig deaths on at least
20 swine farms in New Zealand. Most outbreaks occurred
in areas where selenium-responsive diseases were diag-
nosed in sheep. Outbreaks usually occurred at weaning
time in pigs fed barley-skim milk diets with added cod
liver oil. Removal of the cod liver oil from the diet
and oral administration of inorganic selenium (5 mg)
prevented the occurrence of hepatosis diaetetica. Losses
have also been controlled by selenium administration when
no cod liver oil was being fed.

Michel et al. (1969) studied the nature and patho-
genesis of dietary hepatic necrosis in pigs from seven
Michigan swine herds. The diets on which deficiencies
occurred were typical corn-soybean meal diets without
added vitamin E. The a-tocOpherol and selenium levels of
feed samples from two of the farms were extremely low.
The analyzed values for a-tocopherol were 4.0 and 6.5 mg
per kg diet and selenium concentrations were .04 and
.057 ppm. Losses in all the herds ceased following sup-
plementation of the diets with 22 IU vitamin E per kg of
diet. Trapp et al. (1970) diagnosed a vitamin E-selenium

deficiency in 97 pigs from 37 Michigan swine herds. In

25

most cases, the diets were corn-soybean meal diets sup-
plemented with minerals and vitamins A and D and the
water-soluble vitamins. In addition, arsanilic acid was
used as a feed additive in most cases. The deficiency
was characterized by sudden deaths in feeder pigs and
lesions of hepatic necrosis, icterus, edema, hyaliniza-
tion of arteriole walls and skeletal and cardiac muscular
degeneration. Edema was present in most tissues examined,
especially in the mesentery of the spiral colon, lungs,
subcutaneous tissues and submucosa of the stomach. The
acute death losses due to hepatic necrosis and muscular
degeneration were prevented by supplementing diets with
vitamin E, or injections of selenium-vitamin E, or both,
in affected herds.

Klein et al. (1970) utilized normal and opaque-2
corn to study the effects of added vitamin E and selenium
upon weanling pigs. Supplementation of the diet with
selenium and vitamin E alone or in combination had no
effect on rate or efficiency of gain. Mahan et al.
(1971), Cline et a1. (1973) and Mahan ét al. (1973) con-
ducted two experiments to evaluate the efficacy of
injecting selenium and/or a‘tocopherol in preventing
vitamin E-selenium deficiency syndrome in young swine.
Pigs were obtained from sows kept under confinement con-
ditions and fed natural ingredients. Injections of

vitamin E reduced death losses in young pigs while

26

injections of selenium or selenium plus vitamin E com-
pletely prevented death losses. They suggested that
selenium played a major role in preventing the deficiency
syndrome in the progeny of sows fed natural ingredients.
Groce et a1. (1971) conducted two feeding trials to study
the effects of selenium supplementation of practical
diets on swine health and prevention of selenium and/or
vitamin E deficiency. Classical selenium and/or vitamin
E deficiency lesions were observed in two animals which
died when fed the unsupplemented corn-soybean meal basal
ration. Lesions attributable to selenium and/or vitamin
E deficiency were not observed in any of the groups
receiving supplemental selenium and/or vitamin E in the
diet. Groce et al. (1973b) conducted experiments to
define the minimum dietary selenium requirement of swine
fed corn-soybean meal diets which were low (0.05 ppm) in
natural selenium and contained 4.2 mg d-a-tocopherol per
kg. No difference in average daily gain or gain/feed
could be attributed to the additions of selenium and/or
vitamin E. No death losses recognized as being due to
selenium-vitamin E deficiency occurred, nor were there
gross or histological lesions attributable to dietary
treatments.

Ullrey (1973, 1974) reviewed the selenium deficiency
problem in swine production and animal agriculture. He

stated that the minimum practical level of selenium

27
supplementation from sodium selenite to prevent deficien-
cies in confined growing-finishing swine is 0.1 ppm,
resulting in a total selenium level in many Midwestern
swine diets of about 0.15 ppm. This is 1/50 of the
lowest continuously fed dietary selenium level shown to
produce toxicity in swine. This level of supplementation
appears to be a safe and scientifically sound nutritional
practice.

Mahan et a1. (1974) studied the efficacy of selenium
additions to sow diets over two reproductive cycles in
preventing selenium deficiency in their progeny. During
the second parity, sows receiving the basal corn-soybean
meal diet had significantly smaller litters than sows
receiving the basal diet supplemented with 0.1 ppm
selenium. The histopathologic evaluation of sow tissues
obtained after they had weaned their second litters
revealed no abnormalities on either the basal or selenium
supplemented diets.

Pathology of selenium and/or vitamin

e 1c1ency in swine

 

Gross and microscopic changes. There is considerable

 

variation in the location and appearance of gross and
microsc0pic lesions. The signs and lesions of vitamin E-
selenium deficiency in swine, whether experimentally pro-
duced or occurring naturally, have been reviewed by

Andrews et a2. (1968), Green and Bunyan (1969), Obel

28
(1953), Rosenfield and Beath (1964), Michel et al. (1969),
Trapp et a1. (1970) and Ullrey (1974). In swine, the
syndrome includes hepatic necrosis, nutritional muscular
dystrophy and mulberry heart disease. Pigs weighing 20
to 40 kg often die suddenly and exhibit a bilateral pale-
ness of skeletal muscles. The skeletal muscles most
affected are the gracilis, adductor, quadriceps femoris,
psoas and Zongissimus muscles. Histologically, loss of
striations, vacuolization, fragmentation and mineral depo-
sition in muscle fibers is observed. The liver is often
swollen and pale with focal lesions that give it a
roughened appearance. Histological examination reveals
lobules that have undergone marked degeneration and
necrosis, while adjacent lobules may appear normal.
Damaged lobules exhibit lysis of the hepatic cells and
dilatation of sinusoids with blood which appears as
extensive intralobular hemorrhage. Many of the pigs
exhibit icterus and occasional mottling and dystrophy
of the myocardium. Edema occurs in the mesentery of the
spiral colon, lungs, subcutaneous tissues and submucosa
of the stomach (Michel et al., 1969; Trapp et al., 1970)
and are believed to be useful in diagnosing selenium
and/or vitamin E deficiency in swine. The lesions of
the deficiency in young pigs have been described in many
of the trials discussed above when the deficiency occurred

under experimental or natural conditions.

29

Groce et a1. (1971) described classical selenium
and/or vitamin E deficiency lesions in two animals which
died when fed an unsupplemented corn-soybean meal basal
ration. Chronic fibrosis of the liver was observed in
another pig from the basal group at slaughter. Mahan
at al. (1973) reported that pigs from sows kept under
confinement conditions and fed natural ingredients dis-
played classical lesions of the vitamin E-selenium
deficiency syndrome. Necropsy observations revealed a
white pale striation in the skeletal muscle and in the
heart muscle, a yellow discoloration in the body fat,
degenerative and hobnail livers, enlarged hearts and a
generalized edema of internal tissues.

Mahan et al. (1974) described deficiency lesions in
sows and their progeny over two reproductive cycles.
Vitamin E-selenium deficiency lesions were observed in
the skeletal muscle and gastric area of the stomach in
sows fed semi-purified diets. Esophago-gastric ulcers
and histopathologic deficiency lesions occurred in
progeny of the sows by 56 days of age. The progeny of
sows that died during the experiment exhibited white
striations of the skeletal muscles, hepatosis diaetetica,
enlarged flaccid hearts and a generalized edema of the
internal tissues and organs. Histopathologic deficiency
lesions were observed in the myocardium in most but not

all animals.

30

Clinical pathology of selenium and/or vitamin E
deficiency of swine. Plasma, serum and cellular hemato-
logical measures have been studied in relation to selenium
and/or vitamin E deficiency in swine as possible diag-
nostic criteria. Rahman et al. (1960) observed that
serum albumin to globulin ratios were lower in chicks
with exudative diathesis. This depression was completely
prevented by adding 0.05 ppm selenium to the diet. The
severe edema and exudation observed in exudative diathesis
suggests that one might expect a lower albumin to globulin
ratio but it has not been consistently demonstrated. Klein
et a1. (1970) reported that erythrocyte hemolysis tests,
which have formed the basis for the biological assay of
vitamin E in rats, was not an effective diagnostic cri-
terion of selenium-vitamin E deficiency in swine. Michel
et a1. (1969) and Trapp et al. (1970) indicated that
standard hematological measures were of little use in
diagnosing selenium-vitamin E deficiency in swine.

Plasma or serum enzymes have commonly been used to
assess the development and diagnosis of selenium-vitamin
E deficiency in swine.

Enzyme assays have been used extensively by research
groups in Sweden as diagnostic criteria of selenium-
vitamin E deficiency. Wretlind et al. (1959) reported
that skeletal and heart muscle contained large amounts of
glutamic-oxaloacetic transaminase (GOT) and glutamic-

pyruvic transaminase (GPT) but were very low in ornithine

31
carbamyl transferase (OCT) while liver was very high in
OCT. They suggested that these enzyme parameters might
be useful in differentiating between muscle damage and
liver damage in living animals. Orstadius et al. (1959)
reported elevated plasma GOT, GPT and OCT values in
cases of "liver dystrophy" of pigs. The elevations
occurred early in the course of the disease and declined
gradually with time. In cases of muscular dystrophy
pigs exhibited elevated plasma GOT and GPT levels early
in development of the deficiency with a gradual decrease
with time as was observed in situations of "liver
dystrophy." Michel et al. (1969) reported an increase
in serum OCT levels in pigs with dietary hepatic necrosis.
Lannek et a2. (1960), Augustinssen et a1. (1960) and
Ewan and Wastell (1970) have reported similar changes of
plasma GOT, GPT, and OCT enzymes. They suggested that
plasma GOT and OCT were better indicators of selenium
and/or vitamin E deficiency in swine since plasma GPT
may be elevated in several other swine diseases. Lannek
et aZ. (1961), Orstadius et al. (1963), Ewan and Wastell
(1970), Groce et a1. (1971), Mahan et al. (1971), Wastell
et al. (1972), Grace at al. (1973b), Mahan et al. (1973)
and Mahan et al. (1974) have used serum or plasma GOT
activity to assess the stage of development of selenium-
vitamin E deficiency in swine. Ewan and Wastell (1970)

and Wastell at al. (1972) reported elevated serum lactic

32

dehydrogenase (LDH) activity in dietary induced selenium
and/or vitamin E deficiency in young pigs. They sug-
gested that serum LDH activity may be useful in diagnosing
nutritional muscular dystrophy in swine since Blincoe
and Marble (1960) and Paulson et a2. (1968) have reported
that elevated serum LDH activity is a sensitive indicator
of subclinical nutritional muscular dystrophy in young
lambs.

The discovery of a specific function of selenium as
a component of glutathione peroxidase in animals has been
described by Rotruck et al. (1973) and Hoekstra (1974).
Decreases in gluathione peroxidase appear to explain at
least some of the degenerative diseases induced by
selenium deficiency. The usefulness of this enzyme as
a convenient indicator of selenium adequacy has been
suggested by its relation to the prevention of exudative
diathesis in chicks (Noguchi et aZ., 1973b) and by its
relation to selenium status of rats (Chow and Tappel,
1974; Hafeman et aZ., 1974) and sheep (Oh et aZ., 1974).
However, experiments with swine to determine the rela-
tionship of tissue and blood glutathione peroxidase levels
to selenium adequacy or as a diagnostic aid in selenium-
vitamin E deficiency have not been reported. This enzyme
may become a useful diagnostic criterion of selenium

adequacy of swine in the future.

33

Selenium levels in swine blood and tissues

In establishing the essentiality of selenium, it
has been important not only to understand the metabolic
role of selenium but also to assess the levels of selenium
that occur in animal tissues. Lindberg and Siren (1963,
1965) reported kidney selenium concentrations for normal
pigs (10.98 ppm), pigs with nutritional muscular dystrophy
(3.40 ppm) and pigs with dietary hepatic necrosis (3.33
ppm) on a dry basis. Liver selenium concentrations of
normal pigs ranged from 0.52 to 2.12 ppm while pigs with
muscular dystrophy ranged from 0.13 to 0.28 ppm and pigs
with liver dystrophy ranged from 0.13 to 0.24 ppm expressed
on a dry basis. Lindberg (1968) observed similar tissue
concentrations in later experiments. Lindberg and Lannek
(1965) supplemented a commercial basal diet containing
0.126 ppm selenium with 1.2 ppm selenium as sodium
selenite. Selenium supplementation for 78 days did not
increase kidney selenium concentrations but did increase
liver and muscle selenium concentrations over levels
observed for pigs on the basal diet. Withdrawal of
selenium for 14 days decreased muscle selenium levels
back to control levels and liver selenium levels returned
nearly back to control levels. Andrews et al. (1968)
obtained liver and kidney selenium concentrations of
0.047 and 0.53 ppm, respectively, on a wet basis in

necropsied pigs which had died of hepatosis diaetetica.

34

Sharp et a2. (1970) observed that tissue selenium
levels in growing-finishing pigs were comparable to
tissue levels in deficient and selenium supplemented pigs
previously discussed. They reported that added vitamin
E increased kidney selenium levels while decreasing
liver, muscle and heart selenium levels. This tendency
was observed on both unsupplemented and selenium sulfide
supplemented diets. A liver selenium level of 0.2 ppm
selenium on a dry basis was the minimum level compatible
for protection against selenium responsive diseases and
liver selenium levels were a more sensitive indicator
of selenium status than kidney selenium levels were.

Ewan (1971) studied the effects of vitamin E and
selenium supplementation on tissue composition of young
pigs fed torula yeast diets. Muscle and liver selenium
concentrations of pigs receiving the deficient diet
decreased significantly during the eight week feeding
period. Muscle selenium concentrations were higher in
pigs fed selenium supplemented diets than pigs on unSup—
plemented diets but all diets resulted in lower concen-
trations than the initial muscle selenium concentrations.

Liver selenium levels followed a similar pattern.
Kidney selenium concentrations were highest (7.50 ppm)
in selenium fed animals. Vitamin E had no significant
effect on the level of selenium in the tissues, but
deficiency symptoms were completely prevented by supple-

mentation with vitamin E.

35

Groce et a1. (1971) reported that selenium supple-
mentation (1.0 ppm) of a basal corn-soybean meal diet
significantly increased muscle selenium concentrations
even though the levels on selenium supplemented diets
were lower than levels reported for porcine muscle in
selenium adequate areas. Muscle selenium levels did not
decrease significantly in 30 days but did increase sig-
nificantly by 65 days after the termination of selenium
suppplementation.

Ku at al. (1972) reported a significant linear cor-
relation of 0.95 between dietary selenium level and the
selenium concentration of Zongissimus muscle of pigs
from each of 13 locations in the United States. The
selenium values of the diets ranged from 0.027 ppm to
0.493 ppm on an air dry basis while selenium concentra-
tions of the Zongissimus muscle ranged from 0.046 to
0.521 ppm on a fresh basis. No relationship was apparent
between a-tocopherol concentration and tissue selenium
concentration. Ku et al. (1973) studied the effects of
supplementation of naturally high selenium swine diets
with 0.1 ppm of selenium from sodium selenite. Sodium
selenite additions to the naturally high selenium diets
did not significantly increase Zongissimus muscle or
kidney selenium concentrations and increased liver
selenium concentrations only slightly. Tissue selenium

levels resulting from adding 0.40 ppm of selenium from

36

sodium selenite to a naturally low selenium diet (0.04
ppm) were significantly lower than when a naturally high
selenium diet (0.44 ppm) was fed. They concluded that
the dietary level of naturally occurring organic selenium
compounds is much more significant in influencing the
tissue selenium concentration of the pig than is supple-
mental selenium from sodium selenite. Groce et al.
(1973a) observed that serum and whole blood selenium
levels on seleniferous corn diets were significantly
lower than on selenite selenium diets. Supplemental
vitamin E had no significant effect on serum erythrocyte
or whole blood selenium concentration.

Groce et al. (1973b) conducted an experiment to
define the minimum dietary selenium requirement of swine
fed corn-soybean meal diets which were low (0.05 ppm)
in natural selenium and contained 4.2 mg d-a-toc0pherol
per kilogram. Selenium concentrations increased signifi-
cantly in Zongissimus muscle and liver with increasing
dietary selenium to the supplemental level of 0.1 ppm.
Myocardium selenium was highest with 0.2 ppm supplemental
selenium while kidney selenium did not increase above
that resulting from 0.05 ppm supplemental selenium.
Selenium in whole blood, erythrocytes and serum was sig-
nificantly increased by selenium supplementation, but
there were no differences between supplemental levels.

Selenium withdrawal reduced selenium levels in all

37

tissues as compared to those from pigs fed selenium to
slaughter. Vitamin E supplementation decreased erythro-
cyte selenium concentration and increased kidney selenium

concentrations.

Selenium Balance in Swine

 

There are many factors that affect the absorption
and excretion of selenium. Lindberg and Lannek (1965)
stated 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 overwhelmed, in which case
significant quantities of selenium will be retained and
selenium intoxication may result. Groce (1972) has
reviewed the literature on selenium balance in animals.
Much of the work reported on absorption and tissue dis-
tribution, excretion and secretion of selenium was cited
in this review. The amount of research conducted on
selenium balance in swine has been extremely limited.

Grant et a1. (1961) reported that orally ingested
inorganic selenium followed generally the same pattern
of tissue distribution and excretion as parenterally-
administered selenium materials. Wright and Bell (1966),
using radioactive sodium selenite, found that selenium

was resecreted into the first section of the small

38

intestine and then reabsorbed from succeeding sections
of the small intestine to result in a net absorption of
selenite selenium of 86%.

Buescher et al. (1961) reported that excessive
calcium levels in normal selenium diets did not alter

75Se in

the excretion or tissue distribution of oral
swine. The urinary route of excretion appears to be more
important with inorganic forms than with naturally occur-
ring selenium in food.

Groce et a2. (1971) studied the influence of added
dietary selenium upon selenium excretion and retention.
Their results indicated that young pigs retained a
decreasing percentage of dietary selenium as levels of
supplemental selenium increased but the absolute daily
retention of selenium was similar for both 0.1 and 0.5
ppm of added selenium. Urinary excretion of selenium
increased markedly at the higher level of selenium sup-
plementation. Groce et al. (1973a) conducted balance
trials with young pigs to compare supplements (0.2 ppm
Se) of natural selenium (from seleniferous corn) or
supplements of selenite selenium and a supplement of 22
IU of vitamin E per kg of diet versus no E supplementation.
A higher proportion of selenium from seleniferous corn
was excreted in the urine as compared to selenium from
sodium selenite. Vitamin E supplementation significantly

reduced fecal selenium excretion of pigs fed seleniferous

39

corn. Groce et al. (1973b) studied selenium absorption
and retention on diets containing several levels (0,
0.05, 0.1 and 0.2 ppm) of added selenium from sodium
selenite. The effect of a supplement of 22 IU of vitamin
E per kg of diet was also studied. Selenium retention
was maximized at 0.1 ppm of supplemental selenium.
Vitamin E supplementation appeared to increase urinary
selenium excretion at the supplemental selenium level of
0.2 ppm. Therefore, based on tissue selenium levels and
selenium retention and excretion patterns, 0.15 ppm
selenium was required in a corn-soybean meal diet unsup-
plemented with vitamin E. Serum selenium levels plateaued
at the 0.1 ppm supplemental selenium level, indicating
the possible existence of tissue and serum thresholds
for selenium from sodium selenite in the pig.
Interrelationships of Selenium with Sulfur,
Vitamin E and Other Dietary Factors

It is apparent from the literature already cited that
the metabolic role of selenium in animals is linked with
that of vitamin E, sulfur amino acids and other dietary

factors.

Vitamin E

 

The literature discussed in previous sections indi-
cates that some diseases associated with low concentra-
tions of selenium in the diet (less than 0.1 ppm) are

analogous to those of vitamin E deficiency and are

40
usually observed under conditions of low intakes of
vitamin E. In some cases these syndromes respond fully
to administration of vitamin E while in others selenium
was substantially more effective or induced a further
response. Many of the studies indicate that, although
selenium cannot replace vitamin E in nutrition, it reduces
the amount of tocopherol required and delays the onset

of symptoms of deficiency.

Sulfur amino acids

 

Sulfur amino acids may also influence the need for
vitamin E and selenium. Obel (1953) reported that sulfur-
containing amino acids prevented liver dystrophy in pigs.
However, many of Obel's studies that reported a protective
effect of sulfur amino acids were questioned because the
commercial L-cystine she used was usually contaminated
with selenium. Schwarz (1965) used uncontaminated sulfur
amino acids to show that sulfur amino acids delayed the
onset of dietary necrotic liver degeneration in rats.

He concluded that this effect was mediated by a sparing
action of sulfur amino acids in the vitamin E requirement.
Reid et al. (1968) reported that methionine partially
prevented liver damage in pigs and that selenium contami-
nation was not a significant factor in the protective
action of methionine. On the other hand, sulfur amino
acids do not appear to prevent the development of muscular

dystrophy in lambs (Erwin et aZ., 1961). In general,

41

sulfur amino acids moderate the effects of feeding diets
deficient in these nutrients but cannot substitute for
them. The efficacy of these amino acids may be explained
by their contribution to the antioxidant activity of

tissues.

Polyunsaturated fat

 

The muscular dystrophy producing property of poly—
unsaturated fatty acids has been experimentally demon-
strated by several workers (Obel, 1953; Lannek et aZ.,
1961; Lindberg and Orstadius, 1961; Orstadius et aZ.,
1963). Thafvelin (1960) and Lannek et al. (1960) pro-
duced nutritional muscular dystrophy experimentally in
pigs fed grain obtained from areas where natural out-
breaks of the disease had occurred. Thafvelin (1960)
suggested that the fats of the grains were the cause of
the deficiencies and that in grain causing nutritional
muscular dystrophy, the vitamin E content was reduced.
Swahn and Thafvelin (1962) demonstrated that in grain
which had induced nutritional muscular dystrophy, the
fat was unstable under oxidizing conditions. Oksanen
(1967) indicated that the changes in fatty acid composi-
tion, fat quality and vitamin E content of grain influence
the occurrence of nutritional muscular dystrophy in pigs
and that a low selenium concentration is a prerequisite
for a spontaneous occurrence of nutritional muscular

dystrophy.

42

Synthetic antioxidants

 

Mertz and Schwarz (1958) reported that the synthetic
antioxidant N,N'-diphenyl-p-phenylenediamine was highly
active in preventing "respiratory decline" (indicative of
liver necrosis) when administered intraportally to rats
deficient in selenium and vitamin E. Machlin et a2.
(1959) demonstrated that certain synthetic antioxidants
are also capable of preventing exudates in chicks, even
though they were significantly lower in efficacy than
vitamin E. Hill (1963) reported that ethoxyquin, a
synthetic antioxidant, protected the tissues of the pig
from increased thiobarbituric acid test values and from
increased hemolysis which are usually associated with
low vitamin E status. Tollerz and Lannek (1964) reported
that synthetic antioxidants restored resistance to iron

toxicity under vitamin E deficiency situations.

Iron. Lannek et al. (1962) produced iron hyper-

 

sensitivity experimentally in piglets by feeding the sows
a vitamin E deficient diet during pregnancy and lactation.
The LDSO of iron preparations was reported to be greatly
lowered in vitamin E deficient baby pigs. The resistance
to iron toxicity may be restored by the administration of
a-tocopherol (Lannek et aZ., 1962) and selenium (Arpi and
Tollerz, 1965). Miller et al. (1973) reported that the
administration of a second intramuscular injection of

100 mg of iron from iron dextran gave no evidence of iron

43

toxicity or anaphylaxis. Feeding of oral iron at levels
up to 600 ppm in the diet showed no evidence of iron
toxicity. An intramuscular injection of 1000 mg iron
from iron dextran gave no evidence of iron toxicity. In
these experiments, iron toxicity was not demonstrated in
pigs having low vitamin E-selenium reserves, raised on

diets low in vitamin E and selenium.

Arsenic. Underwood (1971) stated that the beneficial
effects of various organic arsenicals on the growth,
health and feed efficiency of poultry and swine have been
thoroughly established. These effects have been reviewed
by Frost and his associates (Frost, 1953, 1967; Frost et
aZ., 1955). Arsanilic acid, 4-nitrophenylarsonic acid,

3 nitro-4-hydroxyphenylarsonic acid and arsenobenzene are
the four organic arsenic compounds which have been used
and are of value in animal production. No clearly evident
relationship exists between structure and the ability to
promote growth. The arsonic acids are recognized as
growth stimulants for pigs and poultry, but the phenyl-
arsenoxides are more potent than the arsonic acids as
coccidiostats. The action of arsonic acids closely
resembles that of antibiotics and is partially comple-
mentary to it, but the exact mechanism is not known.

Frost et al. (1955) stated that the arsonic acids
differ markedly in tolerance, in coccidiostatic power and

in power to promote growth. In general, toxicity appears

44
to be related to the amount of arsenic that was deposited
in the tissues.

Hanson et al. (1955) studied the value of arsanilic
acid as a growth stimulant for pigs and measured the
arsenic retention in the tissues. Small amounts of arsenic
were retained in the tissues and the amount retained was
related to the level fed. Arsenic retention in the liver
was 1.5 to 2.0 times as great as the storage in the
kidneys. The amount retained in the muscle, fat or skin
was low at all levels of arsanilic acid fed. Removal of
arsanilic acid from the diet resulted in rapid excretion
of arsenic from the liver and kidneys. The rate of excre-
tion from muscle was less rapid.

Mogareidge (1963) studied the differences in availa-
bility to the growing rat of protein bound arsenic and
the inorganic trivalent form. At 16 ppm of dietary
arsenic, both sources gave rise to significant tissue
storage although that from arsenic trioxide was somewhat
higher. The protein bound source resulted in equal
urinary and fecal excretions but with the inorganic
source urinary was greater than 2 times the fecal
excretion.

Overby and Frost (1960) studied the rate of arsenic
excretion of swine receiving arsanilic acid in the diet.
Much more arsenic was excreted in the feces than in the

urine. After the arsanilic acid was withdrawn from the

45
ration, the characteristic excretion level continued for
2 days and then decreased rapidly. This was in agreement
with knowledge of the rate of disappearance of arsenic
from tissues of animals fed arsanilic acid. Unchanged
arsanilic acid was not detected in the urine, but was
present in the feces in an amount representing about 5%
of the arsanilic acid consumed.

Thus it appears that toxic arsenic compounds are
retained in the tissues in greater amounts and are
excreted more slowly than the less toxic forms. The
arsenic of organic compounds such as arsanilic acid is
well absorbed and deposited in tissues of pigs and chicks
in amounts proportional to the level fed. However, it
rapidly disappears from the tissues upon withdrawal and
most is excreted in the feces.

Arsenic has been successfully used to alleviate
selenium poisoning in cattle, dogs, chicks and swine for
many years. A

Moxon (1938) first demonstrated that 5 ppm As in
the drinking water would completely prevent all signs of
selenosis in rats. Moxon (1944) reported that 25 ppm as
sodium arsenate when added to salt partially protected
cattle on seleniferous ranges.

Levander and Baumann (1966a,b) observed that the
excretion of selenium into the gastrointestinal tract via

the bile fluid was markedly increased and retention in

46

the liver, blood and carcass was greatly decreased, when
subacute injections of arsenic were given with an injec-
tion of selenium. I

Ganther and Baumann (1962) reported that arsenic
administration increased the excretion of an injected dose
of selenium into the gastrointestinal tract within an
hour. However, administration of the arsenic and selenium,
more than one hour apart, would not show this effect
(Palmer and Bonhorst, 1957).

Levander and Argrett (1969) compared the effects of
arsenic, mercury, thallium and lead on the metabolism of
selenium. Arsenic inhibited the pulmonary excretion of
volatile selenium compounds by rats injected with subacute
doses of sodium selenate. The biliary excretion of
selenium was stimulated sevenfold by the administration
of arsenic. Arsenic improved the growth of rats chemically
poisoned by selenium, largely prevented the liver damage
caused by selenium and decreased the amount of selenium
retained in the tissues. Urinary selenium excretion was
not affected by As injection in these studies.

With high levels of selenium, arsenic reduces the
loss of selenium in expired air (Olson et aZ., 1963).

Sodium arsenite and arsenate have been found to be
equal in effectiveness of prevention of selenosis. The
arsenic sulfides are not effective and various organic

arsenic compounds have been found to provide only a

47
partial protection against selenosis (Hendricks et aZ.,
1953; Kuttler and Marble, 1961; Wahlstrom et aZ., 1955;
Wright, 1940).

The beneficial effects of dietary arsenic additions
in selenium toxicities produced by selenium salts or
seleniferous grains have been demonstrated in swine by
Wahlstrom et al. (1955, 1956), and Wahlstrom and
Olson (l959a,b) and in poultry (Carlson et aZ., 1954;
Carlson et aZ., 1962; Thapar et aZ., 1969).

The mechanism by which arsenic provides protection
against selenium toxicity is not yet well established.

Muth et al. (1971) suggested that a selenium-arsenic
complementary effect exists in selenium deficiency situa-
tions. The feeding of 1.00 ppm arsenic in the form of
sodium arsenate, when added to a low selenium ration for
pregnant ewes, gave a significant marked protection against
the myopathy commonly associated with selenium deficiency
in lambs.

Although arsenic may be beneficial at low levels,
arsenic can be very toxic and requires proper handling
and use at all times. Frost (1967) and Schroder and
Balassa (1966) have reviewed the literature on arsenic
toxicity. These reviews emphasize that arsenicals are
non-carcinogenic and that wide differences in the toxicity
of different chemical forms of arsenic exist. In general,
inorganic arsenicals are more toxic than the organic

forms. It seems that enzymes are far more susceptible to

48
trivalent than to pentavalent arsenicals and bind similar
levels of As at the point of death. Arsenicals are not
cumulative in most animal tissues. The differences in

toxicity are related to their rate of excretion.

Vitamin E Studies in Swine

 

Although vitamin E is stored in the liver and other
tissues, it is sometimes possible but often difficult to
produce unmistakable deficiency signs by long periods of
dietary vitamin E depletion. For many years it was con-
sidered that swine fed diets composed mainly of corn or
cereal grains had no danger of developing vitamin E
deficiency, since high-quality green feeds, whole cereal
grainsznuithe germ of cereal grains are good sources of
vitamin E (tocopherols). The various tocopherols differ
in their biological activity, with d-a-tocopherol being
the most active. Cereal grains contain about equal
amounts of a-tocopherol and other tocopherols and since
vitamin E is easily oxidized, its concentration deteriorates
in ground feeds.

In recent years, many cases of vitamin E deficiency
have been reported in swine fed diets composed of feed-
stuffs grown over a wide geographic area. Specific infor—
mation regarding the significance of vitamin E in farm-
animal nutrition is especially limited and the dietary

needs for various species are not well defined.

49

Bratzler et al. (1950) mantained 5 barrows on a
vitamin E-low, purified ration for 75 days. Three of the
animals received a supplement of a concentrate of mixed
tocopherols at levels of 2.87, 55.12 and 110.2 mg daily
per kg liveweight. Tocopherol supplementation increased
significantly the total tocopherol content in all organs
and tissues studied. The increases were most marked in
the case of liver and body fat. The gamma-plus delta-
tocopherol content increased significantly only in the
case of whole blood. Tocopherol supplementation
markedly affected the fatty acid composition of the body
fats by increasing the percentage of oleic acid at the
expense of the saturated fatty acids. Hove and Seibold
(1955) reported that 0.01% dl-a-tocopheryl acetate (150
mg of tocopherol per day) added to a synthetic semi-
purified diet had no effect on the growth rate of pigs.
Fatal liver necrosis was observed in growing pigs fed the
basal diet but pigs receiving diets supplemented with
a-tocopheryl acetate failed to show appreciable liver
damage at slaughter.

Rousseau et al. (1957) fed graded levels of tocopherol
0, 1.0, 3.0 and 9.0 mg per lb of liveweight daily added
to basal rations of calves, lambs and pigs for 12 weeks.
Plasma tocopherol was found to increase at diminishing
rates with tocopherol intake whereas liver tocopherol

increased at constant rates with tocopherol intake.

50

Tocopherol intake was found to have no measurable effect
on level of daily gain. Eggert et a1. (1957) fed a puri-
»fied-type, torula yeast diet or the basal diet supplemented
with 40 ppm tocopheryl acetate. Four pigs receiving the
basal diet died within 53 days and showed a marked necrosis
of the liver. No deaths occurred among the pigs receiving
vitamin E and no marked difference in growth rate was
noted in favor of vitamin E supplementation. Forbes and
Draper (1958) utilized 37 baby pigs to produce and study
a deficiency of vitamin E. Only under the conditions of
a stress provided by at least 5% cod liver oil in the diet
was an unmistakable deficiency produced. The deficiency
was not accompanied by changes in EKG or by ability of
dialuric acid to hemolyze red blood cells. Supplementa-
tion of vitamin E (10 mg of dl-a-tocopherol acetate per
100 grams dry matter) prevented the appearance of vitamin
E deficiency symptoms. Garton et al. (1958) reared wean-
ling pigs to about 130 pound liveweight on a basal diet
supplemented with 5 or 10 mg tocopheryl acetate/kg body
weight/day. The animals given tocopherol did not exhibit
changes in serum levels of the vitamin and no liver lesions
were observed in any of the pigs. The serum tocopherol
levels were lower in pigs slaughtered at 200 pounds live-
weight.

Johnson and Alaupovic (1960) administered alpha

tocopherol intraperitoneally to 175 pound pigs. They

51
isolated two metabolites of tocopherol from liver. Neither
metabolite was identical with alpha tocopheryl metabolites
commonly found in urine. Duncan et al. (1960) fed wean-
ling pigs for 8-10 weeks on a basal low-fat, low-tocopherol
diet or a similar diet containing lard (5%) and each diet
was given with or without supplementary dl-a-tocopheryl
acetate. The supplemented pigs received 20 mg tocopheryl
acetate per kg bodyweight per day. At slaughter, the
plasma tocopherol values of the pigs given supplementary
tocopherol were significantly higher than those of unsup-
plemented animals. The effect of tocopherol supplementa-
tion did not depend on whether or not lard was included
in the diet. Thus the absorption of the dietary tocopherol
was not dependent on the simultaneous digestion and
absorption of fat. Histological examination of the livers
of animals, not given supplementary tocopherol, showed no
significant signs of deficiency.

Leat (1961a) investigated the role of tocopherol in
the nutrition of the pig reared from weaning to slaughter
on a low-fat, low-tocopherol diet or this diet supplemented
with 5 or 10 mg per 100 g diet of a-tocopheryl succinate.
There was no difference in growth rate between the groups
and no abnormality in any organs or carcasses at slaughter.
Plasma-tocopherol levels were two to thirteen times higher
in the animals given tocopherol than in the controls.

’There was no difference between groups in the content of

52
unsaturated fatty acids of back fat, plasma, liver or
heart. The peroxide levels in the back fat and liver
lipids of the control animals were four to fifty times
greater than that found in the animals given tocopherol.
He concluded that the major function of tocopherol in
the pig is to protect body lipids from oxidation. Leat
(1961b) reported that supplementation of a basal diet
with 10 mg a-tocopheryl succinate per 100 grams diet
significantly decreased the peroxide content of the body
lipids. He suggested that although the pig does not
require dietary tocopherol for optimum growth up to 200
pounds, its presence will lessen the susceptibility of

body fats to oxidative rancidity.

EFFECT OF ARSANILIC ACID, SELENIUM AND VITAMIN E
ON PRODUCTIVE PERFORMANCE, TISSUE SELENIUM
CONCENTRATIONS AND SELENIUM BALANCE
IN GROWING-FINISHING SWINE

Introduction

 

Selenium-vitamin E deficiency in swine has been
recognized as a practical field problem in confinement-
reared swine in Michigan and other Midwestern states.

The problem has been described by Michel et al. (1969),
Trapp et a2. (1970), Groce et al. (1971, 1973b), and
Mahan at al. (1971, 1973, 1974). Many pigs are being
raised in complete confinement without access to pasture.
Corn and soybean meal are the primary ingredients used

in formulating swine feeds in the midwestern United
States. Swine diets made of corn and soybean meal have
resulted in low intakes of a-tocopherol. The selenium
content of feedstuffs varies widely in different geo-
graphical areas of the United States (Patrias and Olson,
1969; Ku et aZ., 1972). Groce et al. (1971) demonstrated
the efficacy of low levels of supplementary selenium from
sodium selenite in preventing death losses and clinical
Signs or lesions of selenium-vitamin E deficiency in the
growing pig. Groce et al. (1973b) attempted to define

the minimum dietary selenium requirements of

53

54
growing-finishing swine fed corn-soybean meal diets. They
concluded that the minimum practical level of selenium
supplementation from sodium selenite to prevent deficien-
cies in confined growing-finishing swine is 0.1 ppm,
resulting in a total selenium level in many midwestern
swine diets of about 0.15 ppm.

Trapp et al. (1970) observed natural selenium-
vitamin E deficiency cases in swine and reported that
most herds involved were feeding corn-soybean meal diets
supplemented with arsanilic acid as a feed additive.
Arsanilic acid has been demonstrated to counteract
selenium toxicity in swine (Wahlstrom et aZ., 1955).
Trapp et al. (1970) suggested that arsanilic acid may
have been binding selenium and thus enhanced the occur-
rence of a natural selenium-vitamin E deficiency.

The purpose of this investigation was to study the
effects of supplemental arsanilic acid, selenium and
vitamin E on productive performance, deficiency symptoms,
hematology, tissue selenium and selenium balance in
growing-finishing pigs fed diets low in vitamin E and

selenium.

Experimental Procedure

 

Three experiments were conducted in this investiga-
tion. Experiment 1 was designed to study the response of
growing pigs to added dietary selenium (Se), vitamin E

and arsanilic acid. Experiments 2 and 3 were selenium

55
balance studies conducted with young pigs (5 weeks of age)
to study the effects of added vitamin E and arsanilic

acid on the retention and excretion of selenium.

Experiment 1

 

Eighty Yorkshire, Hampshire and Yorkshire x Hampshire
barrows and gilts weighing an average of 8.4 kg were
randomly allotted to eight dietary treatments and housed
10 per pen in a completely enclosed, slotted floor,
environmentally controlled building. The pigs utilized
in this study were offspring of sows which had received
no supplemental vitamin E or selenium during gestation or
lactation. The basal diet (Table l) was a 16% crude pro—
tein fortified corn-soybean meal diet which analyzed
0.036 ppm Se and contained no supplemental vitamin E,
selenium or antibiotics. The following diets were fed
ad Zibitum: (l) basal (Table 1) (0.036 ppm Se); (2)
basal + 0.1 ppm Se as sodium selenite;1 (3) basal + 22
IU E2 per kilogram of diet; (4) basal + 99 ppm arsanilic
acid; (5) basal + 0.1 ppm Se + 22 IU E per kilogram;

(6) basal + 0.1 ppm Se + 99 ppm arsanilic acid; (7) basal
+ 22 IU E per kilogram + 99 ppm arsanilic acid; and (8)
basal + 0.1 ppm Se + 22 IU E per kilogram + 99 ppm

 

1Alfa Inorganics, Ventron Corporation, Beverly,
Massachusetts.

2Myvamix-Type 125. Vitamin E as d-a-tocopheryl
acetate with dextrin. Distillation Products Industries,
Rochester, New York.

56

TABLE 1. COMPOSITION OF BASAL DIETS (EXPERIMENT 1)

 

 

International d
Ingredient ref. no. Grower Finisher
Corn, dent yellow, 4-02-931 79.2 86.7
grain, gr 2 US mn
54 wt (4)
Soybean, seed wo 5-04-612 17.9 10.4
hulls, solv-extd
grnd, mx 3 fbr (5)
(soybean meal)
Calcium phosphate, 6-01-080 1.0 1.1
dibasic, comm(6)
Limestone, grnd, mn 6-02-632 0.9 0.8
33 Ca (6)
Salt, plain white 0.5 0.5
Vitamin-trace mineral 0.5 0.5
premixa 100.0 100.0
Crude protein, %b 16.0 13.0
Calcium, %b 0.64 0.60
Phosphorus, %b 0.50 0.50
Se, ppm as fedC 0.036 0.040

 

3Provided the following per kilogram of diet:
vitamin A, 3,300 IU; vitamin D, 660 IU; riboflavin, 3.3
mg; nicotinic acid, 17.6 mg; d-pantothenic acid, 13.2 mg;
choline chloride, 110 mg; vitamin 812, 19.8 pg; zinc, 74.8
mg; manganese, 37.4 mg; iodine, 2.7 mg; copper, 9.9 mg;
iron, 59.4 milligrams.

bCalculated

CBy analysis

dBasal diet for experiments 2 and 3 also

S7

arsanilic acid. After 12 weeks, the pigs weighed an
average of 52 kg and the basal diet was changed to the
13% crude protein corn-soybean meal diet shown in Table
l. Selenium was withdrawn from diets being supplemented
at the end of 12 weeks of the trial since the Food and
Drug Administration of the U.S. Department of Health,
Education and Welfare had specified a 60-day minimum
withdrawal period before carcasses of those pigs fed
supplemental Se could be utilized for human consumption.
Arsanilic acid was withdrawn from diets being supplemented
8 days prior to slaughter. The pigs were weighed and
feed consumption determined at biweekly intervals. Tap
water (1 part per billion [ppb] Se) was offered ad
Zibitum.

All animals were observed closely for clinical signs
of selenium-vitamin E deficiency throughout the course of
the experiment. Blood samples were collected from the
anterior vena cava from two pigs from each of the 8
treatment groups initially, at two weeks and 8 weeks and
from all pigs two days prior to slaughter. Hemoglobin
(Crosby et aZ., 1954) and microhematocrit (McGovern et aZ.,
1955) determinations were made on heparinized whole blood.
Serum glutamic-oxaloacetic transaminase (SGOT) (Sigma
Technical Bulletin, 1964) was determined on all serum
samples collected. Whole blood and serum samples were

stored at -20 C until Se analyses could be performed.

S8

Liver, kidney and diaphragm muscle samples were obtained
from all pigs at slaughter, placed in plastic bags and
frozen at -20 C until analyzed for Se. Diaphragm muscle
was homogenized with twice its weight of deionized,
distilled water to facilitate uniform sampling and
pipetting. Liver and kidney were homogenized with three
and five times their weight of water, respectively. Homo-
genates were stored at -20 C until analyzed for Se. The
diets were ground twice through a stainless steel screen
with 2 mm diameter openings in a Wiley mill3 and stored
at -20 C until analyzed for Se. Selenium analyses of
blood, serum, tissues and diets were conducted fluoro-
metrically according to the method of Hoffman et al.
(1968) with the following modifications described by
Groce et al. (1971). The glassware used for Se analyses
was segregated and used only for this procedure. Upon
completing a 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 was carried out in 100 ml semi-

micro Kjeldahl flasks4 as described by Hoffman et al.

 

3Thomas-Wiley Mill, Model 50-5. Arthur H. Thomas
Company, Philadelphia, Pennsylvania.

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

59

(1968) after a 12 to 14 hour predigestion in the complete
digestion acid mixture as described by Olson (1969). This
predigestion period reduces charring problems during sub-
sequent digestion. The Z,3-diamino-naphtha1ene solution
and all succeeding preparations containing it were
handled in dim or yellow light and produced a more stable
preparation. In the complexing step the various reagents
were combined in a 250 ml Phillips beaker and swirled by
hand. After the one hour incubation period, the reaction
mixture was extracted with 6 ml of cyclohexane in a 125
ml separatory funnel for 15 minutes. The fluorescence
was read using an Aminco-Bowman SpectrophotofluorometerS
with an excitation monochromator setting of 372 mu and
an emission monochromator setting of 516 millimicrons.
Standards were prepared from Se metal6 dissolved in a
minimal quantity of redistilled concentrated nitric acid
and made up to volume with 0.1 N sulfuric acid. The
fluorometer was zeroed against a reagent blank carried
through the entire procedure.

This method resulted in a linear standard curve up
to 0.5 ug Se which consistently passed through the origin.
A more thorough and complete description of the selenium

analytical procedure is reproduced in Appendix A. The

 

SAmerican Instrument Co., Inc., Silver Spring,
Maryland.

6Alfa Inorganics, Inc., Beverly, Massachusetts.

60

percent of dry matter was determined on diets and tissue
homogenates by drying for 15 hr at 60 C with a vacuum of

740 millimeters.

Experiment 2

 

Six male and six female crossbred and Yorkshire pigs
whose dams had received no supplemental Se or vitamin E
during gestation and lactation were used to study the
effect of added dietary selenium as sodium selenite,
vitamin E as d-a-tocopheryl acetate and arsanilic acid
upon Se excretion and retention. The basal diet was the
16% crude protein corn-soybean meal diet (Table 1) which
analyzed 0.036 and 0.040 ppm Se for phase 1 and 2,
reSpectively, of this balance study. The pigs were
randomly allotted to the following treatments: (1)
basal (Table l); (2) basal + 0.1 ppm Se; (3) basal + 99
ppm arsanilic acid; (4) basal + 0.1 ppm Se + 99 ppm
arsanilic acid.” The pigs weighed an average of 8.3 kg
initially and 8.7 kg at the beginning of phase 2. The
pigs were housed in stainless steel metabolism cages for
an 11 day adjustment period and a subsequent 3 day
collection period when consuming a constant, near ad
Zibitum intake of feed offered as a gruel with deionized
water in three meals per day. Phase 2 began immediately
upon ending of the first collection period. At this time,
22 IU of E were added per kilogram of all diets and the

dietary treatments were identical as in phase 1. A 6 day

61

adjustment period to the diets was followed by a second

3 day collection of urine and feces. Urine was collected
in 6 N hydrochloric acid separate from the feces. The
feces were air dried on trays and were ground before
analysis. Blood and serum were collected initially and
at the end of the first and second collections and stored
at -20 C until analyzed for Se and SGOT. The feces,
urine, whole blood, serum and diets were analyzed for Se

content as described in experiment 1.

Experiment 3

 

Four male and eight female crossbred and Hampshire
pigs whose dams had received no supplemental Se or
vitamin E during gestation and lactation were used to
study the effects of added dietary selenium as selenifer-
ous corn, vitamin E as d-a-tocopheryl acetate and
arsanilic acid upon Se excretion and retention. The
basal diet was the 16% crude protein corn-soybean meal
diet (Table l) which analyzed 0.054 and 0.056 ppm Se,
respectively, for phase 1 and 2 of this experiment. The
pigs were assigned to one of four dietary treatments:
(1) basal (Table 1); (2) basal + 99 ppm arsanilic acid;
(3) basal + 0.1 ppm Se from seleniferous corn; and (4)
basal + 0.1 ppm Se from seleniferous corn + 99 ppm
arsanilic acid. The seleniferous corn was provided by
Dr. Oscar Olson, Department of Agricultural Chemistry,

South Dakota State University, Brookings, and had been

 

62

harvested in 1960 in a seleniferous area of that state.
Selenium analysis indicated that this corn contained 24.8
ppm Se which agreed well with the values reported for
this corn by Groce et al. (1973a). The seleniferous corn
was finely ground and substituted for 0.4% of the basal
diet to provide the supplemental Se in diets 3 and 4.

The pigs weighed an average of 7.6 kg initially and 8.1
kg at the beginning of phase 2. The pigs were housed in
stainless steel metabolism cages for a 10 day adjustment
period followed by a 3 day collection of excreta.

The diets were finely ground and fed three times
daily in near ad Zibitum quantities with deionized water
to form a gruel. Phase 2 began upon completion of the
first collection period. All diets were supplemented
with 22 IU E per kilogram and the dietary treatments
remained the same as in phase 1. An adjustment period
to the diets of 4 days was followed by a second 3 day
collection of excreta. Urine was collected in 6 N
hydrochloric acid separate from the feces. The feces
were air dried on trays and were ground before analysis.
Blood and serum were collected initially and at the end
of the first and second collections. Hemoglobin and
microhematocrits were determined on heparinized whole
blood as described in experiment 1. Serum samples were
sstored at -20 C until analyses for Se could be conducted.

53elenium analyses of diets, feces, urine and serum

63

samples were conducted fluorometrically according to the
method described in experiment 1.

Statistical analyses were conducted utilizing least
squares analysis of variance and two-way and three-way
analysis of variance as outlined by Steel and Torrie

(1960).

Results and Discussion

 

Experiment 1

The performance of pigs in experiment 1 is summar—
ized in Table 2. No significant differences in final
weight, average daily gain or feed conversion could be
attributed to the dietary treatments in this experiment.
Similar results were obtained by Groce et a1. (1971,
1973b). Arsanilic acid supplementation increased final
weights and average daily gains in pigs in these treat-
ments but the main effect was not significant. During
the course of this experiment two male pigs on the basal
+ 99 ppm arsanilic acid treatment died suddenly during
the 8th week of the experiment exhibiting symptoms of
selenium-vitamin E deficiency. In addition, after 12
weeks on experiment, one male and one female pig on the
basal diet (0.036 ppm Se) died suddenly with lesions of
selenium-vitamin E deficiency. The lesions observed in
t:hese pigs will be discussed briefly. The gross lesions

<>bserved upon necropsy of these pigs were a paleness and

64

woucmEoHQmsmcs :o Moxoa nmo.vmv Saucmofimflcmwm “swam: m>wH mo w ..u3 um>fiq

 

.cofluompoucfi m :HEmufl> x Enacoaom Amo.vdu acmowmficmfim m paw muoww enwcoHom

m nasaaa> :H NN

p

pfiom ewfificmmnm Edd moo

9
mm and H.om

 

pcwwmz o>HH
aw

 

wm.a mm.H Nm.H oa.a ov.H av.a om.a am.a a ..82 aosaa
ma.a mm.H ma.a mv.H 44.H a¢.H Hm.H mm.H ma ..u3 ao>aa
mm.o sm.o mm.o om.o am.o am.o am.o am.o momm\aaao
ca.a oa.a om.a om.H ma.H oo.a mo.H Ha.a ma .emmm saaaa
Ho.o mm.o mm.o mm.o mm.o am.o am.o am.o ma .aaaw saaam
”.ma m.mw N.om H.cw o.mm a.om o.am o.mm ma ..uz Haaaa
a.w a.m m.» a.w q.m m.w 4.x m.a ma ..83 HaaaaaH
OH OH a OH w m CH m mafia mo .02
m.m<+ m.m<+ m.m<+ m+ m.m<+ m + mmm+ Human EouH
m+ m+ om+ om+ Hammm H mam Hammm
mm+ Hmmmm Hammm Hmmmm
Hummm

 

mucosumouh xumuofia

 

ma ezmszmaxmv emuHmz mm>HS
nz< moz<zmoamma oHa zoa: one<ezm2maaa=m oHu<

UHAHz<mm< Qz< m ZHZ<HH> .ZDHzmAmm >m<HmHn mo Hummmm

.N

mqm<H

6S

edematous condition in the skeletal muscles. There were
pale areas in the heart muscle and diffusely mottled

and focal hemorrhages or areas of extensive necrosis in
the liver. Esophagogastric ulcers, hyperemia and hemor-
rhage of the fundic portion of the stomach were observed.
Mucous membranes had icterus and the spiral colon and
lungs were markedly edematous. Histopathologic examina-
tion revealed necrotic skeletal muscle fibers with macro-
phages, mineralization, swelling and fragmentation. The

myocardium exhibited subepicardial hemorrhage, fragmenta-

 

IT

tion, loss of striations, focal areas of necrosis and
mineral deposition and excess extrafibrillar and peri-
vascular mononuclear cells. The most apparent microscopic
lesions were those in the liver, consisting of necrosis
and hemorrhage of clusters of adjacent lobules with
increased interlobular fibrous connective tissue deposi-
tion. Interlobular edema was observed in histologic
sections of lung tissue. The cardia of the stomach
exhibited loss of stratified squamous epithelium with
necrosis, fibrin, polymorphonuclear cells and thrombi in
the submucosa. The postmortem diagnostic conclusion in
all cases was hepatosis diaetetica (vitamin E-selenium
deficiency) and esophagogastric ulceration. No sudden
death losses occurred in pigs fed diets containing
:supplemental selenium and/or vitamin E and no gross

Ilesions attributable to treatment were observed at

66

slaughter. Carcass data collected at slaughter were
not significantly affected by any of the dietary treat-
ments and were considered normal values for present day
growing-finishing swine. Liver weight (Table 2) was
not significantly affected by the dietary treatments in
this study but liver weight expressed as a percent of
live weight was significantly (P<.05) lower on unsup-
plemented selenium diets. A significant (P<.05) inter-
action between selenium and vitamin E with regard to
liver weight as a percent of live weight did occur.
Added vitamin E to unsupplemented selenium diets increased
liver weight as a percent of live weight but decreased
this parameter when vitamin E was added to selenium
supplemented diets.

The effects of supplemental, vitamin E and arsanilic
acid on initial, 2 week and 8 week blood parameters are
presented in appendix Table 1. Initial, 2 week and 8
week hemoglobins, hematocrits and SGOT values were not
different among treatments. Serum selenium at 2 and 8
‘Weeks was (P<.01) higher on selenium supplemented diets.

The effects of supplemental selenium, vitamin E and
Errsanilic acid on the final blood parameters are presented
111 Table 3. Selenium supplementation (P<.01) increased
Sermmlselenium levels at the final bleeding even though
selenium had been withdrawn from the diets for 60 days.

13115 finding is contrary to the results reported by

mpofiw popcoEmHQQSm Baum owaficmmhm no pmzofi nHo.vdv xfipcmofiwfiCMfim yahooumEmzw

mpofiw popcoEoHQQSm wflom owawcmmpm co umzofi nHo.vmV AfiuchMwficwfim mm: canoamoEmmu

mpofiw popcoEmHQQSm mm :o umsmfl: fifio.vdv Saucmowwficwwm who: mm enhmmn

mumscm cams hohpm Hamuo>om

 

 

 

 

Hooo. oao. oso. aao. oao. wmo. ammo. saa .om enamm
a.moa a.mm o.mm a.om a.wm m.mm N.mm He\maaca a-m .aoom

an am mm mm am am mafia mo .02

«.mm o.mm ea.oa a.am m.om m.am H.0m a .aaauouasom
m.a m.NH um.mH a.~H N.ma m.mH a.~H as ooa\m .aaaoamoemm

Hm mm an mN am am mmaa mo .02

amZm mm b. mwnphlwwllnmmpu H.o av aoaa

5mm anwum ofifiwcmmu< m amazoEoH am 8mm .mm HmucQEonmzm

 

fia azmszmaxmw mamamz<m<a mooam a<sz zo nHu< quHz<mm<
mz< m sz<aH> .zaazmamm smaamao amaa< mo Humaam . m:m<a

68

Groce et al. (1973b), who reported that serum Se levels
of pigs receiving the basal diet were not different from
those of pigs receiving Se supplemented diets from which
Se was withdrawn 60 days before slaughter. Arsanilic
acid supplementation decreased (P<.01) hemoglobin and
hematocrit values at the final bleeding. The signifi-
cance of this finding is uncertain at this time. SGOT
values were not affected by dietary treatment in this
experiment. All values were in the range of normal values
reported by Wretlind et a2. (1959) and were lower than
values reported by Orstadius et al. (1959) for deficiency
symptoms in pigs. Supplemental vitamin E had no effect
on any of the blood parameters measured in this
experiment.

Tissue selenium levels as influenced by supplementary
selenium, vitamin E and arsanilic acid are presented in
Table 4. Selenium supplementation increased (P<.01)
liver, kidney and diaphragm muscle Se concentrations on
both a wet and dry basis even though selenium had been
\Withdrawn from supplemented diets for 60 days. These
tiéssue Se values are lower than those reported by Groce
9t <21. (1973b) for pigs having received Se followed by
El 60 day withdrawal period. Lindberg and Lannek (1965)
Sllpplemented a Se adequate diet (0.126 ppm natural Se)
With 1.2 ppm Se from sodium selenite which was fed to

growing pigs for 78 days. Skeletal muscle Se

69

.mfimmp Sup mam pm: a anon co mumflp popcoEmHQQSm Baum
ofifificmmhm co swam“: nmo.vmv saucmoflmwcmflm who: mHo>oH asflcmamm oHomse Emmpsmmwmo

.mpmflv woucoEmHmdsm Esflcofiom
co magma: fifio.vmv xfipcmofiwfi:MHm who: wmnxfimcm moammwu Haw pom mHo>ofl Esficonmn

ohmscm came noupo HHmHo>om

 

 

NNOO. aaN.o amom.o mNN.o mmm.o aam.o aaaa.o aha
Hooo. 000.0 ammo.o Noo.o moo.o wao.o amao.o am:
mHomse Ewmpnmmfim
ma ON ma ma NN 0H mafia mo .02
H4H¢.H Nam.m amm.m mmm.m amm.m Haa.o ammo.a Aam
mmao. RHN.H NmN.H sHN.H mm~.a awm.a amoo.H um:
Raceam
«moo. mom.o aom.o me.o amm.o amo.o awoa.o aaa
mooo. oaa.o Noa.o moa.o aoa.o ama.o qu.o “a:
a pm>fiq
mm am am Nm Nm am mmaa mo .02
amZm mm o wx\:H NN o H.o c amaH

 

 

5mm .mfiom ofiaficmmu< m Hmpcmsmdmmsm 5mm .om Hmucosoammsm

 

ma :Lzmzammaxmv Zaa .zDHZmamm mammHa zo aHu< quHz<mm<
az< m zaz<aa> zaHzmamm >m<amao amam< ao aommam .4 mqm<s

70

concentrations were 0.52 and 0.76 ppm for the basal and
supplemental groups, respectively. Supplemental Se was
withdrawn for 14 days and muscle Se concentrations declined
to levels similar to those from pigs on the basal diet.
Grant et al. (1961) observed no difference in muscle Se
concentrations of pigs fed 0.2 ppm supplemental sodium
selenite and those of pigs fed no Se supplement. They
observed little effect of duration of Se supplementation
or length of withdrawal period on tissue Se concentration.
Similar tissue Se values have been reported by Lindberg
and Siren (1963, 1965) and Sharp et al. (1970). Andrews
et a2. (1968) and Sharp et al. (1970) reported tissue Se
levels for pigs dying of selenium-vitamin E deficiency
that were lower than those of pigs on the basal diet in
this experiment. In addition Se supplementation decreased
(P<.05) kidney dry matter percentage. Kidney dry matter
was (P<.05) lower on selenium supplemented versus unsup-
plemented diets (21.7 vs. 22.6%). The significance of
this observation is not apparent, but this difference in
tissue dry matter could be due to minor fluctuations of
fluid balance in the animal.

Arsanilic acid supplementation increased (P<.OS)
diaphragm muscle selenium levels on both a wet and dry
basis. Supplements of vitamin E had no significant

effect on tissue Se levels in this experiment.

71

Experiment 2

 

The main effects of supplemental Se and arsanilic
acid on Se balance and blood Se levels for the two col-
lection periods are presented in Table 5. In the first
collection period, selenium supplementation increased
(P<.01) Se intake, percent Se retention and absolute Se
retention. This increase in Se retention resulted pri-
marily because fecal Se excretion (as a percent of intake)
was markedly (P<.01) reduced, and secondly because urinary
Se excretion was lower on selenium supplemented diets,
but this difference was not statistically significant.
Arsanilic acid supplementation significantly increased
(P<.05) percent Se retention and there was a significant
(P<.01) interaction between selenium and arsanilic acid.
The addition of arsanilic acid to unsupplemented Se
diets increased percent Se retention but decreased per-
cent Se retention when added to selenium supplemented
diets. This interaction can be attributed to the (P<.05)
interaction between selenium and arsanilic acid in percent
fecal Se excretion on unsupplemented Se diets but
increased percent fecal Se excretion on Se supplemented
diets. Serum and whole blood Se levels were higher
(P<.01) on selenium supplemented diets at the end of
phase 1.

In the second collection period (following the addi-

tion of 22 IU of vitamin E per kg of diet), Se intake

72

 

 

ooo. omo. omo. ooo. ooNo. N omooa Ho oam
moo. mmo. omo. oao. moo. H omaoa oo oam
moo. omo. omo. aNo. Nmo. HoHHHaH
Edd .om Eshom
m.N o.N a.oH o.oH m.mH aaaaHao
H.m a.mo m.Hm a.om N.om Haooa
oxmqu mo 1» acowpmhuxo Om
m.H H.HH N.o o.oH on.m Hao\wa .coHoaopoa om
o.m m.oo m.mm o.mo N.Nm oHaHaH H0 a .ooHoaooma om
m.o o.mm o.o~ m.om oo.HH aao\Ma .oxaoaH om
HoHo mx\m .aHs 2H NH - N omaoo
o.o o.HH o.oH m.NH o.oH HaaaHao
o.N m.oo a.oo H.am oo.Nm Hauoa
oxmucfl mo w .aowuopoxm om
o.o H.o o.a o.NH oo.m aao\ma .aOHHaoooa om
a.N o.a 0o.om o.om oo.om oHaHaH H0 a .aoHoaoooa om
o.N o.o o.oH N.mN om.o aao\wa .oHaHaH mm
m .HH> ooooa oz - H omaoo
o o o o mmHa Ho .02
a.m.m oo [pm H.o o sooH

emm,.efium owaficmmu<

 

Ema .om HmucmEonmsm

 

Am bzmszmmxmv uHm 0230» Mme ZH muz<q<m ZDHzmqmm zo QHu<

UHAHz<mm< Qz< m ZHZ<HH> .ZDHzmqmm >m<HmHQ QmQQ< mo hummmm .m mqm<b

73

.N can H mommga mo paw may um mummw woucmsofidmzm Ezmaofiom co mesmH: “Ho.vdv
xfiucmomwficwwm om vooan oHozz cam mm espom .fixmv\mav comucouom .oxmuafl 0mm

Boom oHHmcmmHm vcm Eswcoflmm mo :oHpomHmuam mmo.vmv ucmumchmfim m mm: when» cam
mpomw emucmEdedsm esmcofimm co Hozoa fiHo.vdv mapcmonHcmHm comuohoxo om Hmuom

p
wwom uHHmcmmHm

was Eamamfimm wo cofluumhoucm fiHo.vdv ucmommmcwmm m mm: omega wcm muomw poucoEondsm
coon UHHHCmmHm co Hosmmg mmo.vmv xHucmoHMHcmom moxmucM mo my comucouoh emu

zfiucmomwmcmHm mxmw\mav comucouou om vcm noxmucm mo mo coaucouop om .oxmuam om

mummy voucoEoHQQSm om co macaw: flHo.vdv

n
mouuo cpmvamumm

 

 

 

ooo. oao. mao. moo. omoo. N omaoa mo oam
ooo. ooo. Nao. aoo. momo. H omaoa mo ocm
moo. omo. Hmo. ooo. omo. HaHHHaH
goo .am oooHo oHooz
a.m.m om mo, H.o o EooH

5mm .pwom omfiwcmmn<

 

and mom HancoEonmsm

 

Homoszzooo m mHm<o

74

and absolute selenium retention were increased (P<.01)
by selenium supplementation of the diet. There were no
significant differences in the proportion of Se intake
excreted in the feces or urine. However, the observed
increase in absolute retention must have resulted from
the reduced but not significant (P=.077) reduction in
percent fecal Se excretion resulting from dietary Se
supplementation. Serum and whole blood Se levels were
increased (P<.01) by selenium supplementation of the
diets at the end of the second collection period.

In comparing the data from the two collection
periods in this experiment, the addition of supplemental
dietary vitamin E tended to increase percent urinary
excretion of supplemental dietary selenium and thus
lowered the percentage of dietary Se retained. The
absolute retention of Se increased slightly from the
first to second collections from 12.3 to 16.6 pg per day
on Se supplemented diets which may be a reflection of
the increase in Se intake from 25.2 to 36.3 ug/day. The
comparison of the effect of vitamin E involves a time
span, and thus the effects of supplemental vitamin E are
confounded with the effect of period. These results are
similar to those of Groce et al. (1973b) in which diets
were supplemented with 0.1 ppm Se. Groce et al. (1973a)
reported research that supported the hypothesis that

these differences may truly represent a vitamin E effect

75

since supplemental dietary vitamin E lowered selenite Se
retention and a trend for increased urinary Se loss was
evident in all trials reported. Comparison of the data
from the two collection periods revealed that the effect
of arsanilic acid supplementation of the basal diet on
Se excretion and retention was similar to and parallels
the changes discussed attributable to selenium supplemen-

tation of the diet.

Experiment 3

 

The main effects of dietary Se supplementation (as
seleniferous corn) and arsanilic acid supplementation on
Se balance and serum Se levels for the two collection
periods are presented in Table 6. In the first collec-
tion period, selenium supplementation increased (P<.01)

Se intake, percent Se retention and absolute Se retention.
The increase in Se retention can be attributed to the
decreased (P<.01) urinary Se excretion on Se supplemented
diets and the lower non-significant fecal Se excretion.
Arsanilic acid increased (P<.01) absolute Se retention
and there was a significant (P<.01) interaction between
selenium and arsanilic acid. Arsanilic acid supplementa-
tion decreased absolute Se retention on unsupplemented

Se diets while it increased absolute Se retention on Se
supplemented diets. Serum Se levels had increased (P<.01)
on selenium supplemented diets by the end of the first

collection period.

76

 

m.o o.N o.N o.o mo.o maaaHao
o.m m.oo o.Ho m.om o.Ho Hauom
mxmucm mo w .cowuohoxm om
o.H m.oH mo.oH o.mN Ho.o aao\oa .aomoaoaoa om
N.m H.Nm o.Hm N.mm o.No oHaHaH mo m .aoHoaoooa om
N.N m.om o.oN o.oo oo.oH aao\ma .oHaHaH om
ooHo mH\m .oH> oH NN - N omaoa
m.o o.o o.o o.o oo.HH aaaaHao
H.m m.oo m.oo o.No N.om Haooa
oxmpcm mo » .coHumhuxm om
m.o N.mH UH.HH m.oH Um.m sao\ma .aoHoaoaoa om
o.N N.oo o.No N.om om.om «HaoaH H0 m .aOHaaoaoa mm
N.m N.NN m.mN N.om oN.oH aao\Ma .oxapaH om
m .uH> ooooa oa - H omaoo
o o o o mama Ho .02
a.m.m oo, .o H.o o1 smoH

 

 

sum «poem omfimcmmu<

Ema .om HancoEofimmsm

 

mm Hzmszmmxmv on 0230» m:& 2H muz<q<m EDHZmAmm zo aHu< UHAHz<mm< Qz<

m sz<oH> .Hzmou mooomonmHmmo onzmHmm >m<mmHo omoo< mo mommmm .o mHo<o

N van H mommcd «0 use we muomw woucosmammsm mm :o magmH: fiHo.vmv om Esmom:

vmum umHmcmmHm cam samcoHom mo :omuommmucm
mmo.vmv unmonHcmHm a van muomw popcoEoHdasm om no Hoon mHo.vmv :oHpoHuxo mmmcmmaw

poem uHHmcmmHm
paw enmnoHom mo mmo.vmv :oHuommoucm ucmommmamHm m was open“ one muomw wmom umamcwmum co

mmo.vmv HonMH: use acmumuCoEmHmasm om Eomw magma: nHo.vdV mxmp\wnv compcouo» 0mm

mpowu popcoEoHQQSm mm :o Honwmg nHo.vmV oxmucm omo

mumww voucosoadazm somcoHom no mHo.vmV Hoon :oHuoHoxo Hymnausw

comuummoucm vmum oHHmcmmHm x Enmconm a van :oHumucmEoHdmsm wmom oHHmcmmHm
HI .coHumucoEmHQQSm om mp wouummmm mHo.vmv xHucmumwmcme mxmw\wnu comucouom emu
7

mummp HancoEoHQQSm om co Hogwmg “Ho.vmv Amxmuam mo my coHucoumm om wan oxmucm own

Hommo wnmwcmumm

 

 

moo. mmo. omo. omo. ooNo. , N omaoa Ho mam
Noo. Nmo. Hmo. omo. ooNo. H «mama Ho mam
Noo. oNo. NNo. mNo. oNo. HaHHHaH
Eda .om Esuom
a.m.m mm, o H.pr av. sooH

 

 

Emm.vmom oHHHnmmH< 5mm..om HmucoEonmmm

 

Hamsszzouo o mHo<m

78

In the second collection period (after adding 22
IU vitamin E per kg of diet) Se intake and absolute Se
retention were higher (P<.01) on selenium supplemented
diets. This increase in absolute Se retention is attribu-
table to the decreased (P<.01) urinary Se excretion on
Se supplemented diets and the lower but non-significant
fecal excretion of Se. Arsanilic acid increased (P<.05)
absolute Se retention and a selenium x arsanilic acid
interaction (P<.05) was observed in that arsanilic acid
supplementation resulted in the same absolute Se retention
on unsupplemented Se diets while increasing absolute Se
retention on Se supplemented diets. Furthermore, there
was a significant (P<.05) interaction of selenium and
arsanilic acid in regard to urinary Se excretion. Arsanilic
acid decreased urinary Se excretion on unsupplemented Se
diets but increased urinary Se excretion on Se supplemented
diets. Serum Se levels were higher (P<.01) on Se supple-
mented diets at the end of the second collection period.

Comparison of the data from the two collection periods
in this experiment revealed that dietary vitamin E decreased
urinary and fecal Se excretion on supplemented Se (seleni-
ferous corn) diets and accordingly increased the percent
Se retention and absolute Se retention. The effects of
vitamin E supplementation are confounded with the effect
of period since the two collections were conducted at
different times. However, Groce et al. (1973a) reported

similar results in that added vitamin E increased

79

absolute retention of Se from seleniferous corn (22.5 vs.
31.7 pg per day). The increased Se retention from seleni-
ferous corn in their study, when vitamin E was added to
the diet, was due primarily to decreased fecal Se excre-
tion. The effect of arsanilic acid supplementation of
the basal diet on Se excretion and retention paralleled
the changes observed due to selenium supplementation of
the diet for the two collection periods in this experiment.
The results of experiments 2 and 3 are in excellent
agreement with the results reported by Groce et al.
(1973a). When supplemental Se was from seleniferous
corn, a significant higher percentage of Se ingested was
excreted via the feces and less via the urine as compared
to Se from sodium selenite. They reported a significant
interaction in which added vitamin E reduced the absolute
retention of selenite Se and increased the absolute reten—
tion of Se from seleniferous corn. The increased Se
retention from seleniferous corn, when vitamin E was
added to the diet, was due to decreased fecal Se excre-
tion in their study but primarily due to decreased urinary
and fecal Se excretion in my experiment 3. Fecal Se
could be non-absorbed dietary Se or endogenous Se secreted
into the lumen of the intestine (Wright and Bell, 1966)
and therefore increased Se absorption or reduced endogenous
fecal Se secretion may have been altered by supplemental
vitamin E to cause an increase in Se retention from

seleniferous corn. In addition they observed that serum

80

Se levels were lower on seleniferous corn as compared to
selenite Se and supplemented vitamin E had no significant
effect on serum Se levels. The decrease in serum Se
levels resulting from seleniferous corn may have been
due to decreased intestinal absorption or to increased
clearance of serum Se to tissues. McConnell and Hoffman
(1972) have demonstrated that selenium in the form of
selenomethionine is readily incorporated into rat hepatic
proteins via the methionine pathway. Groce et al. (1971,
1973a,b) and Ku et a2. (1972, 1973) have shown that a
given concentration of Se naturally occurring in feeds
will result in greater Se retention and higher tissue Se
levels than similar dietary Se levels from sodium selenite.

The results of experiment 2 also provide additional
support for the observation of Groce et al. (1973b) that
supplemental dietary vitamin E lowers selenite selenium
retention primarily by promoting the urinary excretion
of selenite Se, since a trend for increased urinary loss
was evident in their trials and in experiment 2 of this
study. Sharp et al. (1970) reported that supplements of
vitamin E to selenite containing diets increased kidney
Se levels and noted that decreased liver and muscle Se
concentrations were associated with the elevation of
kidney Se.

Arsanilic acid was not antagonistic to physiological

requirement levels of dietary selenium in this study.

81

Although hemoglobin and hematocrit values were lower on
arsanilic acid supplemented diets (Exp. 1), the physio-
logical significance of this observation is not clear.
Arsanilic acid increased diaphragm muscle Se concentra-
tions (even though Se had been withdrawn for 60 days) and
had no effect on liver or kidney Se concentrations. The
balance studies demonstrated that arsanilic acid decreases
Se excretion and increased Se retention. These data
indicate that a normal usage level of arsanilic acid is
not antagonistic to selenium at nutritional levels and
support the suggestion by Muth et al. (1971) that a
selenium-arsenic complementary effect exists in selenium
deficiency situations. In their study, feeding 1.00 ppm
arsenic in the form of sodium arsenate, when added to a
low selenium ration for pregnant ewes, gave a significant
marked protection against the myopathy commonly associated
with selenium deficiency in lambs. The data from this
study suggest that discrimination against the use of
arsanilic acid as a feed additive in rations containing
nutritional levels of natural selenium or in rations
supplemented with 0.1 ppm Se from inorganic sodium
selenite for growing-finishing pigs in low selenium areas

of the United States is unwarranted.

Summary

One feeding trial and two balance studies were con-

ducted to study the effects of arsanilic acid, selenium

82

(Se) and vitamin E (E) on productive performance, tissue
selenium and selenium balance in growing-finishing pigs
fed diets low in vitamin E and selenium. Eighty weanling
pigs were assigned to eight dietary treatments in a 2 x

2 x 2 factorial design to evaluate the effects of supple-
menting a basal corn-soybean meal diet (0.036 ppm Se)
with 0.1 ppm Se (as sodium selenite) and/or 22 IU E per
kg of diet and/or 99 ppm arsanilic acid. Selenium sup-
plements were withdrawn from Se supplemented diets 60
days before slaughter. Classic Se and/or vitamin E
deficiency lesions were observed in four animals fed
diets not supplemented with Se or vitamin E. Selenium
supplementation increased (P<.01) liver, kidney and
diaphragm muscle Se concentrations, while arsanilic acid
supplementation increased (P<.05) diaphragm muscle
selenium concentrations. Vitamin E supplements had no
effect on tissue Se levels. The two balance studies were
concerned with Se absorption and retention on diets sup-
plemented with Se as sodium selenite or seleniferous

corn as determined by measurement of Se intake and fecal
and urinary Se excretion. The effects of arsanilic acid
and vitamin E (22 IU E/kg diet) were also studied. Sup-
plemental Se from seleniferous corn increased percent
fecal Se excretion and decreased percent urinary Se
excretion as compared to Se from sodium selenite. Vitamin

E supplementation increased Se retention from seleniferous

83
corn by decreasing urinary and fecal Se excretion.
Arsanilic acid decreased Se excretion and increased Se
retention. Arsanilic acid was not antagonistic to
physiological requirement levels of dietary selenium

in this study.

EFFECT OF DIETARY VITAMIN E LEVEL ON PERFORMANCE,
TISSUE SELENIUM AND SELENIUM BALANCE
IN GROWING-FINISHING SWINE

Introduction

Natural deficiencies of vitamin E and selenium (Se)
have been reported by Michel et al. (1969), Trapp et a2.
(1970), Grace et al. (1971, 1973b) and Ullrey (1973,
1974). When vitamin E was shown to be essential for
small laboratory animals, investigations were carried
out with large animals to determine whether supplementary
tocopherols were required under practical conditions.
Carpenter and Lundberg (1949) reported that swine experi-
ments up to 1949 had shown that tocopherols were not a
limiting dietary factor under field conditions. Prior
to 1973, the National Research Council stated that prac-
tical swine diets are not likely to be deficient in vitamin
E unless the diet contains excessive amounts of highly
unsatnirated fatty acids or oxidized fats. Furthermore,
since' tocopherols are present in almost all cereal grains,
little: research has been conducted to determine the
effecrts of supplemental dietary vitamin E in swine. The
National Research Council (1973) suggests that 11 IU of

vitamin E be added per kg of diet until more specific

84

85

information is obtained on vitamin E and selenium require-
ments of swine.

Preliminary estimates of the dietary selenium require-
ment for the pig have been reported by Groce at al. (1973b).
These studies indicated that the dietary selenium require-
ment for the pig when supplemented vitamin B does not
exceed 11 IU/kg is 0.15 ppm Se. These studies also indi-
cated a need for further research to study the interrela-
tionship of Se with vitamin E. Although vitamin E-
selenium deficiencies primarily affect the young growing
pig, Hartley and Grant (1961) and Mahan et al. (1974)
have reported that deficiencies also occur in the finish-
ing pig and the sow.

The purpose of this investigation was to study the
effects of supplemental Se and 4 dietary levels of vitamin
E supplementation on productive performance, hematology,
tissue selenium and selenium balance in growing-finishing

pigs fed diets low in vitamin E and selenium.

Experimental Procedure

 

Two experiments were conducted in this investigation.
Experiment 1 was designed to study the response of growing
pigs to added dietary selenium and 4 levels of vitamin E.
Experiment 2 was a selenium balance study conducted with
young pigs to study the effects of 4 added vitamin E

levels on the retention and excretion of selenium.

86

Experiment 1

 

Sixty-four Yorkshire, Hampshire and Yorkshire x
Hampshire barrows and gilts weighing an average of 11.7
kg were randomly allotted to eight dietary treatments
and housed eight per pen in a completely enclosed,
slotted floor, environmentally controlled building.

The pigs were offspring of sows which had received no
supplemental vitamin E or selenium during gestation or
lactation. The basal diet (Table 7) was a 16% crude-
protein fortified corn-soybean meal diet which analyzed
.054 ppm Se and contained no supplemental vitamin E or
selenium. The pigs were fed this basal diet or the
basal diet supplemented with 0.1 ppm Se (as sodium
selenite) and vitamin E at 0, 5, 10 or 15 IU vitamin E
per kg of diet in a 2 x 4 factorial design of treatments.
The pigs were weighed and feed consumption determined at
biweekly intervals. Tap water (1 part per billion [ppb]
Se) was offered ad Zibitum. All animals were observed
for clinical signs of selenium-vitamin E deficiency
throughout the course of the experiment. Blood samples
were collected from the anterior vena cava from 5 pigs
from each of the 8 treatment groups initially, at 10
weeks and 2 days prior to slaughter. Microhematocrit
(McGovern et aZ., 1955) and hemoglobin (Crosby et aZ.,
1954) determinations were made on heparinized whole
blood and serum glutamic-oxaloacetic transaminase (SGOT)

(Sigma Technical Bulletin 505, 1964) were determined

87

TABLE 7. COMPOSITION OF BASAL DIET (EXPERIMENT 1)

 

 

. International

Ingredient ref. no. Percent

Corn, dent yellow, grain, 4-02-931 79.2

gr 2 US mn 54 wt (4)

Soybean, seed wo hulls, 5-04-612 17.9

solv-extd grnd, mx 3

fbr (5) (soybean meal)

Calcium phosphate, dibasic, 6-01-080 1.0

comm (6)

Limestone, grd, mn 33 Ca (6) 6-02-632 0.9

Salt, plain white 0.5

Vitamin-trace mineral premix3 0.5
100.0

Crude protein, %b 16.0

Calcium, %b 0.64

Phosphorus, %b 0.50

Se, ppmc .054

 

aProvided the following per killogram of diet:
Vitamin A, 3,300 IU; vitamin D, 660 IU; riboflavin, 3.3 .
mg; nicotinic acid, 17.6 mg; d-pantothenic acid, 13.2 mg;
choline chloride, 110 mg; vitamin B12, 19.8 ug; zinc,
74.8 mg; manganese, 37.4 mg; iodine, 2.7 mg; copper,

9.9 mg; iron, 59.4 milligrams.

bCalculated

cAnalyzed

88
on all serum samples collected. Serum samples were stored
at -20 C until Se analyses were performed. Rectus
abdominus muscle, liver, and kidney samples were obtained
from 5 pigs from each treatment group at slaughter, placed
in plastic bags and frozen at -20 C until analyzed for Se
content. Preparation of tissue samples for selenium.
analyses was as described in Chapter III; Experimental
Procedure - Experiment 1. Selenium analyses of serum,
tissues and diets were conducted fluorometrically accord-
ing to the method of Hoffman et a2. (1968) with the modi-
fications described by Groce et al. (1971) and which were
previously discussed in Chapter III - Experimental

Procedure, Experiment 1.

Experiment 2

 

Seven male and five female crossbred pigs whose dams
had received no supplemental Se or vitamin E during
gestation or lactation were used to study the effect of
dietary vitamin E level as d-a-tocopheryl acetate upon
Se excretion and retention. The basal diet was the 16%
crude protein fortified corn-soybean meal diet (Table 7)
supplemented with 0.1 ppm selenium as sodium selenite
(total Se, 0.172 ppm). The pigs were randomly allotted
to the basal diet or the basal diet supplemented with 5,
10 or 15 IU vitamin E per kg of diet. Average weight of
the 12 pigs initially was 9.2 kilograms. The pigs were

housed in stainless steel metabolism cages for an 8 day

 

89

adjustment period and a subsequent 3 day collection period
when consuming constant near ad Zibitum daily intakes
(375 grams) of the test diets offered as a gruel with
deionized water in three meals per day. Urine was col-
lected in 6 N hydrochloric acid separate from the feces.
The feces were air dried on trays and were ground prior
to analysis. Blood and serum were collected initially,
at the end of the collection and prior to sacrificing the
pigs. Pigs were sacrificed to obtain samples of quadri-
ceps femoris muscle, myocardium, liver and kidney for Se
analyses one day following the completion of the urine
and fecal collection. Serum was analyzed for SGOT, lactic
dehydrogenase (LDH) (Sigma Technical Bulletin 500, 1969)
and creatine phosphokinase (CPK) (Sigma Technical Bulletin
520, 1971) and then stored at -20 C until analyzed for
Se. Hemoglobin and microhematocrit were determined on
heparinized whole blood as previously described. Serum,
tissues, diets, feces and urine were analyzed for Se
content as described in experiment 1.

Statistical analyses of the data were conducted
utilizing one-way and two-way analysis of variance as

described by Steel and Torrie (1960).

Results and Discussion

 

Experiment 1

 

During the first 14 weeks of this experiment no sig-

nificant differences in performance of the pigs were

90
observed. However, at 16 and 18 weeks of the experiment,
dietary supplementation of 5 IU vitamin E per kg of diet
increased (P<.05) pig weight and total gain over the
groups fed 0, 10 or 15 IU vitamin E per kg of diet.
Nevertheless, this observed effect of 5 IU vitamin E
per kg of diet disappeared during the last 4 weeks of
the trial. The performance of pigs for the entire experi- F-_
ment is summarized in Table 8. No significant differences
in final weight, average daily gain or feed conversion

could be attributed to the dietary supplements of Se or

 

vitamin E in this experiment. Groce et a2. (1971, 1973b), .11.
Ku et al. (1973) and Wastell et a2. (1972) have reported
similar results of supplemental Se and vitamin E on

growth and feed conversion of pigs. During the experi-
ment, one pig on the basal + 15 IU of vitamin E/kg of

diet treatment died suddenly during the 18th week of the
trial. Postmortem diagnosis indicated acute bronchopneu-
monia as the cause of death. There were no lesions indica-
tive of vitamin E-selenium deficiency and no gross lesions
of vitamin E-Se deficiency were observed in any pig at
slaughter. The effects of supplemental selenium and
vitamin E on 10 week and final hemoglobin and hematocrit
are presented in Table 9. Selenium supplementation (P<.05)
decreased 10 week hemoglobin values from 11.9 to 10.9

g/100 ml. At the final bleeding, hemoglobin values were

unaffected by selenium supplementation of the diet but.

xmow 3oz .Houmonoom .moHHumsooH
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92

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.O mHm<H

93

vitamin E supplementation of 10 or 15 IU per kilogram of
diet depressed (P<.01) blood hemoglobin levels.

Ewan and Wastell (1970), Groce et al. (1971, 1973b)
and Wastell et a2. (1972) reported that the addition of
vitamin E and selenium alone or in combination to deficient
diets had no effect on the blood hemoglobin concentration.
Selenium and vitamin E had no significant effect on 10
week hematocrits. The hematocrit of blood samples
obtained at the final bleeding were increased (P<.05) by
dietary supplements of 0 and 5 IU of vitamin E/kg of diet
over the hematocrit values of pigs receiving 10 IU per
kg of diet.

The effects of supplemental Se and vitamin E on
serum Se levels during this experiment are presented in
Table 10. Selenium supplementation markedly increased
(P<.01) serum Se concentrations at 10 weeks and at the
bleeding prior to slaughter. Vitamin E supplementation
had no effect on serum selenium concentrations at either
the 10 week or final bleeding which adds additional support
to the observations of Groce et al. (1973a,b) that sup-
plemental vitamin E had no effect on serum selenium levels.
Serum SGOT values were not affected by the dietary treat-
ments in this experiment, but were similar to the normal
values reported by Wretlind et a2. (1959).

Tissue selenium levels as influenced by supplemental

Se and vitamin E are presented in Table 11. Selenium

 

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ohmscm come Hopmo HHmHo>om

 

94

 

 

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95

mmHO. OOO.H mwO.H NOO.H mmH.H coo:

 

 

 

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m sz<mH> oz< onzmHmm maOmmHo omoo< mo Homamm .HH mHHOH

96

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Homoszzooo HH mHm<m

97
supplementation increased (P<.01) rectus abdominus muscle
selenium concentrations on both a wet and dry basis.
There was a significant (P<.05) interaction between
selenium and vitamin E in regard to rectus abdominus
muscle selenium concentration on a wet basis. The addi-
tion of increasing levels of vitamin E to unsupplemented
Se diets decreased muscle Se concentrations while on Se
supplemented diets, muscle Se concentrations increased
with increasing levels of vitamin E. Selenium supplemen-
tation increased (P<.01) liver and kidney Se concentrations
on both a wet and dry basis. The dry matter percentages
of all tissues were unaffected by dietary supplementation
of selenium or vitamin E in this experiment. The vitamin
E main effect was not significantly different for any of
the tissue Se concentrations or dry matter percentages.
Ewan (1971) reported similar findings in that the addi-
tion of selenium resulted in a significant increase in
the selenium content of tissues compared with dietary
treatments containing no added selenium and that vitamin
E had no significant effect on the level of selenium in
the tissues. However, the observation made by Groce et
al. (1971, 1973b) that supplemental vitamin E increased
kidney Se levels was not observed in this experiment.
They also reported that supplemental vitamin E had no
apparent effect on Se levels of other tissues analyzed

which is consistent with the findings of this experiment.

98

Experiment 2

 

The main effects of supplemental vitamin E level on
selenium balance are summarized in Table 12. Supplemental
levels of vitamin E had no significant effects on selenium
excretion or retention in this experiment. Increasing
levels of vitamin E tended to increase fecal Se excretion
and thus decrease absolute and percent Se retention.

Groce et al. (1973a,b) have reported similar results in
that added vitamin E reduced the absolute retention of
selenite Se by increasing fecal and urinary Se excretion.

The effect of dietary vitamin E level on serum Se
and blood parameters are presented in Table 13. The serum
Se and blood hemoglobin levels were not significantly
affected by the supplemental levels of vitamin E at any
sampling time. In addition, initial and final hemato-
crits were unaffected by supplemental level of E, but
prior to being sacrificed pigs receiving 15 IU vitamin
E/kg of diet had lower (P<.05) hematocrits than pigs
receiving diets supplemented with 0 or 10 IU vitamin E/kg
of diet.

The serum enzyme activities are reported in Table
14. Serum SGOT values were unaffected by supplemental
level of vitamin E but were in the normal range of SGOT
values reported by Wretlind et al. (1959). Lactic
<iehydrogenase activity was higher (P<.05) initially in

{Jigs allotted to the 5 IU vitamin E/kg of diet treatment.

99

TABLE 12. EFFECT OF DIETARY VITAMIN E LEVELS OF SUPPLE-
MENTATION ON SELENIUM BALANCE (EXPERIMENT 2)

 

 

Vitamin E, IU/kga
s 10

 

 

Item 0 ’15 S.E.
No. of pigs 3 3 3 3
Se intake, ug/day 61. 63. 60.2 62.6 3.0
Se retention, % 67. 62. 61.3 64.9 2.6
of intake
Se retention, ug/ 41. 39. 36.9 40.7 1.8
day
Se excretion, %
of intake
Fecal 24. 27. 30.6 26.8 2.0
Urinary 8. 10. 8.1 8.3 0.9
aVitamin E as d-a-tocopheryl acetate. Distilla-

tion Products Industries, Rochester, New York.

bStandard error

100

TABLE 13. EFFECT OF DIETARY VITAMIN E LEVELS OF SUPPLE-

 

 

 

 

 

 

MENTATION ON BLOOD PARAMETERS (EXPERIMENT 2)
Vitamin E,§IU/kg
Item 0 5 10 15 S.E.a
No. of pigs 3 3 3 3
Serum Se, ppm
Initial .017 .022 .017 .019 .006
Final .108 .118 .107 .105 .007
Pre-sacrifice .093 .095 .097 .105 .005
Hemoglobin, g/100 ml
Initial 10.9 9.8 10.6 9.8 0.72
Final 10.9 10.3 12.5 10.9 0.99
Pre-sacrifice 9.9 9.6 10.5 11.3 1.12
Hematocrit, %
Initial 35.7 32.4 35.8 33.1 2.40
Final 34.4 32.9 35.1 30.1 1.77
Pre-sacrifice 32.8b 31.0bc 34.1b 26.8C 1.55

 

3Standard error

b’CMeans in the same row with different super-
scripts are significantly different (P<.05).

101

TABLE 14. EFFECT OF DIETARY SUPPLEMENTAL LEVEL OF VITAMIN
E ON SERUM ENZYME ACTIVITY (EXPERIMENT 2)

 

Vitamin E, IU/kg
Item 0 5 10 15 S.E.

 

 

No. of pigs 3 3 3 3
SGOT, S-F unitsb

 

Initial 39.0 58.0 36.7 44.3 6.1
Final 32.3 34.0 31.7 34.0 3.7
Pre-sacrifice 33.0 32.0 26.3 30.7 3.0

LDH, B-B unitsC

 

 

Initial 779e 1311f 8348 7958 124

Final 661 759 733 921 61

Pre-sacrifice 637 711 570 707 37
CPK, Sigma unitsd

Initial 22.7g 63.3h 21.7g 23.3g 7 3

Final 6.0 14.0 6.7 10.7 2.3

Pre-sacrifice 10.3 14.7 9.0 13.7 3.9

 

8Standard error

bSigma Technical Bulletin 505, 1964
CSigma Technical Bulletin 500, 1969
dSigma Technical Bulletin 520, 1971

e’fMeans in the same row with different super-
scripts are significantly different (P<.05)

g’hMeans in the same row with different super-
scripts are significantly different (P<.01)

102

This can be accounted for by the fact that 2 pigs assigned
to this treatment had very high LDH levels in serum.
Both of them being assigned to the same treatment was a
random chance occurrence. LDH values at the final bleed-
ing and prior to being sacrificed were unaffected by
supplemental level of vitamin E. Ewan and Wastell (1970)
stated that the activity of LDH enzyme of pigs fed diets
supplemented with vitamin E alone remained essentially
the same as the enzyme activity of blood of pigs fed both
vitamin E and selenium. Creatine phosphokinase (CPK)
enzyme activities initially were higher (P<.01) on the
5 IU vitamin E/kg treatment due to the two pigs which had
very high serum CPK levels that happened to be randomly
allotted to this treatment as discussed for LDH values.
These two pigs must have been in a marginal state of
selenium and/or vitamin E deficiency since they had
elevated levels of SGOT, LDH and CPK enzymes. However,
CPK levels at the final bleeding and prior to being
sacrificed were not affected by supplemental level of
vitamin E.

Tissue Se levels as influenced by supplemental
level of vitamin E are summarized in Table 15. Dietary
levels of supplemental vitamin E had no significant
effect on Se concentration of any of the tissues analyzed
on either a wet or dry basis. This observation may be

partially explanable since this experiment was only a

103

TABLE 15. EFFECT ON DIETARY SUPPLEMENTAL LEVEL OF VITAMIN
E ON TISSUE Se CONCENTRATIONS (EXPERIMENT 2)

 

Vitamin EJ IU/kg

 

 

 

 

 

 

Item 0 5 10 '15 S.E.a
No. of pigs 3 3 3 3
Quadriagps muscle
Sez ppm
Wetb .059 .058 .059 .057 .003
DryC .282 .290 .274 .266 .013
Myocardium Se, ppm
Wet .106 .128 .112 .132 .011
Dry .551 .653 .559 .654 .059
Liver Se,pppm
Wet .257 .273 .263 .259 .017
Dry .938 .990 .943 .915 .058
Kidney Se, ppm
Wet .988 .968 .874 .950 .085
Dry 5.334 5.008 4.622 5.171 .440

 

aStandard error

bppm Se on a fresh tissue basis

Cppm Se on a dry matter basis

104

13 day experiment and one might not expect to see signifi-
cant changes in tissue Se concentrations in this short
period of time. Groce at al. (1971, 1973b) reported that
supplemental vitamin E had no apparent effect on Se

levels of tissues except for an increase in kidney Se
levels. Ewan (1971) also reported similar observations

in that vitamin E had no significant effect on the level

of selenium in the tissues.

Summary

One feeding trial and one balance trial were con-
ducted to study the effects of supplemental Se and
dietary supplemental levels of vitamin E on performance,
hematology, tissue selenium and selenium balance in
growing-finishing pigs fed diets low in vitamin E and
selenium. Sixty-four weanling pigs were assigned to
eight dietary treatments in a 2 x 4 factorial design to
evaluate the effects of supplementing a basal corn-
soybean meal diet (0.054 ppm Se) with 0.1 ppm Se (as
sodium selenite) and vitamin E at 0, 5, 10 or 15 IU
per kg of diet. There were no lesions indicative of
vitamin E-selenium deficiency and no deaths due to the
deficiency were observed in this study.

No significant differences in overall performance
of pigs during this experiment could be attributed to
the dietary supplements of Se or vitamin E. Selenium

supplementation decreased (P<.05) 10 week hemoglobin

105
values by l g/100 ml. Blood hemoglobin level was
depressed (P<.01) when the diets were supplemented with
10 or 15 IU of vitamin E per kilogram of diet at the
final bleeding. Hematocrits of blood samples obtained
at the final bleeding were increased (P<.05) by dietary
supplements of 0 and S IU vitamin E/kg of diet over pigs
receiving 10 IU vitamin E/kg of diet. Selenium supple-
mentation increased (P<.01) serum Se concentrations,
while vitamin E supplementation had no effect on serum

Se concentrations. Selenium supplementation increased

 

(VF

(P<.01) rectus abdominus muscle, liver and kidney Se
concentrations on both a wet and dry basis. There was
a significant (P<.05) interaction between selenium and
vitamin E on muscle selenium concentrations on a wet
basis. Dietary level of vitamin E supplementation had
no significant effect on tissue Se concentrations.

The balance study was concerned with Se absorption
and retention on a selenium supplemented basal diet
(0.172 ppm Se) with dietary vitamin E added at levels of
0, 5, 10, or 15 IU vitamin E/kg of diet. Supplemental
levels of vitamin E had no significant effects on
selenium excretion or retention, serum Se, hemoglobin,
SGOT, LDH or CPK enzyme activities, or on Se concentra-
tions of quadriceps femoris muscle, myocardium, liver

or kidney on either a wet or dry basis.

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and finishing pigs. J. Anim. Sci. 34:969.

118

Welch, J. G., W. G. Hoekstra, A. L. Pope and P. H.
Phillips. 1960. Effects of feeding fish liver oil,
vitamin E and selenium to ewes upon the occurrence
of muscular dystrophy in their lambs. J. Anim.

Sci. 19:620.

Wretlind, B., K. Orstadius and P. Lindberg. 1959.
Transaminase and transferase activities in blood

plasma and in the tissues of normal pigs. Zbl.
Vet. Med. 6:963.

Wright, C. I. 1940. Effect of selenium on urease and
arginine. J. Pharmacol. Exp. Ther. 68:220.

Wright, P. L. and M. C. Bell. 1966. Cooperative
metabolism of selenium and tellurium in sheep and
swine. Amer. J. Physiol. 211:6.

APPENDICES

 

r

APPENDIX A
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 depending on the agility and
adeptness attained by the technician. There is a l-

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 much 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.

119

120

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 touching 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 mp

Slit widths:

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

Meter sensitivity 50

1| Jill. I .J u

121

Use the small round cuvettes. These are cleaned
with alcohol and dried with acetone between samples.
Periodically they should be cleaned with the cleaning
solution. 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
readings. 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 m1 separatory funnels with Teflon
stopcocks

l 250 ml separatory funnel with Teflon
stopcock

2 100 ml beakers

1 100 m1 graduated cylinder

1 10 m1 graduated cylinder

1 5 ml volumetric pipette for DAN solution

1 1 ml volumetric pipette for standards

122
l 2 m1 volumetric pipette for standards
1 25 m1 burette with Teflon stopcock
2 50 ml burettes with Teflon stopcock
l 250 ml burette with Teflon stopcock

l 10 m1 repipette to greatly speed the adding
of the water and EDTA solution.

The ground glass stoppers of the separatory funnels
must 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
problems of contamination. Once this glassware has been
initially 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
procedure is as follows: (1) Rinse at least 3 times in
flowing distilled water (use a segregated nylon brush
only if necessary - no soap). (2) Pour 250 ml of clean-
ing 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
water. (4) Invert and dry in a reserved section of the

drying room or a forced air oven. Whenever possible

 

 

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123
wash the items in this order: digestion flasks, Phillips

beakers, separatory funnels.

Reagents:
(l) Nitric acid - sp. gr. 1.42 - Redistilled from

glass-use lots of acid-washed boiling beads,
discard first portion of distillate and do not
boil 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).

(2) Sulfuric acid solution - Add 140 m1 selenium-
free H2804 - sp. gr. 1.85 - to deionized dis-
tilled water and dilute to 1 liter.

(3) Ammonium hydroxide solution — Dilute 400 ml
NH4OH - sp. gr. 0.90 - to 1 liter with
deionized distilled water.

(4) Disodium ethylenedinitrilotetraacetate solution
(EDTA) - 0.01M; dissolve 7.4450 g dihydrate in
water and dilute to 1 liter.

(5) 2,3-Diaminonaphthalene (DAN) solution.

Reagent from Aldrich Chemical Company, Milwaukee,
Wisconsin, 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

(6)
(7)

124

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. Note: The shaker

 

should be kept in the darkroom, and all

IMI. -

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 redis-
tilled 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 concen-
trated sulfuric acid (J. T. Baker) with an
equal volume of deionized distilled water.
Add 15 ml of reagent grade 48% hydrobromic

acid per 200 m1 of the mixture. Heat strongly

(8)

(9)

(10)

125

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 HNOS. 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.

Standard selenium solution -

Stock solution - Dissolve 100 mg selenium

metal (Alfa Inorganics - 99.999% pure) in 10

m1 concentrated redistilled nitric acid. Add
28.0 ml concentrated sulfuric acid and dilute
to 1 liter to give 100 ug Se/ml in l N sulfuric
acid. Store in cold room. To prepare a work-
ing solution dilute 1.0 ml of stock solution

to 1 liter with 0.1 N H SO4 to give 0.1 pg

2

Se/ml in 0.1 N H SO .

2 4
Hydrogen peroxide (30%) - Reagent grade (store

in cold room or refrigerator).

126

Procedure

 

Sample Preparation:

 

Grain, feed, feces and other dry samples should be
finely ground to insure good mixing and efficient
predigestion 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 homo-
genates 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 if allowed to
thaw slowly at room temperature. This rapid-thaw pro-
cedure works especially well with the Whirl-paks.
Sampling error seems to be the greatest source of
replication deviation with this method, so due care in

sample preparation is warranted.

Predigestion:

 

All samples (feeds, grains, tissue homogenates,

etc.) except strictly liquid samples (e.g., urine and

127

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 matter) along with 3 glass beads in a 100 ml Kjeldahl
flask. Add in sequence 6.0 m1 redistilled nitric acid,
2.0 ml 70% perchloric acid, and 5.0 ml H2504 (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 contami-

nation during the predigestion 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 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 heat-
ing elements. Heat carefully in initial stages to avoid

loss by foaming. Increase heat as digestion proceeds.

128

Turn the flasks 180° occasionally to "wash" down all
sides vdjfll condensate. Starting with cold heaters,

set rheostat at 5, for 20 minutes, increase to 7 for 20
minutes, then to 9 to finish. ,Take back to 7 for H20Z
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
replaced 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
closely. 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 solution 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 be for feeds and
grains. Replace flask on heater and continue 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

 

129

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 NOW!

Use a repipette to add 20 ml of deionized distilled
water to the digested material, carefully. Mix and
quantitatively transfer the contents of the flask to a
250 ml Phillips beaker using two 10 ml washings and

cover with a watchglass. After you have transferred all

130

12 samples to the Phillips beakers add in turn, with
mixing, 10 m1 disodium EDTA solution, 25.0 ml ammonium
hydroxide solution and 5.0 ml DAN solution. The time
one begins the DAN preparation 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 each reagent.
Boil the flask contents for 2 minutes on the hotplate.
Place on cart and roll into darkroom and let them sit
undisturbed in the 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
contents to a 125 m1 separatory funnel and add 6.0 ml
cyclohexane. Stopper with a Saran-wrapped rubber
stopper and shake for 15 minutes. Let stand for 5

minutes to allow phases to separate. Discard lower

131

aqueous phase and drain cyclohexane solution into 15 ml
centrifuge tube. Centrifuge for 5 minutes, place tubes
in a light 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 pg

selenium and passes through the origin consistently.

Additional notes:

 

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

It has been our experience that when fluorescent
contamination 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 enough to
work with, it should be carried through the rest of the
procedure. We have found that we cannot get consistent
results by digesting samples one day and working them

up the next.

132

In conclusion, I would say that this may seem like
a very lengthy treatment of a relatively simple procedure.
You were forewarned: This is not a routine or simple
procedure. You will spare yourself much agony and
frustration if someone familiar with the analysis walks

you through the first several times.

 

 

 

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