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PATHOLOGY 0F INORGANIC, AND ORGANIC.-
SELENIU‘M Toxmosus m YOUNG-SWINE '

Thesis for the Degree of Ph. Di
MICHIGAN STATE UNIVERSITY
RAWLIN. R. HERIGSTAD,
1972

 

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PATHOINI (I INOIBANIC AND 01mm

SWTQXICOSISINIOUMWE

presented by

hwlin R. Hangout!

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of the requirements for

_&.D.._ degree in m

Mdor professor

Datefl 7- 1972

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ABSTRACT
PATHOLOGY 0F INORGANIC AND ORGANIC SELENIUM

TOXICOSIS IN YOUNG SWINE

By

Rawlin R. Herigstad

A total of 47 young pigs and 6 yearling steers was used to
determine the toxicosis of sodium selenite and selenomethionine and
to compare the lesions produced by both selenium compounds with those
produced by vitamin E-selenium deficiency in pigs. In feeding experi-
ments using rations based either on Torula yeast or dried whole milk
as dietary protein sources, pigs were fed comparable graded amounts
of both selenium compounds in the ration.

In 1 yearling steer, l intravenous injection of 1 mg selenium/
kg body weight as sodium selenite produced death in 8 hours and
gross lesions of massive pulmonary edema, hydrothorax, and congestion
of the gastrointestinal tract, while 0.5 mg selenium/kg body weight
given intravenously into another steer had no effect in 4 days. In
both cattle and swine, intramuscular injections of a selenite solu—
tion resulted in localized muscular necrosis. Two pigs given 3 mg
selenium/kg body weight intravenously as sodium selenite or seleno-
methionine developed fatal selenium toxicosis in 2-1/2 and 14 hours,
respectively. Pulmonary edema was the important lesion of intravenous

selenite toxicosis. Twenty-one pigs fed 20 to 600 ppm selenium as

'.‘..

3;..-

262!

Rawlin R. Herigstad
sodium selenite or selenomethionine developed the clinical signs of
emesis, anorexia, loss of weight, cachexia, depression, respiratory
distress, and coma with subnormal temperature and death. The lesions
of selenosis were similar for both selenium compounds and included
fatty metamorphosis and centrolobular necrosis in the liver; con-
gestion of renal medulla; necrosis in lymphoid follicles; hemorrhagic
necrosis of adrenal cortex; edema and degenerative changes in the
cerebrum, cerebellum and spinal cord; edema and hemorrhagic necrosis
of pancreas; serous atrOphy of body fat and degenerative changes in
skeletal muscles. Lesions in the central nervous system were
generally more common with inorganic selenosis while pancreatic
lesions were associated with organic selenosis. Microangiopathy
was an important lesion of acute selenosis while degenerative
lesions and cachectic changes of a wide variety of organs character—
ized selenosis of longer duration.

The selenium content in liver and kidney, while variable, was
somewhat proportional to the level and duration of selenium in the
feed. The diagnosis of selenium toxicosis in swine depends on both
the nature of the lesions and selenium content of the liver and
kidneys. Selenium toxicosis can be differentiated from vitamin E-
selenium deficiency in swine by the characteristics of the hepatic

lesions.

PATHOLOGY OF INORGANIC AND ORGANIC SELENIUM

TOXICOSIS IN YOUNG SWINE

By

Rawlin R. Herigstad

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Pathology

1972

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and appreciation to my
friend and major professor, Dr. C. K. Whitehair, of the Department
of Pathology, for his patience, guidance, and encouragement through-
out my graduate training.

I am grateful to all members of the Department of Pathology.
The assistance and guidance of Dr. E. R. Miller of the Animal
Husbandry Department and Dr. J. T. Huber of the Dairy Department
were appreciated.

Particular appreciation is extended to Dr. V. L. Sanger of
the Department of Pathology for his gracious and generous assistance
with the ultrastructural studies and to Dr. 0. E. Olson and staff
of Experiment.Station Biochemistry Department, South Dakota State
University, for their interest and cooperation with the selenium
analyses.

During this research, I was supported through Postdoctoral
Fellowship No. 5 F02 AM46384-02(APA) awarded by the National
Institute of Arthritis and Metabolic Diseases, Bethesda, Maryland.

Most of all, I wish to express my sincere gratitude to my
wife, Chris Ann, and daughters, Kelly and Jamie, for their devoted
patience and encouragement during my graduate program and our stay

in Spartan Village.

11

TABLE OF CONTENTS
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . .‘. . 4
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . 4
Sources . . . . . . . . . . . . . . . . . . . . . . . . . 5
Industrial Uses . . . . . . . . . . . . . . . . . . . .'. 5
Metabolism. . . . . . . . . . . . . . . . . . . . . . . . 6
Absorption . . . . . . .
Intermediary Metabolism.

Retention. . . . . . . .
Excretion. . . . . . . .

10

selenium-DafiCiency Disease 0 o o o o o o o o o o o o o o 13

Selenium Toxicosis. . . . . . . . . . . . . . . . . . . . 14

Acute Selenium Toxicosis . . . . . . . . . . . .'. 16
Clinical signs and physiological data . . . l9
Lesions . . . . . . . . . . . . . . . . . . 20
Selenium content of tissues . . . . . . . 21

subacute Selenium Tbxicosis ("Blind Staggers' ') . . 22‘
Clinical signs. . . . . . . . . . . . .'. . 22

Lesions . . . . . . . . . . . . . . . . . . 22

Selenium content of tissues . . . . . . . . 23
Chronic Selenium Toxicosis . . . . . . . . . . . . 23
Clinical signs and physiological data . . . 25
Lesions . . . . . . . . . . . . . . . . . . 26
Selenium content in tissues . . . . . . . . 27

Factors Influencing Toxicosis . . . . . . . . . . . . . . 28‘
Selenium.and Human Health . . . . . . . . . . . . . . . . 30
Selenium and Cancer . . . . . . . . . . . . . . . . . . .' 31
Summary . . . . . . . . . . . . . . . . . . . . . . .-. . 31

OBJECTIWS O O O O O O O O O O O O O O O O O O O O O O O O O O '0 32

111

MATERIALS AND METHODS. . . . . . . . . . . . . .

RESULTS.

Animals, Facilities and Husbandry . . . .

Experimental Design . . . . . . .

Rations O O O O O O O O O O O O O O O O O

Selenium Compounds. . . . . . . . . . .-.

Experimental Parameters and Procedures. .

Postmortem Examination. . . . . . . . . .

AnAlYSIS Of Data. 0 o o o o o o o o o o 0

Preliminary Data. . . . . . . . . . . .

Experiments on Selenium Toxicosis . . .

DISCUSSION .

SUMMARY.

Growth, Duration and Amounts of Selenium

ClinicOpathologic Findings . . . .
Clinical Signs . . . . . . . . . .
Peracute selenium toxicosis
Acute selenium toxicosis. .
subacute selenium toxicosis
Hepatosis dietetica . . . .
Gross Lesions. . . . . . . . . . .

Peracute selenium toxicosis .

Acute selenium toxicosis. .
Subacute selenium toxicosis
Hepatosis dietetica . . . .
Histopathologic Changes. . . . . .

Peracute selenium toxicosis .

Acute selenium toxicosis. .
Subacute selenium toxicosis
Hepatosis dietetica_. . . .
Ultrastructural Changes. . . . . .
Selenium Content in Tissues. . . .

“FERENCES CITED 0 O O O O O O O O O O O O O O O

VITA .

iv

Page
34
34
35
38
38
41
42
44
45
45
48
49
52
58
59
6O
61
61
63
63
67

67
7O

' 7O
' 72

74
8O
81
81
84-
88
97
99

109

Table

10

ll

12

13

LIST OF TABLES

Allotment of pigs according to sex, selenium compound,
and selenium concentration of ration in Experiment A. .

Sex and allotment of pigs according to selenium compound
and selenium concentration of dry ration in Experiment B.

Composition of basal ration fed to pigs in.Experiment A .

Composition of dry basal ration fed to pigs in Experi-
ment B O O O O O O O O O O O O O O O O O O O O O O O O O 0

Summary of results using steers and pigs in preliminary
trials of selenium toxicity . .-. . . . . . . . . . . . .

Total amounts of selenium fed, days'en experiment,

Page

36

37

39

40

46

weights, and growth responses of pigs fed sodium selenite-

or selenomethionine at various selenium concentrations
in the ration in Experiment A . . . . . . . . . . . . .'.

Total amounts of selenium fed, hours on experiment,
weights, and growth responses of pigs fed sodium selenite
or selenomethionine at various concentrations in the

dry ration in Experiment B. . . . . . . . . . . . . . . .

Initial hemoglobins, hematocrits, total and differen—
tial leukocyte counts from pigs in Experiment A . . . . .

Final hemoglobins, hematocrits, total and differential
leukocyte counts from pigs in Experiment A. . . . . . . .

Range of results from selected determinations for
tocopherols in liver and tocopherol, OCT and urea
nitrogen in plasma from various groups of pigs at
initial and final periods during Experiment A . . . . . .

Range of results from.selected determinations for GOT,
OCT and urea nitrogen in serum from'various groups of
pigs at initial and final periods during Experiment B . .

Final weights of livers and kidneys expressed-as per-
centages of final body weights of pigs in Experiment A. .

Final weights of livers and kidneys expressed as per-
centages of final body weights of pigs in Experiment B. .

50

51

53

55

57

59

64

65

Table

14

15

Terminal selenium content in liver and kidney from pigs
fed selenium in Experiment A. . . . . . . . . . . . . . .

Terminal selenium content in liver and kidney of pigs
given selenium in Experiment B. . . . . . . . . . . . . .

vi

Page

85

86

1".

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Figure

10

LIST OF FIGURES
Page

Comparison of control Pig 5 and Pig 6 after 60 experi-
mental days. Notice smaller size, emaciation, rough

hair, tucked-up appearance of abdomen and cocked

appearance of hocks of Pig 6, in foreground, fed 45

ppm selenium as sodium selenite . . . . . . . . . . . . . 62

Pulmonary edema in a pig with peracute selenium
tOXicosis O O O O O O O O O O O I O O O O O O O O O O O O 66

Pale renal cortices and congested renal medullae from

a pig with selenium toxicosis. These were character-

istic but non-specific changes in pigs with selenium
toxicosis . . . . . . . . . . . . . . . . . . . . . . . . 66

Normal liver from control pig. This liver from Pig 133
had a selenium content of 0.21 ppm. . . . . . . . . . . . 68

Very pale liver of acute selenium toxicosis in pig fed
120 ppm selenium in Experiment B. This liver from Pig
13 had a selenium content of 5.62 ppm . . . . . . . . . . 68

Pale mottled liver of acute selenium toxicosis in pig

fed 60 ppm selenium in Experiment B. Compare with

Figures 4 and 5. This liver from Pig 153 had a selenium
content of 15. 9 ppm . . . . . . . . . . . . . . . . . . . 69

Subtly mottled liver of acute selenium toxicosis in

pig fed 45 ppm selenium in Experiment A. Compare with
Figures 4,5 and 6. This liver from Pig 20 had a
selenium.content of 14.6 ppm. . . . . . . . . . . . .'. . 69

Hepatosis dietetica in liver of pig with vitamin E-
selenium deficiency. Notice multifocal and multilobular
hemorrhages (dark areas) seen through Glisson's capsule . 71

Necrosis, edema and cellular lysis in lobular areas
in pancreas from.the pig given selenomethionine intra-
venously. , , , , ,

O O O I O I O O C C O O O I I O O O O 73

Fatty metamorphosis and centrolobular necrosis in liver
from pig with acute selenium toxicosis. . . . . . . . . . 73

vii

Figure Page

11 Comparison of the tips of cerebellar folia between con-
trol pig (top picture) and a pig fed 600 ppm selenium
as sodium.se1enite (bottom picture). Notice the loss of
stroma and granule cells in granular layer of cere-
bellum from pig with acute selenite toxicosis . . . . . . 76

12 Fibrinoid necrosis of wall of arteriole in white matter
of cerebellum from pig with acute selenium toxicosis. . . 77

13 Hemorrhagic necrosis of adrenal cortex from pig with
acute selenium toxicosis in Experiment A. . . . . . . . . 77

14 Comparison of normal bone marrow from control pig (top
picture) and aplastic bone marrow from pig with acute
selenium toxicosis lasting over 1 week (bottom picture).

Both sections were taken from the comparable areas of
ribs. Notice the loss of hematopoietic cells in the
bone marrow from pig with selenium toxicosis. . . . . . . 78

15 Degenerate myofibers in skeletal muscle from pig with
acute selenium toxicosis. . . . . . . . . . . . . . . . . 79

16 Hepatic lobule from pig with subacute selenium toxicosis.
Notice variation in size of hepatocytes, loss of sinusoidal
structure, and difference in staining of peripherolobular
and centrolobular hepatocytes . . . . . . . . . . . . . . 79

17 Necrotic, hemorrhagic lobules and normal adjacent lobule
in liver from.pig with hepatosis dietetica. . . . . . . . 82

18 Fibrinoid necrosis of small blood vessel with perivascular
hemorrhage in the molecular layer of cerebellum from
pig with hepatosis dietetica. Compare with Figure 12 . . 82

19 Hepatocyte from Pig 6 fed 45 ppm selenium as sodium
selenite for 63 days. Notice lipid bodies (arrows) in
cytoplasm C O O O O O _ I O O O O O O O O O O O O O I O I O 83

20 Hepatocyte from Pig 4 with hepatosis dietetica. Notice

osmiophilic lipid bodies (arrows) in cytoplasm. Compare
density of these lipid bodies with those in Figure 19 . . 83

viii

INTRODUCTION

Selenium as an element in animal and human nutrition has para-
doxical biological functions which caused Krehl (1970) to describe .
it as "one of the most maddening, frustrating nutritional minerals F5;
to examine in the entire table of elements." In most animals it is

highly toxic in relatively low amounts by toxicologic standards as

 

‘well as an essential element in trace amounts by nutritional standards. , Li
This knowledge of selenium in.animals has produced a dichotomy of
philosophy between its present use as an additive in animal feeds
and the future effects of its use on public health and the
environment.

WOrkers in South Dakota and Wyoming documented the toxicity of
selenium under natural and experimental conditions nearly 40 years
ago (Franks, 1934a; Rosenfeld and Beath, 1964). Selenium toxicosis
in the natural form primarily involved organic selenium compounds.
Selenomethionine was one of the principal compounds in seleniferous
feed grains (Allaway, 1968). Some question has existed in the
literature concerning the interaction of the components of natural .
foodstuffs with selenium during toxicosis (Muth and Binns, 1964).

Although the toxic effects of selenium, principally inorganic
selenium, have been recorded by many workers, detailed studies on.
the experimental and diagnostic pathology of selenosis in domestic

animals are very limited. Published information on the pathogenesis

2
of selenium toxicosis in swine was not available. In addition, no
experimental work using synthetic organoselenium compounds to produce
selenium toxicosis in swine was located. Moreover, in addition to
limited information on the pathologic effects of abnormal amounts
of selenium, the stigma of the possibility of selenium having carcino-
genic properties has been raised (Anon., 1970).

In 1957, selenium was discovered as the essential component of
"Factor 3" which prevented dietary hepatic degeneration in rats on
a diet of Torula yeast (Schwarz and Foltz, 1957). Since that time
numerous seleniumrresponsive diseases have been described in domestic
animals and man (Hartley and Grant, 1961; Majaj and Hopkins, 1966;
NASANRC Monograph, 1971). In fact, selenium has now been identified
as an essential trace mineral element in cattle, sheep, swine, horses,
chickens, turkeys, quail, dogs and subhuman primates (NAS-NRC Mono-
graph, 1971; Muth et aZ., 1971). Presently, there is an effort in
the scientific community to convince the Food and Drug Administra-
tion to approve selenium as‘a feed additive for swine and poultry
(NAS-NRC Monograph, 1971; Frost, 1971; Frost, 1972).

Although vitamin-E-selenium responsive diseases are more common
today than natural selenosis, occasions of inadvertent selenium
toxicosis have been reported in sheep and cattle following the
therapeutic use of selenium (Morrow, 1968; Lambourne and Mason,

1969; Gabbedy and Dickson, 1969; Shortridge et al., 1971).

If or when.se1enium becomes approved as a feed additive in the
United States, it can be.specu1ated that a concern or need for more
reliable information on.selenium toxicosis not only in swine but
also in.other species of animals will be desired. In addition, if

the widespread use of selenium is permitted in animal feeds, more

3
information is needed on the impact of this development upon the
environment and upon public health and agronomic practices. In
the past the detrimental effects of new drugs and chemicals, such
as mercury and DDT, on the environment and public health were not
fully realized until after many years of extensive use. Thus, there
is public concern, for good reasons, about the widespread indiscrimin-
ate use of potentially toxic chemicals in our food supply and
environment.'

While considerable information is already available on the
pathology of vitamin E-selenium deficiency in a variety of species,
information on the pathology of selenium toxicosis would be valuable
in differentiating these 2 syndromes and might also provide suggestions
as to the biological role of both vitamin E and selenium at the
cellular level.

Thus, additional research information on selenium toxicosis,
not only as it applies to swine, but also other animal species and
public health as well, would seem essential. It would serve as an
important informational resource to the livestock industry, the

biomedical professions and the public welfare.

 

‘13.-

REVIEW OF LITERATURE

The literature on the biological.aspects and agricultural impli-
cations of selenium has been extensively reviewed in several books
(Trelease and Beath, 1959; Rosenfeld and Beath, 1964; Muth et al.,
1967; Mills, 1970; Underwood, 1971; NAS-NRC Mbnograph, 1971) and
review articles (Painter, 1941; Moxon and Rhian, 1943; Schwarz,
1961).

This review of the literature is limited primarily to selenium
toxicosis in animals. Only a fundamental understanding of the
chemistry, sources, industrial uses, and metabolism of selenium is
presented. In addition, the general lesions of vitamin E-selenium

deficiency disease are reviewed.

Chemistry

Baron Jons Jakob Berzelius, a Swedish chemist, discovered
selenium in 1817 and named it after Selene, the Greek goddess of the
moon (Ageton, 1970). Selenium has the atomic number 34 and an
atomic.weight-of 78.96. It is found in naturally occurring materials
such as rocks, soil, plants, and manure in 4 different oxidation
states or valences: -2, the selenides; O, elemental selenium; +4,
the selenites; and +6, the selenates. Elemental selenium and the
selenates tend to resist oxidation or reduction and theoretically
are more stable in a given environment than selenides or selenites.

The selenites as ready electron acceptors are reduced to elemental

"" ‘.'h.r-_‘I

 

5
selenium or organic selenides, while the organic selenides as electron
ddnors are oxidized to those selenium compounds with higher valences
(Allaway, 1968). The many forms of selenium have a complex environ-
mental cycle .

In the periodic table of elements, selenium is in a group VI A
below sulfur. Selenium, sulfur, arsenic and phOSphorus are grouped
together and have some similar chemical and biochemical prOperties.
The organic chemistry of selenium resembles the organic chemistry
of sulfur except that oxidized forms of selenium are more readily
reduced in living systems and organic selenium compounds are less
stable than their sulfur analogs. Many forms of selenium are volatile
at low temperatures which complicate chemical analysis of feeds and

animal tissues (Allaway, 1968).

Sources
Selenium is estimated to be the 69th most abundant element in
the earth's crust (Ageton, 1970). It is rarely found in the elemental
state. This element is associated with sulfide minerals of copper,
iron, lead, and other metals. In the United States, it is mainly
obtained as a byproduct of electrolytic copper refining. High—purity
grades (99.95 to 99.99% selenium) and commercial grades (97 to 99.94%

selenium) of material are used in industry.

Industrial Uses

 

According to a publication of the U.S. Department of Interior
(Ageton, 1970), the current most important use of selenium is in
flat-glass, pressed or blown glass and glassware industries which
accounted for about 272 of the 1968 demand. Selenium removes color

impurities in.g1ass. Equipment for distribution of electrical power

6
such as specialty electrical transformers, semiconductors and related
devices used approximately 232. Another 23% is utilized in xerographic
duplicating machines. The orange-red-maroon pigment, cadmium sulfo-
selenide, which is light stable and heat and chemical resistant,
accounted for another 14% of the 1968 selenium demand. Other uses
of selenium included applications in the manufacture of stainless
steel and in the preparation of pharmaceuticals, such as niacin,
cortisone, fungicides and deodorants. Compounds of selenium are
also used as accelerators and vulcanizing agents in rubber products.
Fat products are hardened by a selenium catalyst. Selenium has had
application in lubricating oils; in extreme-pressure lubricants; in
vegetable oils for painting and printing; in photographic photosensi-
tizers and toners; and in mercury vapor detectors, fireproofing

agents, insect repellents, phosphorescents, and luminescents.

Metabolism

 

The true metabolic function of selenium as a toxicant and as
an essential micronutrient is unknown. Since selenium is present
in varying amounts in all tissues of the body, the true metabolic
function of selenium is probably very complex. Several theories
have been proposed with the initial ones intimately coupled to
vitamin E metabolism. Tappel (1965) demonstrated that selenium was
a scavenger preventing free-radical lipid peroxidation damage similar
to the antioxidant effect of vitamin E. Schwarz (1965a) prOposed
that the dehydrogenases of the tricarboxylic acid cycle, especially
lipoyl dehydrogenase, was the site of inhibition during selenium
and vitamin E deficiency. Although selenium has not been demonstrated

to be a cofactor to this enzyme, it may protect the enzyme in a

7
reduced state.(NAséNRC Monograph, 1971). Diplock at al. (1971),
who no longer regard the antioxidant theory of vitamin E and selenium
as viable, suggest that the active form of selenium may be selenide
and that the selenide may form part of the active center of an unchar-
acterized class of catalytically active non-heme-iron proteins that
are protected from oxidation in vino by vitamin E. Rotruck et al.
(1972) recently suggested that selenium was an integral part of
reduced glutathione peroxidase which catalytically prevents hydrogen
peroxide- or ascorbic acid-induced oxidation of hemoglobin. Thompson
and Scott (1970) indicated that selenium was important in pancreatic
metabolism and lipase production since pancreatichibrosis and
impaired vitamin E absorption resulted in chickens on a purified
selenium-deficient diet. Ganther (1966) reported the in vitro
enzymic synthesis of dimethyl selenide from sodium selenite in
mouse liver extracts. In 1969, Ganther and Corcoran (1969) cone
cluded that selenium was incorporated between 2 sulfur atoms to form
an intramolecular selenotrisulfide linkage in place of the disulfide.
Some of these theories on the chemical and biochemical reactions
and functions of selenium are fundamentally diverse, and it appears
that the true function of selenium will be controversial for some
time to come.

Selenium from plants is usually in the organic form (Painter,
1941) which is apparently present mainly in analogs of the common
sulfur amino acids: selenocystine, selenocysteine, selenomethionine
and selenocystathionine (NAS-NRC MOnograph, 1971). Allaway (1968)
stated that in feed grains most of the selenium is present in the
analog of methionine which is incorporated into proteins of the

plants. The other organic forms of selenium along with some

8
selenate are more commonly found in selenium-accumulator plants:
Astragulus (milk vetch), Xylorrhiza (woody aster), OonOpsis (golden
weed), and Stanleya (prince's plume) species (Rosenfeld and Beath,
1964). Synthetic organic selenium.compounds have only recently
become available commercially. Thus, most of the experimental
studies have used natural feedstuffs with a highly variable selenium
content or purified inorganic selenium salts (Rosenfeld and Beath,

1964; NASéNRC Manograph, 1971).

Absogption

 

Inorganic selenium compounds are synthesized into selenoamino
acids by rumen microorganisms (Rosenfeld, 1962). There is a sug-
gestion that selenite could be reduced to an insoluble unavailable
form by the rumen microorganisms which is considered to be an.important
factor in the susceptibility of ruminants to selenium deficiency
disease (wright, 1967).

The amount of selenium absorbed from the intestine, and its
retention and distribution within the body vary with the species
and with the chemical form and amount of the element ingested
(Underwood, 1971). Selenium compounds are absorbed from the distal
4/5 of the small intestine (Wright, 1967). Using an isotope of
selenite-selenium and a chromium marker, Wright and Bell (1966)
found that the net absorption was approximately 35% for sheep and
852 for swine when the respective rations contained 0.35 and 0.50
ppm.se1enium. Ehlig et al. (1967) compared the absorption of
selenium as selenite or selenomethionine in lambs and reported no
difference when 1 ppm selenium was fed. Direct data on the absorp-

tion of toxic doses of selenium are limited.

9

The mechanism of absorption for ionic selenium has not been
reported, but L-selenomethionine is actively transported across
the intestinal mucosa similar to and in competition with methionine
(MCConnell and Cho, 1967).

Jenkins et al. (1970) indicated that absorbed 75Se is carried
in_the alphaz- and alpha3-globulins of plasma within 2 hours after
a small dose.and also in the albumin fraction of plasma after a
large dose. This albumin activity with the large dose dropped to
the level of the low dose after 28 hours. The selenium from the

small dose was found with the alpha - and gamma-globulin fractions

2
while the seleniumtwith the large dose was inside the red blood

cells associated with globin after 24 hours. This agreed with the
work of Imbach and Sternberg (1966), who found that,3 minutes after
intravenous injection of 75Se isotOpe from selenite into rats, 50%

of the selenium was attached to the precipitable serum proteins
suggesting a surface adsorption to the serum proteins. After absorp—

tion,se1enium is widely distributed in all tissues with the largest

amounts in liver and kidney (Smith at al., 1937b).

Intermediary’Metabolism

Cummins and Martin (1967) found no pathway for in viva synthesis
of selenocystine or selenomethionine from selenite in rabbits. From
the in vitro studies of Ganther and Corcoran (1969), inorganic
selenium compounds are probably firmly bound to sulfur compounds or
amino_acids in the form of selenotrisulfides although a previous
report of McConnell (1963) suggested that selenium.was present in
animal proteins as selenoamino acids. Recently, McConnell and

Hoffman (1972) concluded that selenomethionine is incorporated into

10

polypeptides of enkaryotic cells via the methionine pathway. Awwad
et al. (1967) had demonstrated the in viva convertibility of seleno-
methionine to selenocystine and selenocysteine. In addition to the
above reports, Imbach and Sternberg (1966) found that 75Se isotOpe
from selenite was chiefly in the microsome fraction (65-76%), while
the mitochondria and nuclei averaged 11 and 14%, respectively, in
hepatocytes. This suggested the integration of the isotope into the
ribosomal phase of protein synthesis. Also, 75Se-methionine in the

liver had a more rapid decrease than 75Se from selenite which was

probably related to the elaboration and release of 75Se-labeled
proteins. Thus, it would seem that there are 2 pathways for selenium
metabolism and both involve protein metabolism. One pathway is used
by selenium from inorganic compounds and forms selenotrisulfide
complexes while the other incorporates sulfur-substituted seleno-
amino acids into polypeptides. Further work similar to that of

Ganther (1966) on the enzymic synthesis of dimethyl selenide may

identify the difference between these 2 pathways.

Retention

Underwood (1971)-reviewed data indicating that selenium is more
efficiently retained from selenium-deficient diets than from
seleniumesupplemented diets in several species. This indicates an
adaptive reaction of the animal to the selenium content of the diet.
In addition to this inverse relationship between the percent of
selenium retained in the body for diets of low and high selenium
content, Hopkins et al. (1966) found that the percent of selenium
retained in the liver was relatively constant and did not depend

upon the level of selenium fed. This observation suggested a

 

 

ll
metabolic difference for selenium between the liver and the rest
of the body.

A higher percentage of ingested selenium is retained from
natural organic compounds than from inorganic selenium salts (Smith
et al., 1938). Ehlig at al. (1967) confirmed this observation in
lambs supplemented with 1 ppm selenium as sodium selenite or seleno-
methionine in their feed. Thirteen days after initiation of differ-
ential treatments, tissues from lambs fed selenomethionine had
significantly more selenium than tissues from lambs fed sodium

selenite.

Excretion,

Uhderwood (1971) reviewed reports which indicated that selenium
excretion varies with the species, diet fed, and previous selenium
status. Selenium is excreted primarily by 3 pathways: urine (50-80%),
feces (10-20%), and expired air (10-30%) (Smith et al., 1937b;

Ganther et al., 1966). Urinary excretion is the predominant pathway
of elimination in non-ruminants when physiologic levels of selenium
are fed, but selenium excretion in feces and expired air markedly
increases when toxic levels are fed. Byard (1969) identified trimethyl
selenide as the major excretory product which comprised 60% of

urinary selenium in rats fed subtoxic doses of selenium. Palmer

et a1. (1969) found essentially the same result and termed their
compound "trimethylselenonium", which accounted for 30 to 50% of
urinary selenium in rats injected with selenite. Imbach and

758a from selenite and

Sternburg (1966), using trace amounts of
biliary cannulation in rats, demonstrated that 15.6% of the isotOpe

was excreted in the bile in 3 hours, but there was a significant

12
reabsorption of the isot0pe through an enterohepatic cycle. Ruminants
have a tendency to excrete more of the ingested selenium through the
feces than do non-ruminants. The selenium.compounds excreted in
feces have not been characterized and are generally considered to
be predominantly elemental selenium from reduction by the intestinal
microflora. The selenium product in expired air has been character-
ized as dimethyl selenide (McConnell and Portman, 1952). Ganther
at al. (1966) reported that selenium volatilization can be increased
by increasing the selenium content of the basal diet, by increasing
the protein and methionine content of the basal diet, or by feeding
the appropriate crude materials. Ganther (1966) reported the in
vitno enzymic synthesis of dimethyl selenide from sodium selenite
in mouse liver extracts which is probably an important pathway in
selenium detoxification and metabolism.

Rosenfeld (1964) reviewed data on selenium excretion after
administering toxic doses and concluded that: (1) there was a limit
to-the amount of ionic selenium that can pass through the urinary
tract; (2) administration of toxic doses of selenium reduced the
urinary.excretion with subsequent increase in the feces; (3) the
interference with excretion was probably due to functional damage
to the kidneys; (4) the elimination of selenium by the respiratory
tract increased with increased selenium administration; probably
there was a threshhold that limited further increase in selenium
elimination by the respiratory tract; (5) the selenium that was
retained by tissues and entered in the various metabolic pathways
subsequently was eliminated in the feces or reabsorbed from the

intestinal content for a considerable length of time.

 

13
SeleniumrDeficiency Disease

Selenium and vitamin E have a complex and somewhat unexplained
interrelationship. In some cases the lesions of simple selenium
deficiency have been experimentally produced in animals with adequate
vitamin E and other nutrients in their diet, while in other cases
animals have had natural nutritional diseases which responded to
selenium therapy without any additional treatment. ~Some of the
manifestations of selenium deficiency which fall into one or the
other of the above classifications include: dietary liver necrosis,
poor growth, and alopecia in rats; multiple necrotic degeneration
in mice; hepatosis dietetica, edema and "nutritional muscular
dystrOphy" in swine; exudative diathesis, poor growth, poor feather-
ing, and fibrotic degeneration of the pancreas in chicks; gizzard
myopathy in turkey poults; generalized corporal wasting in Japanese
quail; and white muscle disease of the skeletal and cardiac muscles
and growth.and reproductive inefficiency in ruminants (NAS-NRC
Monograph, 1971). Although the status of selenium in human nutrition
is unestablished, some reports have stated that a low level of
selenium therapy resulted in a favorable response in children with
kwaahiorkor (Schwarz, 1965b; Majaj and Hopkins, 1966).

Trapp et al. (1970), at Michigan State University, outlined the
clinical manifestations and pathology of vitamin E-selenium deficiency
in swine. Sudden death of thrifty feeder pigs was the most character-
istic history. Some pigs had been observed with clinical signs of
icterus, weakness, and reluctance to move. The gross lesions were
multifocal lobules of hepatic necrosis and hemorrhage; bilateral
paleness of skeletal muscles; and edema of the lungs, submucosa of

the stomach, serosa of the spiral colon, and subcutaneous tissues.

 

14

The histopathologic changes in the liver were characterized by cyto-
plasmic and nuclear degeneration and lysis throughout an entire
lobule with massive intralobular hemorrhage. Adjacent lobules often
appeared normal. The microscopic.lesions in the skeletal muscles
varied from Zenker's necrosis, possibly with mineralization, to a
loss.of myofibers and an accumulation of mononuclear cells. Micro-
angiopathy of arterioles in affected organs was observed. These
findings were similar to earlier experimental results of Michel et
a1. (1969).

The suggested requirement for selenium as a.feed additive is
0.1 ppm in swine. Muth and Binns (1964) reported that this dose of
selenium in sheep was somewhere in the order of 1/50 to 1/75 the
minimum.lethal dose when fed in daily oral doses. Earlier Schwarz
(1960) suggested that the ratio between effective dose and toxic
dose based on selenium.from sodium selenite was in the order of
1:100. He was working with rats which are the species most

resistant to selenium toxicosis (Smith at aZ., 1937a).

Selenium Toxicosis

Moxon (1937) reviewed the early history of selenium toxicosis.
In the story of his travels, Marco Polo may have been the first to
describe selenium poisoning in his beasts of burden while traveling
through the mountains of western China. In 1856, Dr. T. C. Madison,
an Army surgeon, wrote a report describing a malaise in cavalry
horses at Fort Randall, Territory of Nebraska. This is credited as
the first authentic report of selenium poisoning although the toxic
factor was undetermined until Robinson (1933) isolated selenium from

toxic feedstuffs. The areas of south central South Dakota and

15
adjacent northern Nebraska which are just west of the Missouri
River and_parts of eastern wyoming have the proper conditions to
grow highly seleniferous vegetation. Early settlers in these areas
had devastating losses of livestock which prompted workers from the
South Dakota Experiment Station and United States Department of
Agriculture to begin a joint effort in 1931 to identify, character-
ize, and answer the agricultural problem that existed. In addition
to these researchers, workers at the Wyoming Experiment Station
studied poisonous plants that were later shown to have a high
content of selenium.

Several potentially toxic minerals, selenium, tellurium,
arsenic, vanadium, and molybdenum, were investigated by the South
Dakota workers. In 1934 K. W. Franke and others began a.series of
papers entitled "A New Toxicant Occurring in Certain Samples of
Plant Foodstuffs" which incriminated selenium as the toxic agent
after Robinson (1933) had identified the presence of selenium in
toxic wheat. It was not until later that Franke and Painter (1938)
were willing to assume that selenium was the sole toxicant in
seleniferous cereals. More recently the question of unidentified
phytotoxins as one of the many complicating factors in selenium
poisoning was raised again (Muth and Binns, 1964).

Selenium poisoning has been divided into 3 forms in livestock:
acute, subacute (blind staggers) and chronic (alkali disease)
(Rosenfeld and Beath, 1964). Experimental selenium toxicosis has
not always produced the same syndrome as under natural conditions
because of the many modifying factors involved (Muth and Binns,

1964). It was extremely difficult to determine the toxic dose of

 

l6
selenium from the literature because the experimental methods,
selenium compounds used, and methods of reporting results have been

so varied.

Acute Selenium Toxicosis
In an early report Franke and MOxon (1936) found that the

intraperitoneal LD in 48 hours for rats was 3.25 to 3.50 mg

75
selenium per kg body weight for sodium selenite and 5.25 to 5.75

mg selenium per kg body weight for sodium selenate. By subcutaneous,
intraperitoneal, or intravenous injection, the minimum lethal dose
of sodium selenite expressed as mg selenium per kg body weight was
from 3.0 to 5.7 for rats, 0.9 to 1.5 for rabbits, and 1.5 to 2.0

for the dog (Smith et aZ., 1937a; Painter, 1941; Anderson and Moxon,
1942). In studies on the toxicity of intravenous sodium selenite,
Heinrich and MacCanon (1957) reported that the 24-hour LD50 in dogs
was between 0.875 and 1.0 mg selenium per kg body weight; the 24-hour
LD100 was between 1.0 and 1.5 mg selenium per kg body weight. From
the conclusions of Franke and Painter (1938), the relative toxicity
of selenium in the different diets fed to rats was in the order:
wheat.> corn > barley > selenate > selenite. Since the natural
forms of selenium were organic, Moxon et al. (1938) studied the
toxicity of B selenodiprOpionic acid, n-prophylseleninic acid,

8 diselinopropionic acid, BB'diselenodipropionic acid and
dibenzyldiselenide and found each of'them to be much less toxic

than selenite to rats. In a subsequent paper, Moxon et al. (1941)
reported that the toxicity of L-selenocystine was similar to selen-
iferous wheat while D-selenocystine was less toxic to rats. In 1949

Klug et al. found that the 48-hour LD for rats was 4 mg selenium

75

17
per kg body weight for selenocystine and 4.25 mg selenium per kg
body weight for selenomethionine. In addition,.se1enocystine was
very toxic to the liver succinoxidase system while selenomethionine
was relatively nontoxic.

Toxicity studies with farm animals are more limited than those
with laboratory animals. The acute toxic oral dose of sodium
selenite in 24 hours calculated as mg selenium per kg body weight g»
was reported as 8.4 to 11.0 for cows, 3.3 for horses and mules, and

13.2 to 17.6 for pigs (Miller and Williams, 1940a; Anderson et aZ.,

.n .b-‘.”.‘

1961). Diener (1961) confirmed the minimum lethal oral dose of

"‘
'rfi‘.

 

sodium selenite reported by Miller and Williams (1940a) and found

q.

‘5

that 17.6 mg selenium per kg body weight killed young pigs in 48

hours. Orstadius (1960) injected sodium selenite subcutaneously

into pigs weighing 30 to 70 kg at single dosages of 2.0 and 1.2 mg ..
selenium per kg body weight, and the pigs died after 4 hours and 5

days, respectively. Dosages of 0.9 to 1.1 mg selenium per kg body

weight.caused no clinical signs of toxicosis. In a report on

selenium toxicosis in calves resulting from an error in preparing

a therapeutic solution of selenium, Shortridge et a1. (1971) found

that one subcutaneous injection of sodium selenite at a dosage of

approximately 0.5 mg selenium per kg body weight was lethal within

24 hours for 18 of 557 stressed calves. This single injection of ‘
selenium resulted in 376 (67%) deaths in these 557 calves over a

deeek period. Eighty percent of the heifers which received both

a brucellosis vaccination and an injection of selenium died, come

pared to 56% of the steers which only had the injection of selenium.

Kuttler at al. (1961) used sodium selenite suspended in peanut oil

containing 2% beeswax which was injected subcutaneously into sheep

 

18
at levels of 0.44, 0.88, 1.76 and 3.3 mg selenium per kg body
‘weight. TWO sheep receiving 3.3 mg selenium per kg body weight and
1 sheep receiving 1.76 mg selenium per kg body weight died in less
than 24 hours. Muth found that 11 of 12 ewes tolerated 3.73 mg
selenium per kg body weight when fed as sodium selenite, 12 of 12
ewes died from 5.03 mg selenium per kg body weight when injected
subcutaneously in a sodium selenite-peanut oil-beeswax suspension, r—
and 12 of 12 ewes died from 0.66 mg selenium per kg body weight
when injected subcutaneously as an aqueous sodium selenite solution

(Muth and Binns, 1964). Binns reported that 8 sheep fed 0.33 to

 

1.15 mg selenium per kg body weight per day as sodium selenite ‘ ‘ EV
remained normal through 90 days, but 2 sheep fed 0.33 mg selenium

per kg body weight per day from seleniferous Astragalus prussii

died on the 5th and 6th day, respectively (Muth and Binns, 1964). o.

In 2 reports, Caravaggi et al. (1970a, 1970b) found that the L050 1
in lambs was 0.455 mg selenium.per kg body weight when sodium
selenite was administered as a single intramuscular injection and
was 1.9 mg selenium per kg body weight when sodium selenite was.fed
as a single oral dose. Neethling et a1. (1968) observed that 4 mg
selenium (as selenium dioxide) per kg body weight when administered
intravenously was fatal to 2 adult sheep in 20 minutes and that 3.4
mg selenium (as selenomethionine) per kg body weight when administered
intravenously was fatal in approximately 10 hours. All reports

have indicated.that metallic selenium is non-toxic because of its
insolubility. Young animals are moresusceptible to acute selenium

toxicosis than are adults when dosages based on body weight are used

(Gabbedy and Dickson, 1969; Shortridge et al., 1971).

19

Clinical signs and physiolggical data. The clinical signs and
physiological data are primarily limited to the dog (Heinrich and
MacCanon, 1957; Heinrich and MacCanon, 1960; Heinrich-and MacCanon,
1961; Heinrich, 1963). Acute selenium toxicosis was characterized
by vomiting, diarrhea, apprehension, respiratory stimulation often
accompanied by rales, and cardiovascular changes such as arrhythmias
and weak, irregular pulse; all of these clinical signs became more
pronounced with increasing dosage. Physiological data after the
intravenous administration of 2.0 mg selenium (as sodium selenite)
per kg body weight included a decrease in systemic blood pressure
and peripheral resistance, an increase in pulmonary arterial pressure
and resistance, and an initial increase and subsequent depression
of cardiac output. Electrocardiographic changes indicated cardiac
damage. Increased minute volume and decreased tidal volume char-
acterized the respiratory stimulation. The significant changes in
the blood components were an increase in hematocrit and plasma
protein, potassium, lactate, and pyruvate, and a decrease in venous
pH. From these studies it was concluded that functional circulatory
failure is a more plausible reason for the cause of death in acute
selenium toxicosis rather than respiratory failure, as reported in
the earlier literature (Death et al., 1935; Moxon and Rhian, 1943).
Previously, Anderson and Moxon (1942) had characterized the changes
in blood from dogs with acute selenium poisoning. These were sum-
marized as a marked increase in hemoglobin and hematocrit; decrease
in inorganic phosphorus, non-protein nitrogen, calcium, ascorbic
acid, and blood sugar; and reduction in the glutathione content of

the blood.

20

In the economic farm.animals, the general clinical signs of
acute selenium toxicosis have been the rapid onset of hyperpnea,
dyspnea, cyanosis, salivation, depression, tremors, hypersensitivity
to external stimuli, normal to slightly elevated temperature,
Opisthotonos, paresis, convulsions, coma, and death (Miller and
Williams, 1940a; Neethling et aZ., 1968; Shortridge et al., 1971).
In addition, vomition was reported in swine. Schneider (1936) F“
cited Gmelin (1824) and credited him with the identification of
a characteristic garlicky odor on the breath of seleniumepoisoned

animals. This observation has been established in the literature

 

“t:

(Moxon and Rhian, 1943) but has been peculiarly omitted from the
more recent reports (Glenn at al., 1964a; Morrow, 1968; Neethling

et al., 1968; Caravaggi at al., 1970a; Shortridge et al., 1971).

Lesions. Descriptions of the gross morphologic lesions of
acute selenium toxicosis in domestic livestock and laboratory animals
have been reviewed (Moxon and Rhian, 1943; Rosenfeld and Beath,
1964) and have recently been reported for sheep and cattle
(Caravaggi et-aZ., 1970a; Shortridge at al., 1971), but the reports
of toxicosis in swine are limited (Miller and Williams, 1940a;
Diener, 1961). The gross lesions are summarized as:» numerous
petechial hemorrhages on serosal surfaces, congestion of the mesen-
teric blood vessels, hemorrhages and edema in the lungs, hydro-
thorax, subendocardial and subepicardial hemorrhages, enlargement
of the heart, enlarged and pale yellow liver, congestion of lymph
nodes, enlarged kidneys, congestion of the renal medulla, distention
of the urinary bladder, hemorrhagic enteritis, and congestion of

adrenals and pancreas. Rosenfeld and Beath (1946a) indicated the

21
general picture in acute selenosis was hemorrhages and smooth muscle
atony, but more recent reports (Diener, 1961; Gabbedy and Dickson,
1969; Caravaggi et al., 1970a; Shortridge et aZ., 1971) emphasized
the histopathologic changes of fatty metamorphosis and necrosis in
liver and kidney as well as myopathy of the heart and skeletal
muscles. Glenn et al. (1964b) reported that the most severe and
consistent lesions of selenate toxicosis in sheep were focal to
diffuse myocardial necrosis and fibrosis, and edema and interstitial

hemorrhages in the lung.

Selenium content of tissues. Dudley (1936) noted that in animals
fed inorganic or organic selenium compounds the selenium was found
to be distributed throughout the entire carcass but in widely vary-
ing proportions. The liver, kidneys and spleen from animals with
selenium poisoning had the greatest concentrations of selenium,
ranging from 4.0 to 25.0 ppm. When lambs were drenched with an
oral dose of 6.4 mg selenium (as sodium.se1enite) per kg body weight,
Gabbedy and Dickson (1969) reported that the hepatic concentrations
of selenium, expressed on a dry weight basis, in lambs dying within
2 days averaged 64 ppm, and in those dying 15 days later hepatic
and renal concentrations of selenium averaged 26 ppm and 7.4 ppm,
respectively. Shortridge at al. (1971) found that 2 calves dying
in 48 to 72 hours had hepatic concentrations of selenium on a wet
basis between 1.82 and 3.11 ppm and renal concentrations between
1.57 and 2.09 ppm, while a calf dying 1 month later had hepatic and

renal concentrations of 0.76 and 0.89 ppm, respectively.

22
Subacute Selenium Toxicosis ("Blind Stgggers")

Although selenium toxicosis from inorganic selenium salts and
organic selenium compounds may be manifested in all stages from
acute to chronic, "blind staggers" was thought to be the result of
more modifying factors such as phytotoxins than any of the other
syndromes involving selenium toxicity (Muth and Binns, 1964).
"Blind staggers", a disease of cattle and sheep, was an inapprOpriate
lay term that has been perpetuated especially by Beath and co-
workers (Draize and Beath, 1935; Rosenfeld and Beath, 1946a;
Rosenfeld and Beath, 1964). This type of poisoning results when
moderate amounts of seleniferous plants have been consumed over a
period of time. The amount of selenium consumed has been highly

variable or virtually undetermined.

Clinical siggg, Rosenfeld and Beath (1964) reviewed 3 stages
of "blind staggers." In the early stage the affected animal wanders,
circles, and disregards objects in its path as if it were partially
blind. Anorexia is present. The body temperature and respiration
are normal. The second stage is characterized by weakness and col—
lapse of the front legs without other evidence of paralysis. The
third stage is paralytic with dysphagia, amaurosis, dyspnea,
abdominal pain, subnormal body temperature, and subsequent death.

The exact cause of death remains unknown.

Lesions. The gross pathologic changes of "blind staggers"
were described by Draize and Beath (1935) and Rosenfeld and Beath
(1946a). This condition is characterized by congestion of the
visceral blood vessels and lymph nodes, hemorrhages in heart and

gastrointestinal tract, stony of the gastrointestinal tract,

 

23
urinary bladder and gallbladder, congestion of the renal medulla,
focal necrosis of liver with cirrhosis, and variable erosions on
the articular surfaces of long bones.

Histologically the changes were fibrosis, congestion and
necrosis of the myocardium with serous atrophy of fat around the
coronary vessels; thickened walls of alveoli in the lungs; fatty
metamorphosis, necrosis and fibrosis in the liver; glomerulonephritis
and tubular degeneration in the kidney; hyperplasia, fibrosis, and
congestion of the spleen; epithelial desquamation, hemorrhages and
frequently ulcerations in the gastrointestinal tract; and necrosis

of the anterior pituitary gland.

Selenium content of tissues. The selenium content of tissues
in the terminal stages of "blind staggers" has been directly related
by Rosenfeld and Beath (1964) to the duration of life of the animal.
If death occurred soon after the disease developed, the concentra-
tions of selenium‘were.high in all tissues. When the disease was
prolonged, the selenium content was considerably lower; the kidney,
liver, and blood contained 2.0 to 5.0, l to 8, and 1.5 to 4 ppm

selenium, respectively .

Chronic Selenium Toxicosis

In chronic selenium toxicosis, it may take several weeks to
months before mortalities are precipitated depending on numerous
modifying factors such as composition of diet, age, sex, and species
of animal. In his first publication on the "new toxicant", F. W.
Franke (1934a) found that feeding toxic grain to rats resulted in
deaths over an extended period. The toxicant was present in the

protein fraction of these grains (Franke, 1934b). Diluting the toxic

24
grain with non-toxic grain so that only 17% was from the toxic
source still resulted in growth depression and death of rats (Franke,
1935a). Alternate feeding of toxic and control diets gave rhythmic
decreases and increases in growth and food consumption (Franke,
1935b). Smith et al. (1937a) reported that chronic.toxicity of
selenium varied in the different species and in different indi-
viduals of the_same species. The rat was the most resistant, and
the cat was the most susceptible of the species examined. Females
of a species were more susceptible than males, and young animals
were more susceptible to chronic selenium poisoning than older
animals (Rosenfeld and Beath, 1964). Smith et al. (1937a) indi-
cated that no acquired tolerance to selenium was demonstrated, but
Heinrich and MacCanon (1957) stated that dogs developed a tolerance
to selenite under suitable conditions. Wahlstrom and Olson (1959)
also observed tolerance to selenium toxicity in pigs from sows that
had been fed selenized rations over a long time.

The levels of selenium, expressed as ppm unless otherwise
indicated, in the ration generally associated with chronic toxicity
have been: 5 to 8 for chickens, 5 to 40 for cattle, 7.2 to 10 for
dogs, 5 to 15 for rate, 10 to 15 for swine, and 10 mg selenium per
day for sheep (Miller and Schoening, 1938; Rosenfeld and Beath,
1946b; Anderson at al., 1961). Miller and Williams (1940b) fed sodium
selenite to 2 horses at the rate of 24 ppm selenium equivalent in
the feed for 24 weeks, 48 ppm for another 37 weeks, and 96 ppm.for
an additional 9 weeks before they died of an atypical selenium toxi-
cosis. Schoening (1936) described 2 pigs that were fed seleniferous
corn containing 10 ppm selenium for about 4 months and had the

clinical signs of toxicosis. Schoening (1936) failed to observe

25
mortality in 2 pigs fed seleniferous corn containing 10 ppm selenium
although the clinical signs began to develop after about 6 weeks.
Seleniferous corn with 10 ppm selenium fed to 3 additional pigs and
corn with 5.ppm selenium fed to 5 other pigs had no visible dele-
terious effect. Maag et al. (1960) fed selenium orally 3 times a
week in varying dosages from 0.55 to 1.1 mg selenium (as sodium
seleniterer kg body weight-to 8 feedlot steers. They reported
that 2 steers died within 8 weeks and 4-more died after 20 to 23
weeks. No cumulative effects from sodium selenite were_demonstrated,

and it was concluded that there were dosages of sodium selenite

 

which an animal can tolerate without ill effects. The classic ' _ E;
lesions of selenium poisoning did not develop; polioencephalomalacia

was noted in 2 steers.

Clinical signs and physiological data. The clinical manifesta-
tions of natural chronic selenium toxicosis have been historically
associated.with another inapprOpriate lay term, "alkali disease."
Domestic animals with this disease were identified by a lack of
vitality, anemia, stiff gait, lameness, rough hair coat, loss of
hair, especially mane and tail in horses, and hoof lesions and
deformities (Rosenfeld and-Beath, 1964). Appetite was markedly
decreased with naturally seleniferous and artificially selenized
feedstuffs in rats, swine, and horses (Munsell st aZ., 1936; Miller
and Schoening, 1938; Miller and Williams, 1940b). Specifically,
in swine, additional clinical signs included loss of hair along the
back, retarded growth, emaciation, lameness and hoof lesions fol-
lowed by the shedding of the hoof wall (Schoening, 1936; Miller and
Schoening, 1938). These clinical manifestations have been thought

to be nearly pathognomonic.

26

Halverson et a1. (1970) determined that the anemia which
occurred with selenite-fed rats was attributable to hemolysis
rather than a defect in blood synthesis. Neethling et‘aZ. (1968)
isolated an abnormal hemoglobin C from sheep erythrocytes after
feeding selenium. They postulated that this abnormal hemoglobin
C which contained selenium was important in the pathogenesis of the
anemia found in the South African ovine diseases, "geeldikkop" and
"enzootic icterus." Rosenfeld and Beath (1946c) reported that.
sheep with selenium poisoning had less protein, sulfur, vitamin A
and ascorbic acid in their livers although these parameters returned
to normal in about 60 days after selenium withdrawal. During
chronic selenosis, adenosine triphosphate and other labile phosphate
compounds were decreased in the liver. Inorganic phosphate was
elevated probably because of an insufficiency in renal function

(Rosenfeld, 1964) .

Lesions. Many of the characteristic findings in chronic
selenosis have been mentioned in the section on clinical signs.
Additional gross changes included a flabby decompensated heart and
an atrophic cirrhotic liver. The renal medulla was congested, and
the spleen was atrophic and dull brownish-yellow in color. Erosions
of the articulating surfaces of long bones and miscellaneous other
changes were present (Draize and Beath, 1935).

The histOpathologic changes included atrophy and fibrosis of
myocardial fibers, an accumulation of yellow pigment near myocardial
nuclei; thickening and fibrosis in the alveolar walls of the lung;
fibrosis, pseudolobulation, and proliferation of bile ducts in the

liver; chronic cholecystitis; fibrosis and obliteration of glomeruli

27
and tubules in the kidney; and fibrosis and hemosiderosis of the
spleen (Rosenfeld and Beath, 1964). In addition, Smith et al.
(1937a) reported gastroenteritis and hematopoietic depression in

rats .

Selenium content in tissues. The reported selenium content
of the tissues from animals dying of chronic toxicosis have been
variable. Rosenfeld and Beath (1945) indicated that liver and
kidney accumulated greater quantities of selenium than did other
body tissues. In sheep on high, medium, and low protein diets,

the selenium concentration in the liver and kidneys of those

 

A.

dying from selenium toxicosis ranged from 9.10 to 33.17 ppm and
3.95 to 13.55 ppm, respectively, when fed between 15 and 30 mg
selenium per day. Death occurred in these sheep after the concen-
tration of selenium in the blood increased above 2 ppm selenium
and selenium feeding continued (Rosenfeld and Beath, 1946a). Dinkel
et al. (1957) confirmed the diagnostic value of the selenium con?
tent in blood and extended their observations to the selenium con—
tent of hair from cattle grazing seleniferous pastures. They
noted that cattle having over 2 ppm selenium in the blood would
likely develop the clinical signs of selenium toxicosis. They also
reported that a selenium content above 10 ppm in hair is a reliable
indication of toxicity to selenium in cattle if the samples of hair
are collected from several animals.

Withdrawal of seleniferous feed for 60 days resulted in normally
non-toxic tissue concentrations of selenium (Rosenfeld and Beath,'
1945; Dinkel et aZ., 1957). However, Munsell et a1. (1936) noted

the effects of selenium poisoning in rats even after the withdrawal

28
of seleniferous feed and the elimination of selenium residues in the

tissues. This emphasized the seriousness of selenium poisoning.

Factors InfluencinggToxicosis

Because selenium occurred in soils with numerous other possibly
toxic elements, arsenic, molybdenum, tellurium, vanadium, and selenium
salts were injected intraperitoneally and were fed to study their
comparative toxicities (Franke and Moxon, 1936; Franke and Moxon,
1937). Selenium salts were the most toxic to rats. The combined
toxicities of 11 ppm selenium from seleniferous wheat with vanadium,
molybdenum, chromium, tungsten, fluorine, arsenic, cadmium, zinc,
cobalt, uranium, and nickel, given as water-soluble salts at 5 ppm
in the drinking water, indicated that these elements, with the
exception of tungsten and arsenic, increased the mortality of rats
(Moxon and DuBois, 1939). Rosenfeld (1964) confirmed increased
selenium toxicity after zinc supplementation. While investigating
the interrelationship between selenium and specific trace elements,
McConnell and Carpenter (1971) reported evidence that there was a
reaction at the site of injection between selenium and zinc or
cadmium but not between selenium and arsenic or tellurium. It
appeared that selenium prevented arsenic and tellurium from reaching
target tissues.

Rosenfeld and Beath (1964) reviewed rather extensive data
indicating that arsenical compounds prevented or reduced selenium
toxicity whenrelatively low toxic amounts of selenium were present
in the diet. Inorganic arsenic salts were too toxic for practical
use. The organoarsenicals, arsanilic acid or 3-nitro-44hydroxyphenyl-

arsonic.acid, were less toxic and have given partial protection

29

against selenium poisoning (Moxon, 1941; Hendrick at al., 1953;
Wahlstrom et aZ., 1955; Dinkel at aZ., 1957; Minyard et al., 1960).

Numerous reports have indicated that increased protein in the
diet partially reduced the incidence of selenium toxicosis (Smith,
1939; Gortner, 1940; Rosenfeld and Beath, 1946b; Rosenfeld and
Beath, 1964). Linseed meal was especially effective when combined
with an arsenical (Wahlstrom at al., 1956), but the amount of lin- FT
seed meal necessary to prevent selenium poisoning is so great that

it is not considered a practical preventative substance (Olson,

.r‘ I! J. .'_'.“‘

1969b). Rosenfeld and Beath (1947) found that beet pectin gave some

 

protection against selenium poisoning from selenite while vitamin

‘F

C and potassium iodide did not. Two months after selenium and beet
pectin feeding was discontinued, ascites due to portal obstruction
in the liver became apparent. a.

Smith (1939) reported that rats fed high fat diets were more
tolerant to selenium than those fed high carbohydrate diets. This
observation was extended by Romanowski et a1. (1958). With sodium
selenite incorporated at a level which permitted growth, rats fed
isocaloric diets with added fat but without added carbohydrate, in,
most instances, exhibited somewhat better growth and more normal
livers than those fed isocaloric diets containing added carbohydrate.
A better understanding of the interrelationship of selenium and
the major dietary components is still needed.

When administered separately methionine and tocopherol were
ineffective against selenium toxicity (Klug et-aZ., 1952; Morse and
Olcott, 1967), but a combination of 0.5% methionine plus 0.01 to
0.05% alpha-tocopherol gave increasingly better protection in rats
depending 0n the level of alpha-tocopherol added (Levander and

Morris, 1970).

30

Bromobenzene has been used in an attempt to increase selenium
excretion following ingestion of seleniferous feed (beon et al.,
1940). As reviewed by Rosenfeld and Beath (1964), some experimental
results have indicated that the administration of bromobenzene
increased the urinary excretion of selenium, but other reports
have failed to confirm this observation.« The inherent toxicity
of bromobenzene must be pointed out. KochAWeser et a2. (1952)
described the acute centrolobular hepatic necrosis observed follow—
ing the parenteral injection of bromobenzene.

There are-no truly effective means of preventing or treating

 

‘L.

selenium poisoning. Prevention through good management is the
most practical answer to the problem of selenium poisoning in

animals (Olson, 1969b).

Selenium and Human Health

Knowledge on actual selenium toxicosis in man has never been
fully documented. The surveys of Smith et al. (1936) and Smith and
Westfall (1937) from seleniferous areas of South Dakota and wyoming
did not identify selenium poisoning as a problem in man although
selenium intake may have contributed to the gastrointestinal dis-
turbances and hepatic dysfunctions that were noted. Urinary excre-
tion was mentioned as a reliable index of selenium intake. Schroeder
at al. (1970) recently estimated that the daily intake of selenium
in~a standard hospital diet was 62 ug. He also noted that wild
animals normally carry a body—burden of selenium 2 to 3 times higher

than that_found in humans.

9.
n

96

31

Selenium and Cancer

 

Frost (1971) indicated that the carcinogenic stigma of selenium
has placed it under the Delaney Clause of the Pure Food Law and
prevented its approval asta feed additive for animals. The experi-
mental evidence on the interrelationship between selenium and
cancer was reviewed (Anon., 1970). Although selenium was considered
a highly toxic element that produced tumors when ingested over long
periods, recent work has suggested that some selenium compounds
may actually inhibit the development of tumors and that there may
be an inverse relation between the level of selenium in the blood
of human subjects and mortality from cancer. More experimental
and epidemiologic-studies are needed to clarify the relationship,

if any, between.selenium and cancer.

Summary

Although the literature on selenium is voluminous, the true
metabolic function of selenium as a micronutrient or a toxicant
is essentially unknown. Many seemingly contradictory and incomplete
findings have been reported. Accurate minimum lethal dosages of
the various forms of selenium for each species of animal, especially
in swine, have not.been recorded for application to current nutri-
tional and agricultural practices. Studies on selenium toxicity as
related to losses from swine diseases are limited.

It is evident from this survey of the literature that additional
information on selenium.toxicosis in swine is needed. This informa-
tion would not only be valuable to the swine and general livestock
industry, but it would also provide additional basic information on

the role of selenium in biomedicine.

"fll

OBJECTIVES

The general objectives of this research were to obtain addi-
tional information on the clinical signs, lesions, and selenium
residues due to feeding young pigs specific amounts of both sodium
selenite and selenomethionine. This research was preceded by
preliminary studies on the effects produced by feeding a vitamin E-
selenium deficient diet to pigs and also by injecting selected
amounts of selenium as sodium selenite into swine and cattle. The
specific goals of the main research were:

1. To determine the general clinical signs and lesions when
young pigs were fed specific amounts of sodium selenite or
selenomethionine.

2. To interpret and record the macroscopic, microscopic, and
ultrastructural changes produced by feeding different amounts of
the compounds in Objective 1.

3. To determine the selenium content of liver and kidney after

 

feeding different amounts of both selenium compounds and to correlate

the selenium content of these organs with the lesions recorded in
Objective 2.
4. To differentiate the pathologic changes of selenium toxi-

cosis from.those of vitamin E-selenium deficiency in pigs.

32

 

 

 

 

 

abeve

33
5. To provide relevant information from a combination of the
above objectives for the diagnosis of selenium toxicosis in swine

and for possible implications in public health.

'U

Pigs
Fact
Thir

 

Wet

 

MATERIALS AND METHODS

Preliminary studies on the toxicity of sodium selenite involved
intramuscular and/or intravenous injections into 3 young pigs and
6 yearling steers. (The steers had been previously used on a
leukemia project.) The essential procedures of these preliminary
studies are included in the results.

Two principal experiments were conducted using a total of 44
pigs which were fed rations based on either Torula yeast (Experiment
A) or whole milk powder (Experiment B) as protein sources. Graded
amounts of either sodium selenite or selenomethionine were added
to each of the above rations to produce various degrees of selenium
toxicosis. In addition to feeding the selenium compounds, each of
these 2 compounds was injected intravenously into 2 individual pigs

from Experiment B to investigate peracute selenium toxicosis.

Animals, Facilities and Husbandry
A total of 50 genetically related Zdweek-old Yorkshire-Hampshire
pigs were purchased from.the L. W. Cheney Farm, Mason, Michigan.
Each pig had had an iron injection at the farm to prevent anemia.
Thirty-eight pigs which were initially assigned to Experiment A
were selected for size and sex from 4 litters in 1 farrowing and
were received in January 1972, while 12 pigs which were assigned
to Experiment B were selected for size from 2 litters in another-

farrowing and were received in March 1972. In addition to the pigs

34

O
f ’1'
g on

va‘.

ea.

In

‘31

35
from the Cheney Farm, four 2dweek-old Yorkshire pigs were obtained
from.a litter which farrowed in March 1972 at Barn 5 (MSU Pathology
Department) and were assigned to Experiment B.

On arrival each pig was weighed and identified by coded ear-
notches. During a Zdweek period of acclimation to the experimental
environment and ration after weaning, 10 of the 38 pigs for Experi-
ment A were selected and removed because of poor appetite, scours
or death. The 16 pigs in Experiment B underwent a similar period
of acclimation without problems.

All pigs were kept at Barn 5 on the MSU Veterinary Research
Farm. They were placed in individual 21 x 36 inch galvanized metal
metabolism cages equipped with wire floors and individual feed and
water crocks. The feed and water cracks were thoroughly washed once
each day. Each pig in EXperiment A had a specifically identified
and weighed feed crock to facilitate measurement of feed consumption.
Ianxperiment B the unconsumed liquid was measured back in a graduated
cylinder.. The daily feed consumption was recorded in both experi-
ments. Each pig was fed and observed 2 to 4 times per day after

initiation of the experiments.

Experimentalspesign.

The pigs in Experiment A.were assigned according to the experi-
mental design given in Table 1. Each group contained a male and a.
female pig; the pigs with better appetites were allotted to groups of
ings fed the higher concentrations of selenium. The experimental
design of Experiment B is given in Table 2. These pigs were consigned
9° Broups of ~2 pigs each so that pigs 13B, 14B, 15B, and 163 from
the litter born at Barn 5 were in different experimental groups.

NO attemptwas made to allot these pigs by sex.

36

Table 1. Allotment of pigs according to sex, selenium compound,
and selenium concentration of ration in Experiment A

 

 

 

 

 

Selenium

added to Sodium selenite Selenomethionine
ration (ppm) Pig no. Sex Pig no. Sex

0 4* M** 31 M

35* F** 5 F

0.1 34 M 13 M

24 F 30 F

5 11 M 12 M

19 F 17 F

10 15 M 8 M

32 F 9 F

20 23 M 3 M

36 F 26 F

45 18 M 7 M

6 F 20 F

100 1 M 14 M

37 F 38 F

 

*

These 2 pigs were not supplemented with 22 IU vitamin E/kg
of ration to produce vitamin E-selenium deficiency; all other pigs
were supplemented.

as
Mbmale; F-female.

37

Table 2. Sex and allotment of pigs according to selenium compound
and selenium concentration of dry ration in Experiment B

 

 

 

 

 

Selenium
added to dry
basal ration Sodium selenite Selenomethionine
(ppm) Pig no. Sex Pig no. Sex
0 33* F** 43 F
133 M** 148* F
60 SB M 63 F
158 M 163 F
120 13 F 28 F
118 M 8B F
600 73 F 108 F
93 M 123 F

 

*After the principals of this experiment had died of
selenium toxicosis, Pigs 3B and 143 were given 3 mg selenium/kg
body wéight intravenously as sodium selenite or selenomethionine,
respectively, to produce peracute selenium toxicosis.

**
F-female; M-male.

38
Rations

The rations of both experiments were calculated to provide 16%
protein in the dry mixed ingredients from information provided by
the suppliers. The basal ration to which the selenium compounds
were added at each feeding for the pigs in Experiment A is given
in Table 3. The general feeding regimen was a modified paired
feeding method where all surviving pigs were fed 150 gm/pig twice
daily for the first 3 weeks, 250 gm/pig twice daily for the second
3 weeks, and 1000 or 2000 gm/pig once daily until the end of Experi-
ment A, which was as long as 13 weeks.

The basal ration to which graded amounts of selenium compounds
were added at each feeding_for the pigs in Experiment B is given
in Table 4. The general feeding regimen was 250 gm reconstituted
milk/pig fed 4 times a day. This amounted to the feeding of 167 gm
dry basal ration/pig/day. No additional water was provided for the
pigs in Experiment B.

There was one irregularity in the feeding regimen in Experiment
B. Following profound anorexia, Pigs 23, SB, 153, 6B and 16B were
force-fed their daily allotments of selenized milk by syringe and
stomach tube during their last 2 days (8 feedings) on experiment,
since the pathologic.changes of selenium toxicosis were primary and

the changes from anorexia were to be avoided.

Selenium Compounds

 

* *
Sodium selenite (bacteriological grade) and seleno DL methionine
(chemically pure) were used as the sources of inorganic and organic

selenium, respectively. Each of these selenium compounds was

 

*
Purchased from Nutritional Biochemicals Corporation, Cleveland,
Ohio 44128.

39

Table 3. Composition of basal ration fed to pigs in Experiment A

'F

 

Ingredients Percent
Torula yeast, Type B (502 protein) 32
Cod liver oil* 5
Mineral mix** 6
Vitamin mix+ TT 2
Cerelose 55

1002

 

*
Provided vitamin D
units/gm.

3, 600 ICU/gm; and vitamin A, 1500 USP

**Prepared by Dr. E. R. Miller, Animal Husbandry Department,
as used previously (J. Nutr., 85, (1965): 347), and contained the
following compounds expressed as percentages of the total mineral
mixture: KCl, 10.0; KI, 0.002; Fe804°2H20, 0.7; CuSO4, 0.1; CoC03,
0.1; MnSO4°H20, 0.1; Znsoa'HZO, 0.4; MgC03, 2.0; NaHCO3, 25.0;~
CaHP04°2H20, 36.0; Ca003, 12.5; and cerelose, 13.098.

+Provided the following compounds expressed as ppm 0f basal
ration: thiamin HCl, 3.0; riboflavin, 6.0; nicotinic acid, 40.0;
Ca pantothenate, 30.0; pyridoxine HCl, 2.0; PABA, 13.0; ascorbic
acid, 80.0; inositol, 130.0; choline, 1300.0; folic acid, 0.260;
d-biotin, 0.050; cyancobalamin, 0.10; and menadione Na bisulfite,
0.065.

HProvided 22 IU vitamin E/kg of basal ration for all pigs
in experiment except Pigs 4 and 35.

40
Table 4. Composition of dry basal ration fed to pigs in Experi-

 

 

 

ment B

Ingredients . Percent
Whole milk powder** 60.5
Cerelose 39.5

W

 

*This dry basal ration was reconstituted for feeding in the
following proportions by weight: 1 part dry basal ration to 5 parts
tap water.

**Supplier's analysis expressed as percentages was: protein,
26.50; lactose, 38.00; fat, 27.00; ash, 6.05; water, 2.00.
accurately weighed and dissolved in distilled water so that stock
solutions contained 1 mg selenium/m1. As needed, volumetrically
measured amounts of each stock solution were diluted with distilled
water to prepare 6 different working solutions for each selenium
compound which contained 0.001, 0.05, 0.1, 0.2, 0.45, and 1.0 mg
selenium/ml. When 1 m1 of each working solution was added to 10 gm
of dry ration, it provided selenized feed which contained 0.1, 5,
10, 20, 45, or 100 ppm selenium, respectively, in Experiment A. A
similar amount of distilled water was added to the ration of those
pigs that received no selenium. In Experiment B, 1 m1 of working
solution (0.1, 0.2, or 1.0 mg selenium/ml) was added to each 10 gm
of reconstituted milk, and this provided concentrations of 60, 120,
or 600 ppm seleniumnwhen calculated on a dry weight basis using
the reconstituted milk. Separate and identified volumetric pipettes
were used to measure the working solutions added to the feed at

each feeding. Following addition of the selenium to the feed,

both were mixed together by stirring.

41

Experimental Parameters and Procedures

 

When the pigs in Experiment A were placed on experiment, blood
(20 ml) from the anterior vena cava was collected into tubes con-
taining heparin, and accurate body weights were recorded to estab-
lish base lines. Additional bleedings were made just prior to
euthanasia of healthy or moribund pigs. Body weights of surviving
pigs were registered at weekly intervals for 2 months and at the
termination of the experiment. From the pigs in Experiment B,
blood (20 m1) without an anticoagulant was collected initially and
terminally as in Experiment A. Initial and final body weights
were also recorded.

From.the blood collected in Experiment A, a small aliquot was
used for determinations of hemoglobin, hematocrit,.and total and
differential leukocyte counts; the remainder was centrifuged and
separated as plasma and packed erythrocytes. The plasma was frozen
and stored for selected determinations of plasma tocopherol by the
Emmerie-Engel method modified by Tsen (1961) and plasma ornithine
carbamyl transferase (OCT) by the method of Sigma Chemical Company
(1965). The 48-hour osmotic fragility of erythrocytes was determined
by the method of Dacie (1953) cited and modified by Stowe (1962).
After evaluating the results of this procedure during the majority
of Experiment A, it was discontinued as an unreliable procedure
in the pig. In Experiment B, the clotted whole blood was centri—
fuged for separation of the serum which was frozen for selectedv
determinations of serum OCT by the method of Sigma Chemical
Company (1965) and serum glutamiceoxaloacetic transaminase (GOT)
by the method of General Diagnostics (1968). In addition to the

above, selected determinations of urea nitrogen were made on either

42
plasma or serum in both experiments by the method of Hycel,
Incorporated (1964).

From specimens of liver and kidney collected at necrOpsy,
selected livers were analyzed for toc0pherols by the Emmerie-Engel
method modified by Tsen (1961); all livers and kidneys from all
pigs were analyzed for selenium by the method of Olson (1969a).*
A11 determinations of selenium were made in duplicate on samples
weighing about 2 gm. The duplicates of each sample were analyzed
on separate days. These results will be reported as average values
of the duplicates.

The clinical signs of each pig were recorded after each
feeding. Body temperatures were taken in pigs that had clinical
toxicosis. When the body temperature was below normal and the pig
appeared moribund, it was.euthanatized by electrocution or bleeding
depending on its state of consciousness. Some pigs with toxicosis
died naturally. Healthy principals and controls were euthanatized
by electrocution at selected periods during the experiments and

at the termination of the experiments.

Postmortem Examination
At necropsy, postmortem weights of liver and kidneys were
registered. A thorough gross examination of all organs was made,
and lesions were recorded. The following tissues were routinely

collected and fixed in buffered 10% formalin for histopathologic

 

*In cooperation with Dr. O.E. Olson, Department of Experiment
Station Biochemistry, South Dakota State University. This coopera-
tion resulted from personal communication with Dr. Olson and his‘
interest in this research.

43
examination by light microscopy: salivary glands, mandibular or
portal lymph nodes, thymus, thyroid, lung, heart, diaphragm, semi-
tendinosus muscle, esophageal and fundic portions of the stomach,
duodenum, jejunum, ileum, colon, pancreas, liver, gallbladder, ovary
or testicle, cerebrum, cerebellum, brain stem, pituitary gland, a
small segment of spinal cord within the 13th or 14th thoracic
vertebra, sciatic nerve, ventral part of the right 3rd rib including
the costochondral junction, medial claw of the right hind foot, and
skin. Selected tissues were also fixed in Zenker's and Bouin's
fixatives. The fixed tissues were routinely embedded in paraffin,
sectioned at 6 u, and stained with hematoxylin and eosin (H & E)
(Luna, 1968). Selected sections of tissues were also stained with
oil red O, Best's carmine, PAS, and Gomori's trichrome.

In addition to the primary postmortem procedures previously
mentioned, 1 mm thick slices of the following organs were collected
in cold buffered 4% glutaraldehyde for transmission electron microscopy
from selected moribund and control pigs: liver, kidney, adrenal
gland, myocardium.from the septum of the left ventricle, diaphragm,
cerebellar folia, spinal cord within the 13th or 14th thoracic
vertebra, and sciatic nerve. After 4 hours of fixation under
refrigeration in glutaraldehyde, the tissues were washed in 6
changes of cold Sorenson's buffer (pH 7.4) and stored in Sorenson's
buffer in the refrigerator to await further processing. Only the
thin slices of liver were processed further. Several (10 to 20)

l mm.cubes of hepatic tissue from 4 selected pigs were post-fixed
in osmium tetroxide, dehydrated, embedded in epoxy resin, sectioned
and examined by the methods described by Cockrell (1969) and Boam

(1970).

44
Analysis of Data
Although the experimental design and results in this research
did not always lend themselves to statistical considerations,
whenever possible, these data were analyzed and observed for sta-
tistically significant differences by the Student's t-test and

Snedecor's F—test as outlined by Lewis (1966).

RESULTS

Preliminary Data

The general results of preliminary trials involving selenium
injections into 6 yearling steers and 3 young pigs are summarized
in Table 5. The clinical signs of peracute selenosis in Steer l
were salivation, respiratory distress, cyanosis and death in 8
hours. The characteristic gross lesions were massive pulmonary
edema; hydrothorax; large_fibrinous clots in thoracic cavity and
trachea; endocardial ecchymoses in the left ventricle; congestion
of the gastrointestinal tract, renal medulla, and urinary bladder;
blood-tinged urine; and meningeal edema over the brain. The.
important histopathologic change was hyaline degeneration in the
arterioles of heart and kidney. Following intramuscular injections
of sodium selenite into 3 steers, the general gross and microscopic
lesions were limited to the sites of injection and were characterized
by muscular necrosis, thrombosis and vascular necrosis, hemorrhage,
edema and an infiltration of neutrophils. For 1 steer, the selenium
solution was neutralized to pH 7, but this did not noticeably change
the character of the muscular lesions.

In Pig 2, the clinical signs of peracute selenosis were dyspnea,
depression, coma and death in 10 hours. The principal gross lesions
were petechiae on the pleura, hemorrhagic lymph nodes, subendocardial
and subepicardial ecchymoses, excessive pericardial fluid and con-
gestion of the renal medulla. Additional changes that were noted

45

46

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9sz a I..-
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new a mamoeaounu Ha\mufin=
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some .amou condone
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muasmom vouoonaa om dowuoufi .us .uooum
om Manon. 15" com

 

 

 

muwoaxou_asfiaoaom mo mamwuu musdflaaaoum ea omen can mucous moan: madness mo huuaaum

.n «Hana

47

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«a

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um

 

 

 

 

 

 

 

 

 

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when m
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muasmom nonsense om downed“ .uz . mam
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A.u.ua00v m ounce

48
histopathologically were congestion of the adrenal cortex and
necrosis of germinal follicles in lymph nodes and Malpighian
corpuscles of the spleen. The intramuscular injection of sodium
selenite into the thigh of Pig 1 produced a reaction similar to

that found in the steers given intramuscular injections of selenium.

Experiments on Selenium Toxicosis

The pigs fed sodium selenite or selenomethionine had con-
siderable variability in their individual response to toxic doses
of selenium. While the objective of producing selenium toxicosis
and vitamin E-selenium deficiency with the same basal ration was
completed, the consumption of the Torula yeast basal ration was not
as good as was anticipated. After 1 or 2 feedings, the appetites
of pigs fed selenium were proportionally depressed with increased
concentrations of selenium in their feed. That is, anorexia became
apparent earlier in the pigs fed 100 ppm selenium than in those fed
45 ppm.. Consequently, the pigs fed 45 ppm selenium ate a larger.
dose of selenium on a body-weight basis before the pigs on the
highest level in their feed. These peculiarities in appetite and
feed consumption must be remembered in evaluating the following
results against practical rations and conditions. The increased
feed consumption expected with natural rations may produce toxicosis
at lower selenium concentrations.

In the pigs fed selenium, clinical toxicosis develOped in 21
of 38 pigs (55.32) fed either sodium.se1enite or selenomethionine
at various levels in the rations of both experiments. In Experiment
A (Torula yeast basal), Pigs l, 37, 6, l8, and 23 which were fed
sodium selenite and Pigs 14, 38, 7, and 20 which were fed seleno-

methionine developed clinical selenium.toxicosis. These pigs

49
represented 9 of 24 pigs (39.12) fed selenium in Experiment A.
In Experiment B (dried milk basal), all pigs fed the selenium COMP
pounds develOped toxicosis either as a result of natural ingestion

or forced feeding of the prescribed amounts of selenium.

Growth, Duration and Amounts of Selenium Fed

A summary of the total amounts of selenium fed, days on experi-
ment, and growth responses for each pig in Experiment A is given in
Table 6. In general, the average daily gain of pigs with clinical
toxicosis was markedly less or negative compared to pigs without
clinical toxicosis. No real difference was identified between the
toxicity of sodium selenite or selenomethionine at levels of 45 and
100 ppm selenium by comparing the average daily weight loss, the
toxic dose, or the number of days to moribund toxicosis. Of the
pigs fed 20 ppm selenium, only 1 developed the clinical signs of
selenium toxicosis.

When the data of individual pigs were examined, it was revealed
that Pig 18 died in 3 days after ingesting only 13.5 mg selenium.

In contrast, Pigs 3 and 36 tolerated a total of 1709 and 1380.5 mg
selenium, respectively, without evident toxicosis. Pig 6 was fed
nearly 1150 mg total selenium but did develOp clinical toxicosis.

A summary of the total amounts of selenium fed, time on experi-
ment, and growth response for each pig in Experiment B is given in
Table 7. The weight changes of all pigs with selenium toxicosis
were markedly less or negative in comparison to the controls. The
interval of time that the pigs fed selenium remained alive and on
experiment was inversely related to the dosage of selenium fed

except for Pig 2B. From the calculated total selenium fed, Pigs ZB

50

 

 

 

Table 6. Total amounts of selenium fed, days on experiment, weights,
and growth responses of pigs fed sodium selenite or
selenomethionine at various selenium concentrations in
the ration in Experiment A

Calculated
Selenium. total Average
added to Selenium selenium -Days on Initial Final daily gain

Pig ration compound fed experi- wt. wt. or loss

no - (ppm) fed (me) ment (8m) (8m) (891/ day)

4* 0 --- --— 4 4675 4540 - 34
35 0 --- --- 84 6660 39600 392
5 o --- --- 91 5015 38925 373

31* 0 -- --- 17 4785 6590 106

24 0.1 3803** 1.48 39 4760 11400 170

34 0.1 $603 1.41 35 4630 11040 183

13 0.1 SeMET** 1.37 38 3355 8810 144

30 0.1 SeMET 1.42 35 4610 10830 178

11 5 Se03 72.8 39 4145 9560 139

19 5 $603 72.6 39 3620 9230 144

12 5 SeMET 69.4 38 3990 9950 157

17 5 SeMET 70.8- 38 3620 9760 162

15 10 Se03 324.0 59 4045 17360 226

32 10 3603 324.5 53 4305 19125 280

8 10 SeMET 324.5 53 6170 21150 283

9 10 SeMET 325.0 59 4110 17180 222
23* 20 SeO3 73.1 32 4660 4160 - 15.6
36 20 Se03 1380.5 84 4990 30800 307

3 20 SeMET 1709.0 84 6135 36450 361
26 20 SeMET 750.7 63 4850 19460 232
6+ 45 $603 1148.8 63 5290 7620 37
18+ 45 Se03 13.5 3 4240 3580 -220
7’r 45 SeMET 74.9 9 4450 4420 - 3.3
20’r 45 SeMET 24.1 5 3910 3000 -l82
1+ 100 Se03 222.0 28 4295 3840 - 16.2

37+ 100 s603 136.0 19 6635 4440 -116

14+ 100 SeMET 84.5 20 5160 3410 - 87.5

38+ 100 seuzr 55.0 11 4835 3300 -140

 

*
These pigs had the lesions of hepatosis dietetica.

**
SeOg-sodium selenite; SeMET-selenomethionine.

+These pigs develOped clinical selenium toxicosis.

51-

 

 

 

 

 

Table 7. Total amounts of selenium fed, hours on experiment, weights,
and growth responses of pigs fed sodium selenite orv
selenomethionine at various concentrations in the dry
ration in Experiment B*

Calculated
Selenium total Average
added to Selenium selenium Hours on Initial Final daily gain-

Pig dry ration compound fed experi- wt. wt. or loss

no . (ppm) fed (me) ment (8m) (8m) (8m/ day)

4B 0 --- --- 29 5330 5505 145
13B 0 --- --- 192 4885 5740 107
SB 60 8e03** 39.2 142 4930 4720 - 35
15B 60 Se03 35.1 152 5460 4480 -157
6B 60 SeMET** 37.6 100 5050 5170 29
16B. 60 SeMET 59.6 150 6170 5850 - 51
1B 120 8e03 10.4 56 4320 3570 -326
11B 120 SeO3 12.8 53 4160 3365 -365
2B 120 SeMET 65.8 220 4530 3740 - 86
8B 120 SeMET 22.0 59 4940 4250 -283
7B ‘ 600 SeO3 27.5 24 3830 3660 -170
9B 600 Se03 43.2 "46 4545 4100 -232
10B 600 SeMET 34.0 29 3715 3770 44
12B 600 SeMET 32.0 46 3500 3460 - 21
Selenium
dosage
injected Selenium Total
i.v.+ compound selenium
(mg/kg injected injected
"BWD Ag_i.v. 11 gi.v. _ __
3B 3 Se03 20.0 2.5 6680 6680 0
14B 3 SeMET 20.6 14 6880 6680 -200

 

*
All pigs given selenium.hecame moribund or died with toxicosis.

**
SeO3-sodium selenite; SeMET-selenomethionine.

+i.v.-intravenously.

52
and 16B were relatively more resistant to selenium than other pigs
in the same groups. Selenium from sodium selenite was
slightly, but not significantly, more toxic than selenium.from
selenomethionine. Also, when Pigs 3B and 14B were given 3 mg
selenium/kg body weight intravenously as sodium selenite or
selenomethionine, respectively, selenium from selenite produced

moribund toxicosis more rapidly than that from selenomethionine.

Clinicopatholggic.FindingE.

The results of hemograms and determinations of hepatic and
plasma tocopherols, plasma or serum GOT, OCT, and urea nitrogen
were not particularly specific for selenium.toxicosis but would
aid in,a differential diagnosis. The hemograms from initial and
final blood samples from pigs in Experiment A are given in Tables
8 and 9, respectively. No pertinent differences could be associated
with selenium toxicosis among the groups of pigs between the 2
sampling periods. The results of selected determinations of
tocopherols in liver and tocopherols, OCT, and urea nitrogen in
plasma from various groups of pigs in Experiment A are summarized
in Table 10. Since these data were so irregular, the results are
presented as ranges of values. In general, the values of hepatic.
tocopherols between control and selenium-fed pigs did not indicate
any abnormal relationship. The values of plasma tocopherols were
slightly higher in the final samples from all groups than were the
values of plasma tocopherols in the initial samples. The Values of
plasma OCT and urea nitrogen from poisoned pigs were elevated above
those.from control pigs although there was considerable variability

among the values from these groups of pigs.

53

 

 

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54

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en 83843 8 662.6
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55

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Hmzmm ON ON

 

 

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R.

 

 

 

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-- -- Nee mee.m eam.H eee.HH ee~.e~ em e.~H nooe eeH H
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1.36683 . 386

Table 10. Range of results from.selected determinations for
tocOpherols in liver and tocopherol, OCT and urea
nitrogeninplasma from various groups of pigs at

57

initial and final periods during Experiment A

 

 

Hepatic Plasma Plasma OCT Plasma urea
Experimental period and tocopherols tocOpherols (Sigma nitrogen
groups of pigs bled (HS/3m) (pg/ml) units/m1) (mg/100 ml)
Initial base-line samples --- 0.65 to 0 to 1050 6.0 to
from pigs l.0(3)* (ll) 31.5(11)
Final samples from pigs 32 to 58 1.0 to 0 to 250 5.8 to
fed 0.1 ppm selenium (4) 1.4(4) (3) 9.0(4)
Final samples from pigs
fed 5, 10 or 20 ppm
selenium without 17 to 87 1.0 to --- ---
toxicosis (10) 2.1(4)
Final samples from
moribund pigs with 42 to 82 1.4 and 75 to 3150 6.5 to
toxicosis (8) 2.2(2) (7) 80.0(6)

 

*
Number in parentheses indicates number of pigs involved in

respective determinations.

58

The results of osmotic fragility of erythrocytes in Experiment
A using the degree of layering hemolysis after 48 hours under
refrigeration indicated that erythrocytes from all pigs in all
groups had layering hemolysis in aqueous solutions of sodium
chloride in the following gradations: no hemolysis, 0.852 saline;
slight hemolysis, 0.7 to 0.6% saline; moderate hemolysis, 0.55 to
0.452 saline; complete hemolysis, 0.35 and 0.2% saline.

The results of determinations of GOT, OCT, and urea nitrogen
in serum in Experiment B are given in Table 11. These data also
had wide variability of values from different groups of pigs;
therefore, only the ranges in values are presented. The high end
of the values of serumGOT and OCT from poisoned pigs was larger
than those from control pigs. Serum urea nitrogen was not elevated

above normal in any of the groups.

Clinical Signs

The clinical signs of selenium toxicosis were arbitrarily
divided into peracute (0-1 day), acute (1-30 days), and subacute
(30-90 days) stages.*l Chronic selenium toxicosis was not observed
although 2 pigs were fed 20 ppm selenium for 84 days (Table 6).
No differences were noted in the clinical toxicosis produced by
sodium selenite or selenomethione.

Control and seleniumrfed pigs without_toxicosis had no clinical
signs except Pig 26 which had a trauma-induced chronic bleeding
ulcer on the bottom of its left hind foot. It was believed that

this pig died from loss of blood and resultant anemia.

 

*
Veterinary Toxicology Training and workshop Notes, Iowa
State University, 1972.

59

Table 11. Range of results from selected determinations for GOT,
OCT and urea nitrogen in serum from various groups of
pigs at initial and final periods during Experiment B

 

Serum Serum

GOT OCT Serum urea
Experimental period and (Trans- (Sigma nitrogen
groups of pigs bled AC units) units/ml) (mg/100 ml)
Initial base-line 22 to 63 0 to 250 1.0 to 5.7
samples from pigs (15)* (7) (8)
Final samples from
pigs fed 0 ppm 17 to 27 0 to 250 1.0 to 5.5
selenium (4) (4) (4)
Final samples from
moribund pigs with 54 to 0 to 7.4 to 25
selenium toxicosis >1050(8) >5000(4) (4)

 

*Number in parentheses indicates the number of pigs involved
in respective determinations.

Peracute selenium toxicosis. Pig 3B given sodium selenite
intravenously had the clinical signs of peracute toxicosis and died
in 2-1/2 hours. This pig began vomiting in less than 15 minutes.
One hour postinjection, profound depression, weakness, and a "thump-
ing" respiration were present. The following measurements were
recorded: rectal temperature, 100.9 F.; respiratory rate, 80/minute;
cardiac rate, 80/minute; and cardiac sounds were faint. After
another hour, the rectal temperature was 98.5 F.; respiratory rate,
only 4/minute; cardiac rate, 180/minute. Although the pig appeared
comatose, the palpebral reflex was present until just before death.

Pig 14B was given selenomethionine intravenously and had a
longer period of clinical signs, but the basic clinical course was
not different. After 1 hour, this pig was depressed, shivering, and

recumbent. There was a tendency toward apisthotonos. The following

60

measurements were taken: rectal temperature, 106 F.; respiratory
rate, 68/minute; and cardiac rate, 80/minute. The elevated tempera-
ture, shivering, and lethargy continued through the 2nd hour when
emesis started and intermittently continued for about 3 hours. The
rectal temperature was 103.5 F.; respiratory rate,68/minute; and
cardiac rate, l60/minute during the 3rd hour after the intravenous
injection. The depression progressed into coma, and the temperature
decreased to 98.5 F. by the 13th hour. The respiratory rate had
slowed to 24/minute. The character of the breathing was regular

but deep and very deliberate.

Acute selenium toxicosis. The clinical signs of Pigs l, 7,-
14, 18, 20, 37, and 38 in Experiment A and all pigs fed selenium
in Experiment B were included in this group. The most characteristic
clinical sign of acute selenium toxicosis was the profound and pro-
tracted anorexia that developed in these pigs after a relatively
short.period of ingesting selenized feed. Some pigs ate only a
very small part of their daily ration. In Experiment A, some pigs
did not eat at all for 4 to 7 days before they became weak and
moribund.

Other clinical signs of acute toxicosis included vomition,
lethargy, accelerated and gasping respiration, coma and death
especially in the pigs of Experiment B. Some pigs had tense
abdominal muscles and squealed abnormally when they were handled.
Ataxia preceded recumbency. Convulsive signs of paddling and
opisthotonos were also noticed. The ears and feet were cold on
palpation. White hoofs appeared somewhat blue due to congestion.

Rectal temperatures decreased from normal to subnormal. Although

61

the pigs in Experiment A had most of the above signs, they did not
usually vomit. Cachexia and loss of body weight was most pronounced
before the terminal period of dyspnea and coma.v Pig 7, fed seleno-
methionine in Experiment A, became paralyzed before moribund signs
developed. In addition, Pig 2B, with Hampshire markings, lost the
hair from the white belt after several days of anorexia but the
hair in the black areas was unaffected. In general, the duration

of the moribund signs varied from 1 day to several days.

Subacute selenium toxicosis. Pigs 6 and 23 from Experiment A
were included in this classification, and both were euthanatized
when their general condition had deteriorated to a near terminal
state of cachexia. During the initial 3 weeks of the experiment,
these pigs ate their daily allotment of selenized feed fairly well.
After the amount of feed was increased for the controls, Pigs 6 and
23 refused to eat the increased amount of daily ration. Pig 23
became severely cachectic while Pig 6 began eating slightly better
after_another 3 weeks.

The clinical signs in Pigs 6 and 23 were mainly anorexia and
cachexia. These pigs were small and had rough hair coats, yellow-
tinged skin in unpigmented areas, and muscular atrophy and weakness

(Figure 1). No loss of hair or lesions of the hoofs were noted.

Hepatosis dietetica. Pigs 4 and 31 developed the lesions of
hepatosis dietetica during the course of this research. No selenium
had been fed to either pig, but Pig 31 had been supplemented 22 ppm
vitamin E in its feed. Pig 4 was observed 2 hours before death,
and the clinical signs were minimal. Both pigs had eaten their

previous meal, and both died suddenly and unexpectedly.

62

 

 

Figure 1. Comparison of control Pig 5 and Pig 6
after 60 experimental days. Notice smaller size, emaci-
‘ation, rough hair, tucked-up appearance of abdomen and
cocked appearance of hocks of Pig 6, in foreground,
fed 45 ppm selenium as sodium selenite.

63

Gross Lesions

There were no essential differences between the gross lesions
of selenium toxicosis in pigs poisoned with either sodium selenite
or selenomethionine. No lesions in.the gastrointestinal tract could
be attributed to selenium toxicosis. Nearly all of the pigs in
both experiments had some degree of mycosis in the esOphagus and
esophagogastric area of the stomach. Some pigs had eSOphagogastric
ulcers which were unrelated to selenium toxicosis. Pigs in Experi-
ment B that were force-fed their daily allotment of selenized milk
developed foreign-body pneumonia in the anterior ventral portions
of their lungs.

Organ weights of livers and kidneys, expressed as percentages
of final body weights, from pigs in Experiment A and Experiment B
are given in.Tab1es l2 and 13, respectively. Although there was no
apparent difference between the pigs with or without toxicosis as
groups, it is suggested that-the livers of Pigs l and 23 were slightly
atrophic, but the degree.of cachexia and dehydration markedly

influenced these results.

Peracute selenium toxicosis. The pig given sodium selenite
intravenously developed massive pulmonary edema (Figure 2). The
heart had subendocardial ecchymoses. The renal cortex was normal,
but the medulla was slightly congested. The appearance of the liver
was normal which might have been expected since this pig lived only
2-1/2 hours. I

The pig given selenomethionine intravenously had a yellowbbrown
mottled liver. The renal cortex was pale, and the medulla was con-

gested (Figure 3). No other gross lesions were seen.

64

Table 12. Final weights of livers and kidneys expressed as per-
centages of final body weights of pigs in Experiment A

 

 

 

 

 

Sodium selenite Selenomethionine
Selenium Selenium
added to added to
Pig ration Liver Kidney Pig ration Liver Kidney
no - (ppm) (1) (2) no - (ppm) (74) (Z)

4 0 3.27 0.71- 5 0 2 01 0 55
35 0 2 13 0.52 31 0 4 28 0 84
24 0.1 2.55 0.56 13 0.1 2.88 0.51
34 0.1 3.22 0.61 30 0 l 2.73 0.52-
11' 5 3.30 0.62 12 5 2.68 0.56
19 5 3.25 0.64 17 5 2.86 0.54
15 10 3.16 0.63 8 10 2.83 0.68
32‘ 10 3.57 0.74- 9 10 2.46 0.62
23 20 1.44 1.09 3 20 3.07 0.67
36 20 3.26 0.63_ 26 20 3.34 0.84

6 45 2.34 0.87 7 45 3.55 0.73
18 45 3.76 0.77 20 45 2.90 0.72

l 100 1.74 0.76 14 100 2.30 0.64

37 100 6.36 1.24 38 100 2.30 0.72

 

65

Table 13. Final weights of livers and kidneys expressed as per-
centages of final body weights of pigs in Experiment B

 

 

 

 

 

 

Sodium selenite Selenomethionine
Selenium Selenium
added to added to
Pig dry ration Liver Kidney Pig dry ration Liver Kidney
no - (ppm) (X) (7!) no - (ppm) (2) (2)
13B 0 2.56 0.56 4B 0 2.36 0.46
5B 60 3.67 0.80 6B 60 2.34 0.64
15B 60 3.04 0.67 16B 60 3.13 0.60
1B 120 4.45 0.95 2B 120 3.05 0.80
11B 120 3.82 0.88 8B 120 3.03 0.79
7B 600 2.90 0.74 10B 600 3.35 0.59
9B 600 3.24 0.60 12B 600 3.82 0.73
Selenium Selenium
injected. injected
i.v. (mg/ 1m. (1113/
kg BW) kg BW)

 

BB 3 2.27 0.52 143 3 2.60 0.56

 

66

 

Figure 2. Pulmonary edema in a pig with peracute
selenium toxicosis.

 

IIHJIHIJHIIH [HIJHJIHJ u 11 '
j 1‘15} 1 ski 1 7* i1 ‘13.} Lunlléhluiuwmuu£11+11114111121+1111w1

Figure 3. Pale renal cortices and congested
renal medullae from a pig with selenium toxicosis.
These were characteristic but non-specific changes
in pigs with selenium toxicosis.

67

Acute selenium toxicosis. Selenium toxicosis in pigs fed the
largest oral dosages of selenium was characterized by a liver which
was pale yellow throughout its parenchyma compared to a normal
liver (Figures 4 and 5). In acute toxicosis from a lower dosage
of selenium, the liver had an obvious yellow4brown mottling on its
external (Figure 6) and cut surfaces, but a few livers had to be
inspected closely to detect this change (Figure 7). The gallbladder
was smaller than normal, and the bile was so thick that it could
not be expressed through the common bile duct. The renal medulla
was congested. Distention of the urinary bladder with urine
resulted in a turgid appearance of the ureters. In Experiment A,
hemorrhage into the adrenal cortex was common. The hemorrhages,
which were few, on the heart, lungs, and serosal surfaces were asso-
ciated with agonal changes because hemorrhages were not noted in.
moribund pigs that were euthanatized. Hemorrhagic.pancreatitis,
was more commonly associated with toxicosis from selenomethionine.

The cachectic changes were serous atrophy of fat in the coronary
groove of the heart, in the perirenal area, and in the subcutis of
the head. Some pigs also had edema of the pancreas, intestinal
mesentery, spiral colon, and meninges of the brain. Skeletal
muscles were occasionally pale and edematous. Atrophy of the
thymus was also noted. Emaciation of the carcass was most pro-

nounced in these pigs that survived the longest time.-

Subacute selenium toxicosis. The 2 pigs in the classification
of subacute selenium toxicosis had more,advanced lesions of toxi-
cosis and cachexia with a similar distribution as those previously
described under acute selenium toxicosis. The livers of these pigs.

were slightly atrophic and less mottled.

68

 

Figure 4. Normal liver from control pig. This
liver from Pig 133 had a selenium content of 0.21 ppm.

 

 

 

ens-c I 1 l A S i 7 I 9 '0 u I .
\ n n! n ‘
- . n i .11 "” uj -. mummuuumummmnum

Figure 5. Very pale liver of acute selenium toxi-
cosis in a pig fed 120 ppm selenium in Experiment 3.
This liver from Pig 13 had a-selenium content of 5.62
ppm.

69

, um um HIHVIH IIH um rm um nu H11 “H 'm nn 1' m ’z 1 z ' w ' " '4 -
Lle 2} I J I ‘I I a; 1- a; s-Ii; 1111131 mug“ 11.11.1(11‘Lm-,m‘wu-u;n£-;+.u.1uuufiu1uuuiiu.minim \

 

Figure 6. Pale mottled liver of acute selenium
toxicosis in pig fed 60 ppm selenium in Experiment 3.
Compare with Figures 4 and 5. This liver from Pig 153
had a selenium content of 15.9 ppm. '

 

Figure 7. Subtly mottled liver of acute selenium
toxicosis in pig fed 45 ppm selenium in Experiment
A. Compare with Figures 4, 5 and 6. This liver from
Pig 20 had a selenium content of 14.6 ppm.

70
Hepatosis dietetica. The acute dietary liver necrosis in 2
pigs was characterized by multifocal to diffuse areas of lobular
hemorrhage, irregularities in Glisson's capsule, and hepatic swel-
ling (Figure 8). Both pigs had hemorrhagic ileitis. One pig had
subcutaneous hemorrhages behind the shoulders. It also had pale,
edematous skeletal muscles. The other pig had epicardial hemor-

rhages as well as hemorrhagic portal and mesenteric lymph nodes.

Histopathologic Changes-

Although the livers of euthanatized pigs fed no selenium and
those fed 0.1 ppm selenium were considered normal, there were
subtle histological changes of undetermined significance in the
livers of pigs fed 5, 10, and 20 ppm selenium without clinical
toxicosis in Experiment A. These livers had peculiar hepatocytes
with basophilia of the cytoplasm near the nucleus and a lacy
appearance of the peripheral cytoplasm. The hepatocytes were
swollen, and the hepatic sinusoids were usually obliterated. The
above changes were more pronounced in the pigs fed 10 or 20 ppm
selenium.

There were histologic changes in other tissues of the experi-
mental pigs that were not related to selenium.toxicosis. The epi-
thelium of the esophagus and esophagogastric portion of the stomach
with mycosis had hyperkeratosis, acanthosis, fungal yeast and mycelial
forms, and foci of neutrophils in the keratin, and accumulations of
lymphocytes in the submucosa. The esophagogastric ulcers, when,
present, were characterized by denudation of the epithelium with
fungal organisms, neutrophils, lymphocytes, and necrotic changes

in the submucosal connective tissue. Histologic sections of the

71

 

Figure 8. Hepatosis dietetica in liver of pig with
vitamin E-selenium deficiency. Notice multifocal and
multilobular hemorrhages (dark areas) seen through
Glisson's capsule.

72

pulmonary lobes with foreign-body pneumonia were characterized by
a bluish-pink foreign material in the bronchioles and alveoli with
massive infiltration of neutrophils into the affected areas.’ Some
lobulestwere extensively involved while other lobules were rela-
tively normal.

Although the gross lesions of selenium.toxicosis from.sodium
selenite or selenomethionine were, in general, similar, there were
some differences noted in the histopathologic changes. The extent

of changes and organs involved were somewhat different.

Peracute selenium.toxicosis. In the pig given sodium selenite
intravenously the pulmonary edema was characterized microscopically
by extensive edema in the alveoli and interlobular septa with
resultant distention of these structures. No cellular infiltrates
were present. The heart had endocardial hemorrhage around the
Purkinje fibers without visible changes in these fibers. The
spleen had necrosis of lymphocytes in the Malpighian corpuscles.
Germinal follicles in the lymph nodes and other lymphoid tissues
had increased pyknotic nuclei and cellular fragmentation. No micro-
scopic lesions were noted in the liver.

In the pig given selenomethionine intravenously, the most
striking histopathologic change was in the pancreas. The lesion was
characterized by necrosis, edema, and cellular lysis of entire
pancreatic lobules while other lobules had fairly normal acini,
but the nuclei were pyknotic (Figure 9). The sections of lung were
considered normal. The centrolobular hepatocytes of the liver were
swollen and had increased cytoplasmic eosinophilia compared to the
peripherolobular hepatocytes. Fatty metamorphosis of the liver was

not.apparent.

73

 

Necrosis, edema and cellular lysis in
H & E stain. x 180.

Figure 9.
lobular areas in pancreas from the pig given seleno-

methionine intravenously.

 

Fatty metamorphosis and centrolobular

Figure 10.
necrosis in liver from pig with acute selenium toxi-

H & E stain. x 180.

cosis.

 

acute
of en
incre
fat v
24 he
lobul
died
cytes
durat
pigs
in pi
the f
perip
cytes
the t
live:
swell
KUpfj

toxic

both
than!
large
rhage
of 1),-
large

Del-v0

74

Acute selenium toxicosis. In general, livers from pigs with
acute selenium.toxicosis were characterized by fatty metamorphosis
of entire hepatic lobules and by varying degrees of necrosis and
increased centrolobular eosinophilia (Figure 10). This hepatic
fat was identified by oil red O stain. Livers from pigs that died
24 hours after the initial ingestion of selenium had less centro-
lobular degeneration or necrosis than did those from pigs that
died after 50 hours in Experiment 3. The fat vacuoles in hepato-
cytes were, to a certain extent, slightly smaller with the shorter
duration of toxicosis. Also, the hepatic fatty metamorphosis in
pigs fed selenomethionine had smaller fat vacuoles than it did
in pigs fed sodium selenite at the same dosage. In Experiment A,
the fatty metamorphosis of the liver had a lacy appearance in the
peripheral cytoplasm with basophilia near the nucleus of hepato-
cytes. Fatty metamorphosis was more extensive at the periphery of
the hepatic lobules than in the centrolobular areas. A few of the
livers from poisoned pigs fed selenomethionine had only cloudy
swelling of the hepatocytes and obliteration of hepatic sinusoids.
Kupffer cells were quite prominent in all livers with acute selenium
toxicosis.

Although the liver appeared to be the primary target organ for
both selenium compounds, there were several other significant
changes in other organs. Some of the pancreases from pigs fed
larger dosages of selenomethionine had extensive necrosis, hemor-
rhage and edema (Figure 9). This was not noted in the pancreases
of pigs fed similar amounts of sodium.se1enite. But with the
larger dosages of sodium selenite, histologic lesions of the central

nervous system were more common. The granular layer of the cerebellum

 

.L
'1
u

75
had pyknosis of granule cells while at the tips of the cerebellar
folia the granular layer had a peculiar loss of both stroma and
granule cells (Figure 11). Edema or clear.spaces were also noted
around glial cells in the white matter of the cerebellum. The
cerebral cortex had edema and possible early laminar necrosis
between the layers of gray and white matter. Two pigs, 1 fed
sodium selenite and 1 fed selenomethionine, had extensive polio-
myelomalacia in different areas of the spinal cord. Hyaline
degeneration and fibrinoid necrosis of arterioles was a common
lesion in the brains as well as other organs of pigs fed either
selenium compound (Figure 12).

Other histopathologic changes were adrenal cortical hemorrhage
and pyknotic nuclei in the zona glomerulosa and zona reticularis
(Figure 13). Pyknotic acidophils and pallid staining character of
the cells in the anterior pituitary were noted in some pigs.
Lymphocytes in the germinal follicles of lymph nodes and in the
thymus and spleen were pyknotic and fragmented. The number of
hematopoietic cells in the bone marrow'was markedly depleted in
pigs fed larger dosages of selenium for more than 1 week (Figure
14). The remaining cells in the bone marrow were mature polymorpho-
nuclear cells. Skeletal muscles and diaphragm had degenerate myo-
fibers without infiltration of mononuclear cells (Figure 15). The
heart did not, in general, have significant histologic changes.

No renal lesions, other than a mild hydropic change in the epithelium
of the tubules, could be identified. The skin did not have any
significant changes except edema or serous atrophy of fat in the

subcutis. Hooves were considered histologically normal.

76

I r
' -..~

I ~‘.
t.£?€‘

‘VH

 

     

- ,' > '. ”:3, . ‘
,-..‘,, NI. , .
.~ r . ,
7"! ’ ' 44' .e r.

w- 7‘

 

Figure 11. Comparison of the tips of cerebellar
folia between control pig (top picture) and a pig fed
600 ppm selenium as sodium selenite (bottom picture).
Notice the loss of stroma and granule cells in granular
layer of cerebellum from pig with acute selenite

toxicosis. H & E stain. x 70.

77

 

Figure 12. Fibrinoid necrosis of wall of arteriole
in white matter of cerebellum from pig with acute
selenium toxicosis. H & E stain. x 720.

. N" ‘4 . 1"
.10- ,- v- §
-' ~§a~>
' :‘M\G\ ‘ I ‘ W“
. ..

; Ci‘k:’i:‘~ 5» .

- 1' " r v, .\
‘33-7-1‘1“ ‘ ---.
1"\“‘ \
$.34 ‘g, '-
. « \
."J ,m x
H. .

 

Figure 13. Hemorrhagic necrosis of adrenal cortex
from pig with acute selenium toxicosis in Experiment
A. H & E stain. x 70.

78

 

Figure 14. Comparison of normal bone marrow
from control pig (top picture) and aplastic bone mar-
row from pig with acute selenium toxicosis lasting
over 1 week (bottom picture). Both sections were
taken from the comparable areas of ribs. Notice the
loss of hematopoietic cells in the bone marrow from
pig with selenium toxicosis. H & E stain. x 180.

79

 

Figure 15. Degenerate myofibers in skeletal
muscle from pig with acute selenium toxicosis. H 8 E
stain. x 180.

 

Figure 16. Hepatic lobule from pig with subacute
selenium toxicosis. Notice variation in size of hepato—
cytes, loss of sinusoidal structure, and difference.in
staining of peripherolobular and centrolobular hepato-
cytes. H 8 E stain. x 180.

80

Subacute.se1enium toxicosis. The livers of pigs with subacute
selenium toxicosis were characterized by degenerative and regenerat-
ive hepatocytes, loss of sinusoidal architecture without loss of
lobular structure, increased sinusoidal and peripherolobular con-
nective tissue, and increased numbers of lymphocytes~and plasma
cells in the periportal and peripherolobular areas (Figure 16).
The-hepatocytes at the periphery of the hepatic lobules were
slightly larger than normal and had basophilia in the cytoplasm-
while in the centrolobular areas the hepatocytes.were very large,
as if coalesced, and had foamy, lightly eosinophilic cytoplasm.
Mitotic figures, multinucleated hepatocytes and nuclei with
multiple nucleoli were observed. Although cellular degeneration
and pyknosis of nuclei were present, they had to be looked for
carefully because the large regenerative hepatocytes hid the
degenerative ones. The structure of hepatic lobules remained
intact, but there was marked variation in the size of hepatic
lobules. Fat vacuoles were demonstrated in some hepatocytes with
oil red O stain; however, fat was not nearly as abundant as in the
fatty metamorphosis of acute selenium toxicosis. Cirrhosis had
not developed in these livers with subacute selenium.toxicosis.

Additional histologic lesions in the pigs with subacute
selenium toxicosis were similar to the more prolonged cases of
acute selenium.toxicosis. These included hemorrhagic necrosis of
the adrenal cortex, edema of pancreas and brain, myopathy, cellular
depletion of the bone marrow, degenerative changes and vacuolation

of the anterior pituitary.

81

Hepatosis dietetica. The livers from pigs with dietary liver
necrosis were characterized by hepatic lobules with extensive.
nuclear pyknosis, karyorrhexis and cytolysis of hepatocytes, loss
of.sinusoidal architecture, and massive hemorrhage into the
affected lobules (Figure 17). Some adjacent-hepatic lobules
appeared relatively normal. There was also interlobular prolifera-
tion of connective tissue and bile ducts.

In the molecular layer of the cerebellum from 1 pig, there was
fibrinoid necrosis of the walls of small blood vessels and peri-
vascular hemorrhage (Figure 18). The skeletal muscles had early
changes of myopathy characterized by hyalinization, loss of stria-
tions and fragmentation of myofibers. One section of heart also

had hemorrhage between the myocardial fibers.

Ultrastructural Changgs'

After examining 114 electron photomicroscOpic exposures (2 or
3 embedded blocks and 3 or 4 grids from each pig) of liver from
Figs 6, 37, 4 and 30 to compare the ultrastructural changes of
selenium toxicosis with hepatosis dietetica, it is suggested that
there is a difference bewteen the osmiOphilic prOperties of large
lipid bodies in the cytoplasm of hepatocytes in selenium.toxicosis
and hepatosis dietetica. The cytOplasm of hepatocytes from control
Fig 30, fed 0.1 ppm selenium, did not have these lipid bodies. In
Pig 6, with selenium.toxicosis, the cytOplasmic lipid bodies in
hepatocytes were smaller and less electron-dense (Figure 19) than.
those in Pig 4 with hepatosis dietetica. The large, densely osmio-
philic lipid bodies in hepatocytes of Pig 4 contained smaller, less

electron-dense bodies (Figure 20). The cytoplasmic membrane was

82

.I-‘: ft '-
55“?"7"

 

Figure 17. Necrotic, hemorrhagic lobules and
normal adjacent lobule in liver from pig with hepa-
tosis dietetica. H & E stain. x 180.

 

Figure 18. Fibrinoid necrosis of small blood ves-
sel with perivascular hemorrhage in the molecular
layer of cerebellum from pig with hepatosis dietetica.
Compare with Figure 12. H & E stain. x 720.

6'9'

83

    

    

“. .1 . 9...“. , .
En", ~ , .'
..’ _., '. av ‘ -.

.w . ., . .

e from Fig 6 fed 45 ppm selenium
Notice lipid bodies
uranyl acetate and lead

     

-‘ ‘i: 7“ ~ u '9‘
Figure 19. Hepatocy
'as sodium selenite for 63 days.
(arrows) in cytoplasm. Stain:

citrate. x 12,000.

 

e from Pig 4 with hepatosis

Notice osmiophilic lipid bodies (arrows)
Compare density of these lipid bodies
uranyl acetate and

Figure 20. Hepatocyt
dietetica.
in cytoplasm.
with those in Figure 19. Stain:
lead citrate. x 12,000.

84
more distorted with hepatosis dietetica probably because of crowding
by the numerous erythrocytes. Swollen mitochondria and vacuolation
of endoplasmic reticulum.were seen in the hepatocytes with both

selenium toxicosis and hepatosis dietetica.

Selenium Content in Tissues

The mean terminal selenium content, expressed as ppm selenium
(wet weight basis), in liver and kidneys from each pig in both
Experiment A and Experiment 3 is tabulated in Tables 14 and 15,
respectively. In Experiment A, pigs fed 5 ppm selenium or more
without clinical toxicosis had ranges of selenium residues of 2.27
to 18.0 ppm.in livers and 2.19 to 20.1 ppm in kidneys. The pigs
with clinical toxicosis had ranges of selenium residues of 6.7 to
36.2 ppm in livers and 2.73 to 22.4 ppm in kidneys. In Experiment
3, the.pigs with fatal toxicosis had ranges of selenium residues
of 5.62 to 48.2 ppm in livers_and 2.56 to 15.2 ppm in kidneys.

Statistical analyses of the above data substantiated the fol-
lowing conclusions when statistical differences were identified
at the 0.05 or less level of probability. In Experiment A, the
selenium content in livers from pigs with selenite toxicosis was
not significantly different than that in livers from pigs with
selenomethionine toxicosis. The selenium content in kidneys from
pigs with selenite toxicosis was not significantly different than
that in kidneys from pigs with selenomethionine toxicosis. Also,
there was.no significant difference in the selenium content between
livers and kidneys from.pigs with selenite toxicosis or from.pigs

with selenomethionine toxicosis.

85

Table 14. Terminal.selenium content in liver and kidney from pigs
fed selenium in Experiment A

 

 

 

Selenium
added to Selenium Selenium concentration

Pig ration compound (ppm, wet-wt.)
no. (ppm) fed Liver Kidney
4*. 0 -- 0.11 0.37
35 0 --- 0.20 1.27
5 0 --- 0.20 0.90
31* 0 --— 0.09 0.43
24 0.1 8e03** 0.42 1.41~
34 0.1 $603 0.39 1.05.
13 0.1 SeMET** 0.22. 0.90,
30 0.1 SeMET 0.23 0.89
11. 5 Se03 2.45 2.19.
19 5 $603 2.27 2.64~
12 5 SeMET 4.66 6.64-
17- 5 SeMET 5.13 6.58
15 10 $603 3.36 4.07
32 10 3603 3.88 3.14
8 10 SeMET 9.13 10.1

9 10 SeMET~ 10.7 10.7~
23+ 20 SeO3 36.2 9.52
36 20 SeO3 8.04 16.3

3 20 SeMET 6.90 20.1
26 20 SeMET 18.0 17.4
6: 45 5603 8.19 11.9
18+ 45 $603 6 70 2.73
7 45 SeMET 21.5 15.7
20+ 45 SeMET' 14.6 8.21
1+ 100 SeO3 36.0 10.3
37+ 100 $603 27.4 17.2
14:‘ 100 SeMET 24.3 22.4
38 100 SeMET 14.2 11.0

 

*

These pigs had the-lesions of hepatosis dietetica.
**

SeO3-sodium selenite; SeMET-selenomethionine.

+Thesepigs developed clinical selenium toxicosis.

86

Table 15. Terminal selenium content in liver and kidney of pigs
given selenium in Experiment 3*

 

 

 

 

 

 

Selenium
added to Selenium Selenium concentration
Pig dry ration compound (ppm, wet wt.)
no. (ppm) fed Liver Kidney
4B. 0 --- 0.26 0.73
133 0 --- 0.21 0.45
53 60 Se03** 6.05 4.44
153 60 SeO3 15.9 5.09
68 60 seMET** 15.3 12.1
163 60 SeMET 20.6 15.2
13 120 SeO3 5.62 2.65
113 120 SeO3 9.60 2.98
23 120 SeMET 48.2 14.1
83 120 SeMET 20.8 8.08'
73' 600 $603 9.10 2.56
93 600 SeO3 9.54 3.35
103 600 SeMET 28.5 14.4
123 600 SeMET 26.2 11.1
Selenium
injected Selenium
i.v. (mg/ compound
kg BW) injected
33 3 SeO3 10.2 7.28
143 3 SeMET 11.3 10.8

 

*
All pigs given selenium.hecame moribund or died with
toxicosis.

**
SeO3-sodium selenite; SeMET-selenomethionine.

87

In Experiment 3, the selenium content of both livers and kidneys
from pigs with selenomethionine toxicosis was significantly greater
(P<0.01) than that in these organs from.pigs with selenite toxicosis.
Also, the selenium content of livers from pigs with selenium toxi-
cosis (both compounds) was significantly greater (P<0.025) than-that
in kidneys, but the selenium content in these livers had signifi-
cantly more variation (P<0.01) than did the selenium content in “‘
kidneys.

In a comparison of Experiment A and Experiment 3, there was no

significant difference in the selenium content in livers from pigs

1

with selenite toxicosis or in livers or kidneys from pigs with
selenomethionine toxicosis between Experiment A and Experiment 3.
However, the selenium content in kidneys from.pigs with selenite
toxicosis in Experiment A.was significantly greater (P<0.025) than

that from pigs with selenite toxicosis in Experiment 3.

DISCUSSION

This research supplied important new information on the pathology
of selenium toxicosis in young pigs which was the primary purpose
of these studies. Specifically, several clinical features and gross
and microscopic lesions were determined and recorded for both
inorganic and organic.selenium.toxicosis in 2 experiments. In-
addition to characterizing the lesions of selenium toxicosis using
both sodium selenite and selenomethionine, these lesions were come
pared with those of vitamin E-selenium deficiency or hepatosis
dietetica in the same experiment by feeding a Torula yeast basal
ration. This latter disease was previously studied in more detail
in this department by Michel et al. (1969).

Individual pigs had variability in their response to the
amounts of selenium.admdnistered. Some of this variability may
have been due to appetite, selenium tolerance and other inherent
biological variation of each pig.

Consumption of the Torula yeast basal, semipurified diet was
not as good as had been anticipated; however, the Torula yeast-
basal diet provided a ration to study the lesions of both selenium
toxicosis and vitamin E-selenium.deficiency in the same experiment.
A much higher daily consumption of a practical or natural ration
might be expected. With higher consumption, selenium toxicosis
might have been induced at lower selenium levels in the feed than
those used in Experiment A. However, a natural ration may have

88

89
afforded more protection against toxicosis than did the semipurified
ration.. In Experiment 3, the consumption and palatability of the
milk basal ration with added selenium probably was better than
might be expected with a selenized natural ration. Consequently,
the pigs in Experiment.3 ingested larger amounts of selenium more
rapidly thanucould be expected under natural conditions. The pigs
used in this research were quite young and perhaps were more suscept-
ible to selenium toxicosis than older pigs would have been. For
the above reasons, the information collected in this research must
be used judiciously in its application to naturally occurring
selenium toxicosis in swine.

The preliminary experiments in pigs and steers were informative
and useful in designing the main experiments and in standardizing
techniques. The amount of total selenium that could be administered
intravenously to either species was unexpectedly large although the
selenium dosages administered on a body-weight basis were quite small
compared to those of the common veterinary drugs. The rather severe
local reaction to intramuscular injections of moderate amounts of
selenite solutions was interesting and suggested the need for
multiple inj ections.

The results of the main experiments indicated that selenium
from either sodium selenite or selenomethionine was highly toxic
to young pigs. In Experiment A, Fig 18 died 3 days after consuming
only 13.5 mg total selenium (3.2 mg selenium/kg body weight) in its
feed. In Experiment 3, the calculated mean lethal dose was 9.4 mg
selenium/kg body weight in pigs fed sodium selenite and 13.6 mg
selenium/kg body weight-in pigs fed selenomethionine. One intra-

venous injection of 3‘mg seleniumlkg body weight as either sodium

 

90

selenite or selenomethionine into 2 pigs resulted in fatal toxicosis
in 2-1/2 hours and 14 hours, respectively. The above results indi-
cated that under the conditions of this research the minimum lethal-
dose of selenium as sodium selenite was lower than the 13.2 to
17.6 mg selenium/kg body weight reported by Miller and Williams
(1940a) and confirmed by Diener (1961). The toxicity of seleno-
methionine in Experiment 3 more nearly paralleled the above reports.

No real difference was noted in the toxicity of selenium from
sodium selenite or selenomethionine in Experiment A; however, in-
Experiment 3 it appeared that selenium as sodium selenite was
slightly more toxic than selenium as selenomethionine. In earlier j
work the 48-hour LD75 in rats for selenomethionine and sodium
selenite was 4.25 and 3.25 to 3.50 mg seleniumlkg body weight,
respectively (Franke and Moxon, 1936; Klug et al., 1949). Thus,
selenomethionine should probably be considered less toxic than
sodium selenite although no statistically significant difference
was evident in this research. This is contrary to the toxicity
of organic selenium compounds found in natural cereals inasmuch as
Franke and Painter (1938) indicated that the relative toxicity of
selenium in the different diets was in the order: wheat > corn >
barley > selenate > selenite. 'This~order of relative toxicity of
selenium.assumed that selenium was the sole toxicant in selenized
cereals. Some reports have mentioned a concern about unidentified
phytotoxins with selenium during natural selenium toxicosis (Muth
and Binns, 1964).

Stowe (1962) indicated that vitamin E-selenium deficiency
disease in swine could be expected when the.serum levels of

tocopherols were below 0.65 ug/ml. On this basis, the pigs in

91
Experiment A were in a state of marginal vitamin E deficiency.
Hepatosis dietetica observed relatively early in 2 pigs that were
not fed selenium.could be explained by marginal levels of plasma
tocopherol. This marginal vitamin E deficiency may have enhanced
selenium toxicosis in pigs fed selenium in the early part of
Experiment A. While Morss and Olcott (1967) could not demonstrate
an effect of tocopherol supplementation on acute oral toxicity of r
selenium in rats, vitamin E deficiency may increase susceptibility
to selenium.toxicity. In addition to the vitamin 3 status, the

selenium was probably absorbed more efficiently in these marginally

 

deficient pigs (Wright, 1967). i
Selenium toxicosis in this research produced both hepatic and
muscular degeneration. Thus, the elevated levels of serum or
plasma GOT and OCT that were observed could be expected but could
not be directly cqrrelated with the lesions of selenium toxicosis,‘
especially in acute cases. For the diagnosis of selenium toxicosis,
plasma or serum levels of these enzymes were nonspecific. Michel
et al. (1969) reported that the levels of serum OCT were elevated with
dietary liver necrosis (vitamin E-selenium deficiency) in the pig.
Orstadius (1960) found that plasma levels of GOT were increased but
plasma levels of OCT were not increased and concluded after post-
mortem examination that muscles were more vulnerable in selenium
toxicosis than other organs including the liver. The results of
the present research did not totally support the conclusions of
Orstadius.
In addition to the above clinicapathologic.findings, hemograms
were not changed.and, therefore, would be of little specific value

in the diagnosis of selenium toxicosis in the pig but would aid in

92
a differential diagnosis. Diener (1961) also made a similar
observation.

The selenium content in livers and kidneys was highly variable
in pigs fed selenium with or without selenium toxicosis. There were
selenium.concentrations in liver and kidney of healthy pigs fed
selenium that were greater than those in pigs that died of selenium
toxicosis. Thus, there must have been a significant mechanism for
tolerance to selenium in these pigs. Wahlstrom.and Olson (1959)
previously observed tolerance to selenium in pigs from sows fed
selenium during gestation.

Although selenium accumulates in the body and death results,
there was no concentration in liver or kidney below 20 ppm selenium
that identified selenium toxicosis under the conditions of this
research. Selenium.analyses of these organs will readily differen-
tiate between pigs fed selenium and pigs not~fed selenium. In a
comparison of the selenium content of livers and kidneys from.pigs
with selenium toxicosis, kidneys, as a group, had less variation
in their selenium content than did livers which indicated that
kidneys gave the more reliable or accurate estimation of selenium
status.. But in addition to this, the ratios of selenium content
between livers and kidneys generally were: 1-4:l in pigs with
selenium toxicosis and 1:1-4 in pigs without selenium toxicosis,
respectively. Glenn.et al. (1964c) also observed a similar rela-
tionship between the. selenium content of liver and kidney from sheep
‘with selenate.toxicosis. Thus, as an aid in establishing a diag-
nosis of selenium toxicosis, the selenium content in liver and
kidney from a suspected animal should be evaluated not only by the
amount in each organ.but also by the relationship of the amounts

between these 2 organs.

 

193

In general, the clinical signs of anorexia, emesis, loss of
weight, weakness, depression, subnormal temperature and respiratory
distress were in agreement with those of earlier reports on a fewer
number of pigs (Miller and Williams, 1940a; Diener, 1961). The:
exceptions were that diarrhea was not common and rectal temperatures
were elevated early in the course of peracute and acute selenium
toxicosis.

Since the pigs readily consumed l or 2 feedings of the selenized
rations, the anorexia that develOped was probably due to a systemic
reaction of selenium rather than the unpalatability of the rations.
The loss of hair that occurred in l pig in Experiment 3 resulted 59
only after profound toxipathic anorexia. Thus, it can be specu-
lated that selenium toxicity may induce general and specific malnu-
trition which results in the classic clinical manifestations of
chronic selenium toxicosis such as loss of hair and hoof lesions.

Olson (1969b) mentioned that the rate and manner of selenium adminis-
tration was probably important in determining the resultant-clinical
manifestations since some experimental reports have failed to con-
firm the natural manifestations of selenium toxicosis.

The pathologic changes of selenium toxicosis in this research
involved many different organs. In general, the important histo-
logic lesion was microangiopathy which probably contributed to the
production of the lesions found in the lungs and brains of pigs with
peracute and acute selenosis. In acute selenosis of longer duration
and in subacute selenosis, the degenerative changes in the liver and
other organs contributed to the_demise of these pigs. Although the
liver appeared to be the primary target organ from.gross examina-

tion, histologic lesions were found in cerebrum, cerebellum, spinal

94
cord, pituitary gland, pancreas, lymph‘nodes, thumus, bone marrow, adrenal
gland, diaphragm and skeletal muscles. These histopathologic changes
had a wider distribution than previously described by others
(Schoening, 1963; Miller and Schoening, 1936; Miller and Williams,
1940a; Diener, 1961). This distribution of lesions indicated that
the pathogenesis and cause of death in selenium toxicosis may be
more complex than previous.research has suggested. It is believed
that the hepatic lesion alone was not severe enough to result in
death.

Histopathologic changes in the central nervous system have not
been previously reported. Laminar edema between the white and-gray
matter of the cerebral cortex in a few pigs with selenite toxicosis
gave some credibility to the previous association of polioencephalo-
malacia in cattle with the "blind staggers" of selenium poisoning
in cattle (Maag at al., 1960). In addition, the loss of granule
cells in the cerebellar granular layer-was similar to the cerebellar
lesion of chronic methylmercury toxicosis in calves reported by
Herigstad et a1. (1972). A relationship between the metabolism of
selenium and methylmercury has been suggested by others (Ganther
at al., 1972).

Selenium toxicosis and vitamin E-selenium deficiency have some
comparable changes. The severe lesions in the pancreas especially
with selenomethionine toxicosis are considered both interesting and
important because pancreatic changes have also been.suspected but
not clearly established in vitamin-E-selenium deficiency (Thompson
and Scott, 1970). The vascular lesions of selenium toxicosis are
comparable to the microangiopathy of vitamin E-selenium deficiency

in swine mentioned.by Trapp et a1. (1970). Edema, myOpathy, and

l'
7

 

g!

95

hepatic lesions are common.to both syndromes. Jubb and Kennedy
(1970) theorized that massive necrosis occurred in the liver with
selenium toxicosis as a result of nutritional deficiency since
selenium apparently replaces sulfur in sulfur-containing amino acids
and possibly produces a conditional deficiency in essential amino
acids. If this be true, it must be remembered that the massive
hemorrhagic.hepatic necrosis of vitamin E-selenium deficiency has "*
also been considered to be a complex nutritional deficiency involving
vitamin E, selenium, and sulfur amino acids. Although the biochemical
pathogenesis of bath selenium toxicosis and vitamin E-selenium
deficiency disease remains undetermined, ultrastructural studies 3
of advanced hepatic lesions in this research suggested that there
may be a difference in the composition of lipid bodies in the cyto—
plasm_of hepatocytes from livers of pigs with selenium toxicosis
or hepatosis dietetica. These ultrastructural studies should be
expanded to a separate project in and of themselves.

In_a differential diagnosis of selenium toxicosis and vitamin
E—selenium deficiency in swine, several differences can be empha-
sized. The clinical signs of selenium toxicosis were more prolonged
while sudden death characterized vitamdn E-selenium deficiency.
Anorexia and resulting cachexia was usually profound in selenium
toxicosis. On gross examination, the liver was pale to normal in
selenium toxicosis. In vitamin.E-selenium deficiency, if hepatosis
dietetica was present, the liver had multifocal areas of lobular
necrosis and hemorrhage with possible irregularities in Glisson's
capsule. The cachectic changes of serous atrophy of fat were
usually quite characteristic of selenium toxicosis of relatively

short duration. Histopathologically, the liver in selenium

 

96
toxicosis was characterized by fatty metamorphosis and centrolobular
eosinophilia and necrosis of all lobules without much congestion
while in vitamin E-selenium deficiency the liver was characterized
by massive lobular necrosis and intralobular hemorrhage with some
lobules remaining relatively normal. The selenium residues were
over 2 ppm selenium (wet weight basis) in selenium toxicosis while
they were less than 0.5 ppm selenium in liver with vitamin E-
selenium deficiency.

Selenium probably will become approved as a feed additive for
livestock. As with other minor.chemicals and some drugs, the full
impact of this practice on practical.livestock production, agronomic
practices and public health probably will not be known for a number
of years. Selenium is clearly established to be an essential micro—
nutrient for livestock. Likewise, in small absolute amounts it can.
be very toxic to livestock. With judicious use in rations, selenium
can be very valuable in minimizing livestock losses from selenium
deficiencies. If the‘past misuse of.other chemicals is any indica-
tion of the future, some misuse of selenium might be anticipated.

In the past the prophylactic and therapeutic use of selenium
preparations has been limited, but a number of reports have been
published attesting to the nature of livestock losses due to inad-
vertent errors in formulating, mixing or administering these selenium
preparations. One might,wonder about the livestock losses that have
been unidentified or have gone unreported. The information from

this research will be most useful in diagnosing selenium toxicosis

in livestock and equally important--or perhaps even more essential-—

in determining what is.not selenium toxicosis.

 

 

SUMMARY

A total of 47 young pigs and 6 yearling steers was used to

 

determine the toxicosis of sodium selenite and selenomethionine and F.-
to compare the lesions produced by both selenium compounds with

those produced by vitamin E-selenium deficiency in pigs. In feed-

ing experiments using rations based either on Torula yeast or

dried whole milk as dietary protein sources, pigs were fed comparable’ 1&3

graded amounts of both selenium compounds in the ration.‘

In 1 yearling steer, l intravenous injection of 1 mg selenium/
kg body weight-as sodium selenite produced death in 8 hours and
gross lesions of massive pulmonary edema, hydrothorax, and conges-
tion of the gastrointestinal tract, while 0.5 mg selenium/kg body.
weight given intravenously into another steer had no effect in 4
days. In both cattle and swine, intramuscular injections of a
selenite solution resulted in localized muscular necrosis. Two pigs
given 3 mg selenium/kg body weight intravenously as sodium selenite
or selenomethionine developed fatal selenium toxicosis in 2-1/2 and
14 hours, respectively. Pulmonary edema was the important lesion
of intravenous selenite toxicosis. Twenty-one pigs fed-20 to 600
ppm selenium as sodium selenite or selenomethionine developed the
clinical signs of emesis, anorexia, loss of weight, cachexia,
depression, respiratory distress, and coma with subnormal temperature
and death. The lesions of selenosis were similar for both selenium
compounds and included fatty metamorphosis and centrolobular

97

98

necrosis in the liver; congestion of renal medulla; necrosis in
lymphoid follicles; hemorrhagic necrosis of adrenal cortex; edema
and degenerative changes in the cerebrum, cerebellum and spinal
cord; edema and hemorrhagic necrosis of pancreas; serous atrophy
of body fat and degenerative changes in skeletal muscles. Lesions
in the central nervous system were generally more common with
inorganic selenosis while pancreatic lesions were associated with 3““5
organic selenosis. Microangiopathy was an important lesion of 2
acute selenosis while degenerative lesions and cachectic changes 1

of a wide variety of organs characterized selenosis of longer

 

duration . 1;;
The selenium content in liver and kidney, while variable, was-

somewhat proportional to the level and duration of selenium in the

feed. The diagnosis of selenium toxicosis in swine depends on_both

the nature of the lesions and selenium content of the liver and

kidneys. Selenium toxicosis can be differentiated from vitamin

E-selenium deficiency in swine by the characteristics of the

hepatic lesions.

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VITA

VITA

The author was born in Glendive, Montana, on July 13, 1940, as
the son of Mr. and Mrs. Raymond I. Herigstad. He was raised on the
family ranch and received his primary and secondary education in
the public schools of Glendive, Montana. His education was con-
tinued at Mbntana State University, Bozeman, Montana (1958-59,
1960-61) and Colorado State University, Fort Collins, Colorado
(1961-65). He received his D.V.M. degree in June, 1965.

Dr. Herigstad was in general veterinary practice at Mitchell,
Nebraska, from June, 1965, to December, 1968. He began his graduate
studies in pathology at Michigan State University in January, 1969.
He was an instructor of pathology from January, 1970, through June,
1970, in the Michigan State University Veterinary Pathology Diag-
nostic Laboratory. From July, 1970, through June, 1972, he was
supported by NIH Postdoctoral Fellowship. He received his M.S.
degree in pathology from Michigan State University in 1971.

The author is a member of the American Veterinary Medical
Association and the American Association of Bovine Practitioners.
He has received recognition by the following academic and scientific
societies: Phi Eta Signs (1959), Phi Zeta (1964), Phi Kappa Phi
(1971), Sigma Xi (1971 and 1972).

The author married Chris Ann Fisher in 1964 at Genoa, Colorado.

They have 2 daughters: Kelly May, 6, and Jamie Jo, 4.

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