TEE PAWGLQGY 0F VITAMiN E-SELENI‘W
BEFFSEENCY iN SWHE AS INFLUENCE!)
BY EXERCESE AND CONHNEMENT

Thesis for the Degree of M. S.
MICINGAN STATE UNIVERSITY
KENNETH M. AYERS
1970

THEF'S

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LIBRARY

Michigan State
University

 

 

 

mm; L" ":"Jlllis’ l

ABSTRACT
THE PATHOLOGY 0F VITAMIN E-SELENIUM DEFICIENCY IN SWINE

AS INFLUENCED BY EXERCISE AND CONFINEMENT

By

Kenneth M. Ayers

Eight pigs were used to study the pathology ofia vitamin E-selenium
deficiency, as influenced by exercise. The basal vitamin E-selenium
deficient diet was a purified ration containing adequate amounts of other
nutrients. It was composed of soya protein, cerelose, and cod liver oil.
The protein content of the ration was-decreased during the experiment,
and a stress agent, silver acetate, was fed to all pigs during the last
9 days of the experiment. .

Pigs fed the basal ration died suddenly after an average of-63 days,
and exhibited cardiac degeneration, liver necrosis, and muscular dystrophy.
Exercise did not significantly influence the clinical signs or severity
of the lesions. Pigs fed the basal ration and subjected to exercise did
display subendocardial hemorrhages, however, whereas the other pigs did
not. The administration of vitamin E and selenium provided complete

protection against the lesions noted.

THE PATHOLOGY OF VITAMIN E-SELENTUM DEFICIENCY_IN SWINE

AS INFLUENCED'BY'EXEKCISE"AND'CONFINEMENT

By

Kenneth M. Ayers

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1970

Dedicated to
My Lovely Wife

and Daughter

11

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to Dr. Allan
Trapp and Dr. C. K. Whitehair, for their guidance in the conduct of this
research and in the preparation of this manuscript.

The writer's gratitude is extended to all staff members of the
Department of Pathology for their counsel and assistance in this study.

The writer's gratitude is extended to Dr.'G. R. Carter for his"
assistance In this work.-

Finally, the author gratefully acknowledges the support given him

and the sacrifices made by his wife Jane.

iii

TABLE OF

INTRODUCTION AND OBJECTIVES .

LITERATURE REVIEW . . . . . .
Vitamj-n E. O O O O O O 0
History . . . . . . .

Absorption and Distribution .

Metabolic Role.

Selenium .

History . . . . . .

Tissue Distribution an

Metabolic Role.

Vitamin E Deficiency in Swine

Mulberry Heart Disease .

Field Occurrence.

Experimental Mulberry Heart

Dietary Hepatic Necrosis

Muscular Dystrophy . .

Serum Enzymes.

MATERIALS AND METHODS . .

Experimental Animals and Desi

Housing. .

Method of Exercise .

Rations and Feeding Practices

Supplements.

iv

CONTENTS
Page
. . . . . . . .‘. . . . . . . . l
. . . . . . . . . . . . . . . 2
. . . . . . . . . . . . . . . 2
. . . . . . . . . . . . . . . 2
. . . . . . . . . . . . 3
. . . . . . . . . . . . . . . . 3
. . . . . . . . . . . . . 5
. . . . . . . . . . . . . . . . 5
d Excretion . . . . . . . . . . 5
. . . . . . . . . . . . . . . . 6
. . . . . . . . . . . . . . . 6
. . . . . . . . . . . . . . . 7
. . . . . . . . . . . -.- . . . 7
Disease . . . . . . . . . 8
. . . . . . . . . . . . . . . 8
. . . . . . . . . . . . . . 9
. . . . . . . . . . . . . 10
. . . . . . . . . . . . . . 12
gn. . . . . . . . . . . . . . . 12
. . . . . . . . . . . . . . 12
. . . . . . . . . . . . . 12
. . .V. . . . . . . . . . . 13
. . . . . . . . . . . . . . 13

Page~

Serum Enzyme Determinations. . . . . . . . . . . . . . . . . 13
Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . 15
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Serum Enzymes. . . . . . . . . . . . . . . . . . . . . . . . l9
Pathology. . . . . . . . . . . . . . .'. . . .7. . . . . . . 19
Cardiac lesions . . . . . . . . . . . . . . . . . . . 19
Hepatic necrosis. . . . . . . . . . . . . . . . . . . 24
Muscular dystrophy. . . . . . . . . . . . . . . . . . 26
Additional Findings. . . . . . . . . . . . . . . . . . . . . 26
Ascites and hydrothorax . . . . . . . . . . . . . . . 26
Generalized venous congestion . . . . . . . . . . . . 26

Edema and hemorrhage of the gallbladder . . . . . . . 26

Pulmonary lesions . . . 23

DISCUSSION. . . . . . . . . . . . . . . . . . . .'. . . . . . . . . 29
Clinical Signs and Mortality . . . . . . . . . . . . . . . . 29
Protein Content of Ration. . . . . . . . . . . . . . . . . . 30
Growth . . . . . . . . . . . . . . . . . .-. . . . . . . . . 30
Effect of Silver Acetate . . . . . . . . . . . . . . . . . . 31
Serum Enzymes. . . . . . . . . . . . . . . . . . . . . . . . 31
Method of Exercise . . . . . . . . . . . . . . . . . . . . . 32
Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . 32-

Cardiac lesions . . . . . . . . . . . . . . . . . . . 32
Hepatic necrosis. . . . . . . . . . . . . . . . . . . 33
Hydropic degeneration of the liver. . . . . . . . . . 33
Muscular dystrophy. . . . . . . . . . . . . . . . . . 33
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

VITA O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O 40

 

 

Iable

Essa
Bxpe
Iota
Seek

Week

LIST OF TABLES
Table Page
1 Basal ration .-. . . . . . . . . . . . . . . . . . . . . . . 14
2 Experimental design. . . . . . . . . . . . . . . . . . . . . l6
3 Total and average daily weight gain. . . . . . . . . . . . . l8
4 Weekly SGOT values.(Sigma-Frankel units) . . . . . . . . . . 20

5 weekly LDH values (IOUO) o o o o a o o a o o o o ’0 o o o ‘0 o 20

vi

LIST OF FIGURES

Figure Page
1 Mottling of the myocardium and flaccid appearance of
right ventricle. Pig fed basal ration . . . . . . . . . . 22
2 Subendocardial hemorrhage. Pig fed basal ration and
exercised on treadmill . . . . . . . . . . . . . . . . . . 22
3 Subendocardial hemorrhage. Pig fed basal ration and

exercised on treadmill . . . . . . . . . . . . . . . . . . 23

4 Cardiac myOpathy. Note the local increase in muscle
nuclei. Pig fed basal ration. Limited exercise . . . . . 23

5 Hepatic necrosis. Pig fed basal ration. Arrows point
to fI'aCtures o a o o o o a o o o o o o o o o o o o 'o a o o 25
6 Hepatic necrosis. Note pyknotic nuclei, hemorrhage and
hydropic degeneration. Pig fed basal ration . . . . . . . 25
7 Muscular dystrophy. Note lysis of fiber and local

increase in muscle nuclei. Pig fed basal ration.
Limited exercise . . . . . . . . . . . . . . . . . . . . . 27

vii

 

 

 

Vitamin
with respect
I'nere have be
and the meta‘:
reported in I
of vitamin E
however. pert
and selenium
animals IEd <
Strass of 8)“

The Obje
the influence
diet; (2) ,0
Vitamin E‘seJ

dietary "S tn

INTRODUCTION AND OBJECTIVES

Vitamin E and selenium have been intensively studied in recent years
with respect to their importance in the nutrition of man and animals.
There have been numerous publications pertaining to sources, requirements,
and the metabolism of these nutrients. Clinical diseases have been-
reported in most species of animals, which respond to the administration
of vitamin E and/or selenium. The description of these diseases,
however, pertains to field problems in which the amounts of vitamin E
and selenium are undetermined. Information on the nature of lesions in
animals fed deficient amounts of these nutrients, and subjected to the‘
stress of exercise, is limited.

The objectives of this experiment were as follows: (1) to evaluate
the influence of exercise on swine fed a vitamin E-selenium deficient
diet; (2) to determine the effect of strict confinement on pigs fed a
vitamin E-selenium deficient diet; and (3) to evaluate the effect of

dietary "stress" in pigs fed a vitamin E-selenium deficient diet.

Iona-L's!!!

 

 

Vitamir
research in
syndrome hax
eerily to t'r.

Wine .

7 “8th

LITERATURE REVIEW

Vitamin E and selenium have been the subject of a great deal of
research in recent years. Many different aspects of the deficiency
syndrome have been studied and reported upon. This review pertains pri-
marily to the special pathology of vitamin E and selenium deficiency in

swine.
EEfiEEEiLlE

History. Evans and Bishop (1922) discovered the importance of vitamin

E in nutrition. They reported that female rats maintained on a diet
using casein as a source of protein had a high incidence of reproductive
failure. The condition could be prevented by feeding fresh lettuce,
dried alfalfa, or high levels of butterfat. Vitamins A,;n,wex C were
ineffective in preventing the disorder. In later work they isolated
from wheat germ 011, an alcohol, which possessed all of the properties
of vitamin E (Evans et aZ., 1936).

G. M. Calhoun, a professor of Creek at Berkeley, and a friend of
Evans, suggested that the new vitamin be named tocOpherol. The word is
derived from the Greek tacos, meaning childbirth, and phero, meaning
to bear or carry. The suffix "OZ" indicates the compound is an alcohol.

Tocopherol is composed of a basic chromane ring, on which methyl
groups are located at various points (Vasington at al., 1960). There are
7 methyl derivatives, of which alpha-toeopherol is the most abundant and

the most active.

3
Absorption and Distribution. The mechanism of absorption of vitamin E
appears to be similar to the other fat soluble vitamins (Wise at al.,
1962). Its absorption is probably linked with fat and is facilitated by
the bile. The gastrointestinal lymphatics may play a role in absorption
(Roels, 1967). Absorption appears to be incomplete, as large amounts are
excreted in the feces.

The administration of toc0pherol in the free form rapidlyresults in
high blood values. When administered in the acetate form, absorption is
much slower, probably as the result of partial saponification of the
compound in the intestine. The intact ester is then partially absorbed
and hydrolyzed in the tissues (Wiss et aZ., 1962).

The heart, lungs, spleen, liver, and mammary gland are sites of
major concentrations of vitamin E (Mason, 1942). Wise et al. (1962)
reported that much of the tissue vitamin E is concentrated in the mito-

chondria and microsomes.

Metabolic Role. The exact nature of the metabolic role of vitamin E is
unknown. Much of the research to determine its role has been concerned
with the ability of tocopherol to prevent the peroxidation of certain
essential compounds in the body, such as fatty acids, other vitamins,
and enzymes. The antioxidant theory is based on this ability, and Tappel
(1962) is one of the theory's most ardent supporters. He suggests that
vitamin E acts solely as a biological lipid antioxidant. If tocopherol
does function as a lipid antioxidant, then other antioxidants should be
able to replace vitamin E in preventing the deficiency. Diphenyl-P-
phenylene-diamine (DPPD), an antioxidant structurally unrelated to
tocopherol, can replace vitamin E in preventing encephalomalacia in
chicks (Singsen et aZ., 1955). Crider (1961) later reported that DPPD

will prevent fetal resorption in rats fed a vitamin E deficient diet.

4

The presence of.fat in the diet, especially polyunsaturated fats,
plays an important role in the production of many of the vitamin E
deficiency syndromes. Singsen at al. (1955) mentioned that a simple
vitamin E deficiency per se does not cause encephalomalacia in the chick.
The addition of unsaturated fats such as cod liver oil to a vitamin E
deficient diet will produce the disease, however.

The second part of Tappel's theory suggests that the fat undergoes
peroxidation, and that the resulting lipid peroxides damage the lipid
. component of lysosomal, microsomal, and mitochondrial membranes. Tappel
has measured increased lipid peroxide levels in the tissues of vitamin
E deficient animals.

If the lysosomal membranes were damaged, and their proteolytic
enzymes were subsequently released, these enzymes could conceivably cause
tissue damage by autolytic digestion. Bunyan et a2. (l967a,b), working
in England, described increased tissue levels of these hydrolases in
vitamin E deficient chicks exhibiting muscular dystrophy and exudative
diathesis. He has also reported increased-levels in vitamin E deficient
rats displaying testicular degeneration.

If_vitamin E acts as a lipid antioxidant, a deficiency of the
nutrient would cause decreased levels of unsaturated fats in the tissues
and increased tissue levels of lipid peroxides. Bunyan ettaZ. (1967c)
has reported that vitamin E deficiency does not depress total polyun—
saturated fatty acid levels in rat kidney, heart, Spleen, brain, adrenal,
and adipose tissue during experimental periods of up to 13 months. It
has also been reported that adipose tissue levels of lipid peroxides in
vitamin E deficient rats remain low unless large amounts of unsaturated
fats are fed for long periods of time (Bunyan st aZ., 1968). Bunyan

at al. (l967a) reported that there was no rise in lipid peroxides

5
in dystrophic chick breast muscle, cerebellum, cerebrum, or adipose
tissue. Similarly, lipid peroxide levels in chicks with exudative diath-
esis were not altered. In contrast to Tappel, these British workers
concluded that vitamin E is not concerned with lipid peroxidation in viva.
Other workers have suggested that vitamin E may be involved in ion
transport and cellular respiration. A discussion of these hypotheses

is included in the section on dietary hepatic necrosis.
Selenium

History. Prior to the 1950's, selenium was thought to be important only
as a toxic element found in the soil of certain parts of the United
States. In 1957, it was reported to be interrelated with vitamin E and
effective in preventing many of the syndromes attributed to vitamin E
deficiency (Schwarz, 1962). A year later, Muth (1963) reported that

0.1 ppm selenium protected lambs and calves against white muscle disease.
Selenium at a level of 0.3 ppm was reported to prevent exudative diath-

esis in chicks (Patterson at al., 1957).

Tissue Distribution and Excretion. Wright and Bell (1966) reported that

 

an oral dose of radioactive labeled selenium produces peak plasma and
whole blood concentrations 12 hours after administration. Less than 20%
was present in the circulation 4 hours after an intravenous dose. Oral
administration results in 152 of the dose being excreted in the feces
and 7.52 being excreted in the urine. Following an intravenous dose,
212 is excreted in the urine and 2.72 is excreted in the feces. The
greatest concentration of selenium following intravenous administration

is in the kidneys.

6
Jones and Godwin (1963) found a preferential.concentration of
selenium in the nuclei of most tissues.
McConnell (1963) reported that selenium can pass the placental
barrier in dogs. He also found that 90% of the selenium in a lactating

bitch's milk is bound to protein.

Metabolic Role. Olcott-et a2. (1961) suggested that selenomethionine may
act as a lipid antioxidant in viva. Hamilton and Tappel (1963) mentioned
that selenium was 500 times more active as an antioxidant than alpha-
tocOpherol. They also reported that there did not appear to be any
synergism between the two nutrients. They concluded that selenium acts
independently in reacting with free radical intermediates of lipid
peroxidation, thus breaking the chain reaction.

Schwarz (1958b) suggested that selenium and vitamin E were each
essential to the proper function of two separate, but equivalent,
metabolic pathways. Either one of these pathways was sufficient to

maintain an animal's health.

Vitamin E Deficiency in Swine

 

The gross and microscopic lesions of vitamin E deficiency in the
pig may assume any one or a combination of many forms. The important
syndromes include: "yellow fat" (Davis and Gorham, 1954), liver
necrosis (Hove and Seibold, 1955), muscular dystrophy (Marr at aZ.,
1956), anemia (Nafstad, 1965), mulberry heart disease (Lamont et aZ.,

1950), cardiac and extracardiac vascular-degeneration (Grant, 1961).

7

Mulberry Heart Disease

 

Natural Occurrence. Lamont (1950), in Ireland, was the first to
describe a naturally occurring syndrome, which he called mulberry heart
disease (MHD). The myocardium of affected pigs was deep purple, and
there were subepicardial hemorrhages of varying size scattered over its
surface. Harding (1960), working in England, described the microscOpic
appearance of naturally occurring MHD. He observed areas of congestion
and hemorrhage in the myocardium. In addition, there was pyknosis of the
muscle nuclei, hyaline degeneration, and loss of cross striations in the
myocardial fibers.

Donnelly (1969), working in Ireland, has described three separate
clinical types.of MHD. Type I occurs in 80% of all clinical cases, and
is characterized by sudden death. Type II occurs in 17% of affected
pigs. Prior to death, skin pallor, cyanosis, palpebral edema, respira-
tory distress, and ataxia are observed. Type III is characterized only
by a short period of ataxia before death.

A Swedish worker, Grant (1961), has made an extensive study of
naturally occurring MHD. Sudden death was the most common clinical sign
reported. Gross lesions included subepicardial petechiae and ecchymoses,
subendecardial hemorrhages, mottling of the myocardium, hydrothorax,
and ascites. Histopathologic examination revealed congestion, hemorrhage,
and myocardial degeneration. Sections stained by the periodic acid-
Schiff method demonstrated an accumulation of PAS-positive material in
the walls of capillaries, precapillary arterioles, and arterioles. Since
the latter was the primary lesion, he named this disease "microangiopathy"
(MAP). Extracardiac MAP was found in the lungs, skin, kidneys, stomach,
skeletal muscle, liver, and adrenal capsule. He concluded that MHD and

MAP are one disease.

T?

 

 

 

1:!

8
(Experimental Mulberry Heart Disease. Grant (1961) produced MHD (MAP)
with various experimental diets, and has demonstrated"the protective
effect of selenium and tocopherol. Pigs fed a diet consisting of a semi-
synthetic soya meal, with cod liver oil, developed MAP. Pigs fed the
same diet to which sodium selenite (0.2 mg./kg. of ration) was added
were fully protected. Pigs fed grain and high levels of corn oil
developed MAP. TocOpherol supplementation effectively suppressed the

development of MAP in pigs fed this diet.

Dietarngepatic Necrosis

 

Quin and Shoeman (1933), in the United States, made one of the
earliest reports of what was probably dietary hepatic necrosis (DHN).
They investigated an outbreak in a herd of swine in Iowa and described
the livers as being covered with spots of hemorrhage. They were unable
to determine the cause of the outbreak.

Daft et a2. (1942) reported the occurrence of DHN in rats fed a
casein-base diet. The addition of methionine 0r cystine in sufficient
amounts prevented the disease.

Dietary hepatic necrosis has been produced in swine fed a vitamin
E deficient diet containing cod liver oil (Hove and Seibold, 1955).

Michel at al. (1969), using a fat-free torula yeast diet, produced
DHN in swine. Supplementation with methionine or low levels of vitamin
E did not completely prevent the disease. High levels of vitamin E,
additional protein, or selenium offered complete protection.

The basic biochemical disturbance in dietary hepatic necrosis is
unknown. Schwarz (1962) has reported that liver in the latent phase of
DHN cannot maintain normal respiratory activity in vitro. He has named

this phenomenon respiratory decline. He theorizes that the cause of this

9
decline is a blockage of sensitive sulfhydryl sites, by trace element(s),
on an enzyme necessary for respiration in mitochondria. The origin of
the trace element(s) is unknown.

McLean (1963) suggests that the basic lesion is faulty ion transport.
Normal rat liver slices lose potassium in cold saline solution and reac-
cumulate potassium.when placed in warm, oxygenated Ringer's solution.
Liver slices from rats fed a vitamin E deficient diet are unable to
reaccumulate the potassium lost during cooling. In addition, oxygen
uptake in the cooled liver slices decreases. The addition of vitamin E
to the diet abolishes these defects, whereas selenium does not.. If liver
slices from vitamin E deficient rats are incubated as soon as they are
cut, the ionic composition does not change, and oxygen uptake does not
decrease. McLean suggests that the defects in vitamin E deficient tissues,
that are caused in vitro by the stress of cooling, may be produced in
vitro by some other stress. Vitamin E deficient tissues may not be able

to recover from situations that stress the ion transport mechanism.

Muscular Dystrophy

 

Metzger and Hagan (1927), at Cornell, were the first to report
muscular dystrophy (MD) in animals. They investigated an outbreak that
occurred in a flock of sheep, but were unable to determine the cause.
Marr at al. (1956) reported 3 outbreaks of MD in sheep in the north of
Scotland.

Diet plays an all-important role in the production of the disease.
Century and Horwitt (1960) have reported that the addition of 72 cod
liver oil, 72 linseed oil, or 15% corn oil to a vitamin E deficient diet
will produce the disease readily in rats. The addition of 152 coconut

oil or 0.2% corn oil failed to produce the disease.

10

A diet using purified soya meal as a source of protein has been used
to produce muscular dystrOphy in swine (Ewan et al., 1969).

Young et a1. (1961) reported that the disease in lambs is not related
simply to the length of time their dams were fed a diet known to cause
muscular dystrophy, but was dependent on the stage of gestation or lac-
tation during which it was fed. The greatest incidence of MD in lambs
occurred when the dystrophogenic ration was fed to ewes during late ges-
tation and early lactation.

High levels of sulphur in the diet may negate the protective effect
of selenium and increase the incidence of muscular dystrophy (Hintz and
Hogue, 1964).

The amount of activity the muscle is subjected to appears to play an
important role in the production of the disease. Pappenheimer and Goettsch
(1940) performed unilateral sciatic nerve sections on young rats nursing
dams fed a vitamin E deficient diet. Muscular dystr0phy was later found
in the gastrocnemius of the leg with the intact nerve. The gastrocnemius
of the leg on which a nerve section had been performed displayed only
those changes associated with nerve section.

Young and Keeler (1962) immobilized the left foreleg of lambs born
to ewes fed a diet known to cause muscular dystrophy. Muscular dystrOphy

was later observed, but the muscles of the left foreleg were unaffected.

Serum Enzymes
Serum enzyme determinations are useful as an aid in the diagnosis
of many diseases, and are often of value in prognosis.
Serum glutamic oxaloacetic transaminase (SGOT) levels have been used
to aid in the diagnosis of muscular dystrophy. The enzyme is found in

most body cells, but its concentration is highest in muscle (Medway et-

11
al., 1969). Blincoe and Marble (1960) have reported marked increases of
SGOT in sheep with white muscle disease.

Serum lactic acid dehydrogenase (LDH) levels are often used in man,
in conjunction with SGOT determinations, to aid in the diagnosis of acute
myocardial infarction (Medway st aZ., 1969). Blincoe and Marble (1960)
have reported that LDH values are markedly elevated in sheep with white

muscle disease.

MATERIALS AND METHODS

Experimental Animals and Design

 

A single litter of-6 male and 2 female Yorkshire-cross pigs was used.
They were purchased from a local farmer and were 12 days old when the
experiment began. Prior to the start of the experiment, the pigs were
identified by ear-notching. They were weighed at the beginning and end
of the experiment, and at intervals during its-course. Their final
weights were determined at the time of death or as soon after death as
possible. The pigs were observed twice daily for signs of illness.

The general experimental design is given in Table 2.

Housing

Pigs 1 through 6 were maintained on a concrete-floored 4' x 12'
pen. The pen was in a heated, isolated room. Pigs 7 and 8 were main—

tained in 1.5' x 2' metal metabolism cages.

Method of Exercise
Pigs 1 through 4 were exercised daily for 30 minutes on a motor
driven treadmill. The circumference of the treadmill was 90 inches and,
when in operation, it revolved at a speed of 25 rpm. The total distance
traveled by each pig in the 30~minute exercise period was approximately
1 mile. Pigs 5 and 6 were allowed to move freely within their pen.

Movement was restricted in Pigs 7 and 8 by the confines of the cages.

12

13

Rations and Feedinngractices

 

All pigs were fed twice daily. Those in individual cages were fed
and watered in separate bowls. Water was supplied to the penned pigs by
an automatic device. The caged animals were watered individually. They
emptied their bowls frequently and were often without water during part
of the night.

The feed was prepared in a Hobart commercial mixer,* in amounts cal-
culated to last 1 week. The prepared feed was stored in polyethylene
bags at room temperature.

During the last 9 days of the experiment, silver acetate was added
to the basal ration as a 0.15% (weighrfvolume)'solution.

The basal ration and the time length each variation was fed are

given in Table l.

 

Supplements
The supplements used in this experiment included:. vitamin E (aqueous)
and selenium. One hundred I.U. of aqueous vitamin E and 0.2 mg. of
sodium selenite were administered to Pigs 1 and 2 by intramuscular injec-

tion, 3 times weekly.

Serum Enzyme Determinations

 

Blood samples were collected from the anterior vena cava at weekly
intervals during the course of the experiment. Terminal samples were,
collected prior to euthanasia of the supplemented animals and as soon
after death as possible in the unsupplemented pigs. The samples were

then centrifuged, and the serum.was withdrawn and frozen. They were thawed

 

*Hobart Manufacturing Company, Troy, Ohio.

Table 1.‘ Basal ration

l4

 

Day 1 to 41

fi'

Day 42 to 48

Day 49 to 67

 

Purified Soya Mea1* 25%

Cerelose 60%
Cod liver oil 5%
Cellulose 5%
Mineral Mix** 4%
Vitamin M1x*** 25 ml ./
kg. ration

Purified Soya Meal 10%

Cerelose 79%
Cod liver oil 5%
Cellulose 5%
Mineral Mix 4%
Vitamin Mix 25 ml./
kg. ration

Purified Soya Meal 5%

Cerelose 80%
Cod liver oil 5%
Cellulose 5%
Mineral Mix 4%
Vitamin Mix 25 m1./

kg..ration

 

*General Biochemicals, Chagrin Falls, Ohio.

protein.

**Mineral Mix:

Approximately 82%

Calcium carbonate 26.8760%
Calcium phosphate monobasic 8.5110%
Cupric sulphate 0.0268%
Ferric citrate 2.4620%
Magnesium sulphate 9.1380%
Manganese sulphate 0.03132
Potassium iodide 0.07l6%
Potassium phosphate dibasic 37.8SOO%
Sodium chloride 15.0060%
Zinc chloride 0.0223%
Cobalt chloride 0.0050%
100.0000%
***Vitamin Mix:
Thiamine mononitrate 0.300 gm.
Riboflavin 0.600 gm.
Pyridoxine 0.200 gm.
Calcium pantothenate 3.000 gm.
Niacin 4.000 gm.
Para-amino-benzoic acid 1.300 gm.
Biotin 0.005 gm.
Folic acid 0.026 gm.
Inositol 13.000 gm.
Ascorbic acid 8.000 gm.
Choline chloride 130.000 gm.
Vitamin 312 (0.1% triturated) 10.000 gm.
Vitamin D2 (500,000 I.U./gm.) 0.044 gm.
Menadione sodium bisulfite 0.400 gm.
Absolute ethyl alcohol 250.000 ml.
Distilled water q.s. 2500.000 ml.

15

it

later and lactic acid dehydrogenase and serum glutamic oxaloacetic

transaminase* values were determined.

Pathology

The number of days each pig was maintained on the experimental
regimen is given in Table 2. The animals were necrOpsied at the end
of the indicated time period.

The supplemented pigs were euthanatized by electrocution. A care-
ful gross necrOpsy was performed on each pig. Tissues collected for
microscopic study included: stomach, duodenum, jejunum, ileum, spiral
colon, lung, heart, tongue, intercostal muscle, diaphragm, and masseter
muscle. Sections were also taken from the superficial gluteal, quadri-
ceps femoris, longissimus dorsi muscles, adrenal, kidney, urinary blad-
der, liver, gallbladder, ovary, testicle, brain, and skin. Blood and
tissue (liver) samples were collected and frozen for future analysis of
vitamin E and selenium content.

The tissue specimens for histopathologic examination were fixed
in 10% neutral buffered formalin. They were processed in an automatic
processing machine,** sectioned at 611, and stained with hematoxylin
and eosin and by the periodic acid-Schiff method, as described in the

Armed Forces Manual of’HistoZogic and Special Staining Technics (1960).

 

*Sigma Kit No. 505, Sigma Chemical Company, St. Louis, Mo.

**Autotechnicon, Technicon Company, Chauncey, New York.

16

Table 2. Experimental design

 

‘1

fl

 

Pig No. Treatment Exercise Days on Experiment

1 Basal + E-Se Treadmill*' 67+

2 Basal + E-Se Treadmill*‘ 641

3 Basal Treadmill*‘ 64++
4 Basal Treadmill*' 631+
5 Basal Penned** 66++
6 Basal Penned** 611+
7 Basal Caged*** 63++
8 Basal. Caged*** 654+

 

*3O minutes/day
**Free movement in pen
***Metabolism cages

IEuthanatized

++Died during the course of the experiment

RESULTS

The animals ate well during the greater part of the study period.
When the protein level of the ration was lowered to 5%, however, their
appetites decreased. Pigs 1 through 6 gained weight rapidly, were well
muscled, and had smooth, glossy hair coats. Pigs 7 and 8 gained very
slowly_and had very dull and rough hair coats. The total and average
daily weight gains are given in Table 3. During the last week of the
experiment, all pigs developed a diarrhea. Bacteriologic cultures of
the kidney, lungs, and spleen were negative for pathogens in Pigs l,

2, 3, 4, 6, and 7. Cultures of the ileum of the above animals were

also negative for pathogens. In Pig 5, there was a heavy overgrowth

of all organs and ileum with Proteus sp. In the viscera of Pig 8, there
was a moderate growth of Proteus vulgaris and a nonhemolytic Strepto-
coccus sp. salmonella sp. were isolated from the ileum of this pig.

It is presently being serotyped.

The supplemented animals had no difficulty running on the treadmill
at any time during the experiment. About midway in the study period,‘
Pigs 3 and 4 began to tire after approximately 20 minutes of exercise.
After a short period of rest they would appear normal. During the last
week of the experiment, Pig 3 would collapse and refuse to rise after
approximately 20 to 25 minutes of exercise. After a short period of rest
it would appear normal.lv

Death occurred suddenly in-the unsupplemented pigs. The pig would
appear normal at the evening feeding and be found dead the next morning.

17

18

 

 

Table 3. Total and average daily weight gain
Pig Starting Total Wt. Days on Avg. Daily
No. Treatment Wt. (kg.) Gain (kg.) Experiment gain (kg.)
1 Basal + E-Se (treadmill) 5.20 17.05 67 .26

2 Basal + E-Se (treadmill) 2.04 4.23 64 .07

3 Basal (treadmill) 5.59 12.59 64 .20

4 Basal (treadmill) 3.00 12.90 63 .21

5 Basal (penned) 5.21 12.49 66 .19

6 Basal (penned) 3.18 14.54 61 .24

7 Basal (caged) 3.63 6.82 63 .ll

8 Basal (caged) 4.36 8.82 65 .14

 

l9
Pigs 3 and 5 were observed just prior to death. They displayed ataxia,

respiratory distress, and collapse just prior to death.

Serum Enzymes

 

Nonhemolyzed blood samples were difficult to obtain. Serum glutamic
oxaloacetic transaminase (SGOT) and lactic acid dehydrogenase (LDH) values
were not determined in those samples exhibiting hemolysis. The SGOT
values were within normal limits in the supplemented pigs. The terminal
SGOT levels in Pigs 3, 4, 7, and 8 were markedly elevated. The SGOT was
slightly elevated in the last blood sample taken from Pig 6 before its
death. The weekly SGOT values are given in Table 4.

The LDH values were very high and variable in all animals. These

values are given in Table 5.

Pathology

Grossly there was mottling of the myocardium, liver necrosis,
generalized venous congestion, edema of the gallbladder, hydrothorax,
and ascites. Histopathologic examination revealed cardiac myopathy,
muscular dystrOphy, and pulmonary lesions. The following describes the

general gross and histopathologic appearance of the lesions.

Cardiac Lesions.

figggg. The hearts of the supplemented pigs were normal on gross examina-
tion. In those animals not supplemented with vitamin E and selenium,

the right ventricle appeared collapsed and thin on cut section. The sur-
face of the myocardium was mottled with pale and dark red areas (Figure 1).
These extended through the entire thickness of the myocardium. There were

numerous petechiae on the epicardial surface of the right ventricle of

20

 

 

 

 

 

Table 4. Weekly SGOT values (Sigma-Frankel units)
Week ' Week Week Week Week Week

Pig No. l 2 3 4 5 6 Terminal
1 34 H H H 22 54 28
2 30 H H H H 30 H
3 18 H H H 30 36 250
4 26 50 H H 30 60 240
5 18 50 H H 22 36 H
6 32 H H H 20 60 H
7 34 22 H H H H 220
8 44 H 36 54 134 224 240

H - hemolysis
Table 5. Weekly LDH values (I.U.)
Week Week Week Week week Week

Pig No. l 2 3 4 5 6 Terminal
1 900 H H H 1400 1440 H
2 750 H H H H 1390 1720
3 1180 H H H 1240 1720 1840
4 1280 1540 H H 1280 1660 1840
5 1320 1500 1400 H 1180 1030 H
6 1560 H H H 1440 1780 H
7 1160 1180 H H H H 1820
8 1560 H 1180 1280 1800 1800 1840

 

H - hemolysis

21
Pig 8. Numerous subendocardial hemorrhages were found in the left ven-
tricle of Pigs 3, 4, 5, and 6 (Figure 2). They caused the overlying

endocardium to bulge and appeared to extend into the myocardium a

few mm.

Microscopic. There was rowing of the sarcolemmal nuclei and slight con-
gestion of the myocardium of the pigs supplemented with vitamin E and
selenium.

The dark red areas observed grossly in the myocardium of the unsup-
plemented animals were extremely congested microscopically. There was
also hemorrhage between the muscle fibers in these areas. Those areas
that were pale grossly displayed slight congestion and little hemorrhage
between the muscle fibers. The subendocardial extravasations caused the
overlying endocardium to bulge, and the hemorrhage extended into the
myocardium, separating the muscle fibers slightly (Figure 3). The
hemorrhage also caused separation and displacement of the Purkinje fibers
in the area. There was a dramatic increase in the number of sarcolemmal
nuclei, and many appeared pyknotic. The majority of the nuclei were
arranged in linear rows. The sarcoplasm of many of the muscle fibers
appeared to be undergoing a granular form of degeneration. Within a
muscle cell, there would be many small, spherical, slightly eosinophilic
granules surrounding the nucleus. There were many focal areas in which
individual fibers or groups of fibers were undergoing fragmentation and
myolysis, and in these areas there were aggregations of sarcolemmal»
nuclei (Figure 4). The most severe lesions were observed in Pig 8. Very
little of the myocardium was unaffected. In addition to the changes.

listed above, there were many focal areas of calcification and fibrosis.

22

M“. f..
0‘ 0 JV I. ....

e o
,I\..D|

 

Mottling of the myocardium and flaccid appear-
Pig fed basal ration.

Figure l.
ance of right ventricle.

 

Pig fed basal

Subendocardial hemorrhage.

ration and exercised on treadmill.

Figure 2.

23

 

Figure 3. Subendocardial hemorrhage. Pig fed basal ration
and exercised on treadmill. Hematoxylin and eosin stain. x 400.

 

Figure 4. Cardiac myOpathy. Note the local increase in
muscle nuclei. Pig fed basal ration. Limited exercise.
Hematoxylin and eosin stain. x 400.

24
Sections stained by the periodic acid-Schiff method did not display

the microangiopathy described by Grant (1961).

Hepatic Necrosis

 

EEQEEJ The livers of those animals supplemented with vitamin E and
selenium were normal. The livers from the nonsupplemented pigs were
enlarged and friable. There were numerous red spots of varying size
scattered over the surface and in the parenchyma. There were 4 fractures,
each approximately 5 cm. long, on the surface of the liver of Pig 7
(Figure 5). There were large blood clots adhering to the fractured

areas .

Microscopic. In the livers from the animals supplemented with vitamin

E and selenium, the cytOplasm of the hepatocytes failed to stain with
hematoxylin and eosin. The lesion was not due to fat content, as an

oil red O stain was negative. Suitably fixed tissues were not available
for glycogen stains, but the lesion was suggestive of hydropic
degeneration.

In the livers from the nonsupplemented group, some lobules displayed
only a vacuolar degeneration of the hepatocytes and congestion of the
sinusoids. In other lobules, there was centrilobular necrosis.and
pooling of blood around the central vein (Figure 6). Hepatocytes'
peripheral to this were undergoing a vacuolar degeneration and the
sinusoids were congested. In many lobules, the architecture was com-
pletely obliterated by necrosis of the hepatocytes and a filling of the
lobule with blood. The necrosis was limited by the interlobular con-
nective tissue. In addition to these changes, there was a moderate

increase in the interlobular connective tissue-in Pigs 7 and 8.

25

‘
939'

,,.
A, 3.:
v “’

 

Figure 5. Hepatic necrosis. Pig fed basal ration.
Arrows point to fractures.

 

 

Figure 6. Hepatic necrosis. Note pyknotic nuclei, hemor-
rhage and hydropic degeneration. Pig fed basal ration. Hema-
toxylin and eosin stain. x 400.

26

Muscularggystrophy

 

Gross. There were no gross lesions of muscular dystrOphy in the sup-
plemented animals. The skeletal muscles of Pigs 3, 5, and 8 were slightly

pale.

MicroscoPic . There was no apparent pattern of involvement in the muscles
of the nonsupplemented animals. In some sections, only edema and sepa-
ration of the fibers were observed. In other sections, there was an
increase in the number of sarcolemmal nuclei, nuclear rowing, and focal
areas of myolysis. In these areas, there were aggregations-of sarcolemmal

nuclei (Figure 7).
Additional Findings

Ascites andHydrothorax. There were no significant collections of fluid
in the thorax or abdomen of the supplemented animals. There were 15 to
20 ml. of serosanguineous pleural fluid in Pigs 3, 4, 5, 6, 7, and

8. Approximately 20 ml. of serosanguineous fluid were found in the peri-
toneal space of Pigs 4, 6, and 8. There were approximately 60 ml. of
serosanguineous fluid in the peritdneal space of Pigs 3 and 5. There

were 110 ml. of sanguineous fluid in the peritoneal space of Pig 7.

Generalized Venous Congestion. In the nonsupplemented animals, there was
moderate to severe congestion grossly and microscopically in the follow-
ing organs: brain, posterior 2/3 of the small intestine, spleen, spiral

colon, lungs, kidneys, adrenal, liver, and stomach.

Edema and Hemorrhage of the Gallbladder. The gallbladders from the sup-
plemented animals were normal grossly and microscopically. Those from

the nonsupplemented animals were opaque and distended with bile on gross

27

 

Figure 7. Muscular dystrophy.' Note local increase in
muscle nuclei. Pig fed basal ration. Limited exercise.
Hematoxylin and eosin stain. x 400.

28
examination. The bile was very viscous, the wall of the structure was
thickened on cut section, and there was a line of hemorrhage at the
gallbladder-liver junction. MicroscOpically, the wall was very

edematous.

Pulmonary Lesions. On gross examination, the lungs of all pigs exhibited

 

some degree of pathologic alteration. In general, there was emphysema,
congestion, focal areas of atelectasis and, in some pigs, consolidation.
Microscopically, there were alternating areas of emphysema and atelectasis
and severe congestion. In Pigs 3, 4, 5, 6 and 7 there was pulmonary

edema and inflammatory infiltrates consisting of primarily lymphocytes.

DISCUSSION

Clinical Sigps and Mortality

 

Clinical signs were not a consistent feature of this experiment.
This is in agreement with the reports in the literature. The diarrhea
observed in Pigs 1, 2, 3, 4, 6, and 7 is difficult to explain, as all
organs and the ileum were negative for pathogens by bacteriologic culture.
In Pig 8, the diarrhea was very likely due to the salmonella that were
present. The salmonella organism may have played a role in the patho-
genesis of the diarrhea in Pig 5, but this would be difficult to prove,
as the only organism found was Proteus sp. A heavy overgrowth of one
organism may mask the presence of another, however.

The tiring observed in Pigs 3 and 4 when exercised may have been
due to a weakness in the skeletal muscles or the result of the decreased
output of a failing heart.

Sudden death, and the signs of ataxia, respiratory distress, and
collapse observed in Pigs 3 and 5 prior to death, suggest that acute
circulatory failure was the most probable cause of death in all animals.
The fractures of the liver in Pig 7 and the large quantity of bloody
fluid in its abdomen, indicate that in this animal a decrease in blood
pressure may have played a secondary role in its death. The cardiac
myopathy would almost certainly interfere with muscular contraction,
while subendocardial hemorrhage, by separating and displacing the Purkinje
fibers, would probably induce conduction defects. The condition of the
liver must also be considered in determining the cause of death. Acute,

29

30

massive liver necrosis would interfere with the liver's ability to per-

form a variety of vital functions.

Protein Content of Ration

 

The basal ration containing 25% protein was fed to all pigs for 41
days. At the end of this time, all pigs appeared clinically normal. It
was thought that perhaps the high protein level may have been exerting
a protective effect. Consequently, it was decided to lower the protein
level to 5%. At this point all pigs had been eating well. When the
protein level in the ration was decreased to 5%, however, there was-a
decrease in appetite. It was difficult to determine if supplementation
with vitamin E and selenium affected appetite as both the supplemented
and nonsupplemented pigs ate from the same pan.

The low protein level in the ration may also have played a role in

the pathogenesis of the ascites and hydrothorax observed in these animals.

Growth

Supplementation with vitamin E and selenium did not appear to en-
hance the growth rate. - This supports the work of Ewan at al. (1969).
The slowest growth rate was observed in an animal supplemented with
vitamin E and selenium. This animal, however, was the "runt" of the
litter, in that its weight at the beginning of the experiment was approxi~
mately half that of its siblings. Growth rate also seemed-to be depressed
in the nonsupplemented animals that were housed in metabolism cages.
There was an approximately 50% reduction in average daily gain in these
pigs. The "stress" of close confinement may have been a factor in sup-

pressing their growth rate.

31

Effect of Silver Acetate

 

Dietary liver necrosis has been observed in rats fed a vitamin E
deficient diet, to which silver acetate was added (Diplock at al.,
1967). The effect of silver acetate in this experiment is difficult to
evaluate. The day after it was first added to the ration, the first
death occurred. The lesions in this animal were quite severe, and it
would seem unlikely that the silver exerted any major influence in their
development. The effect of silver acetate in the remainder of the pigs
is impossible to evaluate, as all were being fed silver in the basal

ration.

Serum Enzypes

 

Serum enzyme determinations were hampered by the difficulty in
obtaining nonhemolyzed blood samples.’ This increased susceptibility to
hemolysis was observed in all animals at various times. This suggests
that vitamin E-selenium deficiency increases the fragility of the erythro-
cyte and makes it more susceptible to hemolysis. In support of this
finding, Fitch (1968) has reported increased susceptibility to hemolysis
in erythrocytes of primates fed a vitamin E deficient diet. Regardless
of its pathogenesis, any degree of hemolysis will interfere with
SGOT and LDH analyses. The markedly elevated SGOT values in the non-
supplemented pigs were probably the result of cardiac and skeletal
myopathy, as well as liver necrosis. Blincoe and Marble (1960) have
reported elevated SGOT values in sheep with white muscle disease. The
SGOT values in Pigs 3, 4, 6, and 7 increased quite late in the experiment.
In contrast, there was a more gradual increase in Pig 8, which suggests
that the lesions in this animal were of a more chronic nature. The

microscopic appearance of the heart and liver verified this finding.

32
The extremely high LDH values observed in all pigs may have been

due to trace hemolysis that could-not be detected visually.

Method of Exercise

 

Certain deficiencies in the design of the treadmill were encountered
during the latter part of the experiment. The animals survived longer
than was expected and, as a result, were almost too large to exercise
on the treadmill. They were able to st0p the treadmill at will, but
gentle coaxing prevented this. Perhaps a more powerful motor would have
prevented this problem.

Since the pigs were exercised daily, the possibility must be con-
sidered that they became conditioned to the exercise. In future experi-
ments, it might be wise to wait until the latter stage of the experimental

period before exercising the animals.

Pathology

Cardiac Lesions. The results of this experiment suggest that supplemen-

 

tation with vitamin E and selenium affords pigs complete protection
against the lesions produced in nonsupplemented animals. Grant (1961),
in a series of experiments, reached the same conclusion. The results
also suggest that some form of exercise enhances the development of sub-
endocardial hemorrhage. There was very little difference in the severity
of lesions between those pigs that were exercised on the treadmill and
those given limited exercise. This suggests that perhaps the amount of
exercise the pigs were given on the treadmill was insufficient to cause
any major difference in severity of lesions. The chronic nature of the
lesions in Pig 8 suggests that perhaps close confinement may be a greater

stress than exercise.

33
Hepatic Necrosis. The results of this study suggest that supplementation
with vitamin E and selenium completely protects pigs against hepatic
necrosis of dietary origin. Michel at al. (1969) has reported that high
levels of vitamin E or selenium will prevent hepatic necrosis in pigs fed
a vitamin E deficient diet. A very characteristic feature of the lesion
in this experiment was the limitation of the necrosis by the interlobular
septa. Chronic changes do occur, as is indicated by the proliferation

of connective tissue in the interlobular septa of Pigs 7 and 8.

Hydropic Degeneration of the Liver. The failure of the hepatocytes of
all livers to stain with hematoxylin and eosin, oil red O, or by the
periodic acid-Schiff method, suggests that this lesion was.hydropic
degeneration. This lesion may have been the result of the low protein
level in the ration during the latter part of the experiment. This is

in agreement with the report of Hove and Seibold (1955). The addition

of silver acetate to the ration may also have played a role in the patho-

genesis of this alteration.

Muscular Dystrophy. The results of this experiment suggest that supple-
mentation with vitamin E and selenium also fully protects pigs against
nutritional muscular dystrOphy. The degree of muscular dystrophy in the
experimental animals was not severe. Other workers have reported muscular
dystrophy in swine fed a vitamin E deficient diet in which soya bean

meal was the source of protein (Ewan et al., 1969).

There did not appear to be any difference in severity of lesions
between the pigs that were exercised on the treadmill and those given
limited exercise. Perhaps the amount of exercise the treadmill group
was subjected to was insufficient to produce any major difference in

severity. The pigs housed in metabolism cages had about the same degree

34

of muscular dystrOphy as those that were exercised. This also suggests
that close confinement may be as great a "stress" as exercise.
These results suggest that the basal ration may not be as dystro-

phogenic as other rations reported in the literature.

SUMMARY

Eight pigs were used to study the pathology of a vitamin E—
selenium deficiency, as influenced by exercise. The basal vitamin E-
selenium deficient diet was a purified ration containing adequate
amounts of other nutrients. It was composed of soya protein, cerelose,
and cod liver oil. The protein content of the ration was decreased
during the experiment, and a stress agent, silver acetate, was fed to
all pigs during the last 9 days of the experiment.

Pigs fed the basal ration died suddenly after an average of 63
days and exhibited cardiac degeneration, liver necrosis, and muscular
dystrophy. Exercise did not significantly influence the clinical signs
or severity of the lesions. Pigs fed the basal ration and subjected
to exercise did display subendocardial hemorrhages, however, whereas
the other pigs did not. The administration of vitamin E and selenium

provided complete protection against the lesions noted.

35

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Young, S., Hawkins, W. W., and Swingle, K. F.:. Nutritional muscular
deficiency: importance of ewe's diet at certain stages of gesta-
tion and lactation. Am. J. Vet. Res., 22, (1961): 412-415.

Young, S., and Keeler, R. F.: Nutritional muscular dystrophy in lambs--
the effect of muscular activity on the symmetrical distributions
of lesions. Am. J. Vet. Res., 23, (1962): 966-971.

 

VITA

Kenneth M. Ayers was born in Waukegan, Illinois, on 5 August, 1939.
He is a graduate of Pasadena Senior High School of Pasadena, Texas. He
completed his preveterinary and veterinary studies at Texas A & M Uni-
versity. He received his D.V.M. on 5 August, 1966.

Following graduation from veterinary school, the author entered
the United States Air Force as a First Lieutenant in the Veterinary
Corps. After completing his basic officer training at Sheppard AFB,
Texas, he was assigned to Royal Air Force Station Bentwaters, near
WOodbridge, England. He served there as Chief of Veterinary Services
from 28 October, 1966, to 2 August, 1969. Dr. Ayers was promoted to
the rank of Captain on 27 March, 1967.

On 29 September, 1967, Captain Ayers married the former Jane Mary
Black of Felixstowe, Suffolk, England. They have a daughter, Misty
Michele, aged 18 months, and are expecting the arrival of their second
child in September 1970.

In the fall of 1969, the author entered the graduate school at
Michigan State University, under the sponsorship of the Armed Forces
Institute of Technology, to pursue a Master's degree in veterinary
pathology. After graduation, he will begin an assignment at the Armed

Forces Institute of Pathology in Washington, D.C.

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