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THESIS

This is to certify that the

thesis entitled

ASPECTS OF VITAMIN E
AND SELENIUM NUTRITION IN
RELATIONS TO GASTRIC ULCERS IN SWINE

presented by

David M. Bebiak

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Animal NUtI'itiOIl

flfifm

Major professor

 

Date 20 1980

0-7839

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£15,; _.. ~

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MIChigan State
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ASPECTS OF VITNMIN E AND SELENIUM

NUTRITION IN RELATION TO GASTRIC ULCERS IN SWINE

BY

David M. Bebiak

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

DOCTOR OF PHILOSOPHY _

Department of Animal Husbandry

1980 .

ABSTRACT

ASPECTS.OF VITAMIN E AND SELENIUM
NUTRITION IN RELATION TO GASTRIC ULCERS IN SWINE

By
David M. Bebiak

Two experiments involving 70 weanling pigs were conducted to
evaluate the effects of vitamin E (E) and selenium (Se) on the incidence
and severity of gastric ulcers: Pigs were randomly assigned from litters
to two dietary groups, (1) a basal ulcerogenic diet (BUD) composed of
corn starch, soybean meal and corn oil adequately fortified with minerals
and vitamins except Se and E and (2) the BUD + .2 ppm Se + 44 IU E/kg
from d, z-alpha-tocopheryZacetate.. Data were collected from animals at
S, 10 and 20 weeks of age. The effect of E-Se supplementation was
determined by growth rate, serum levels of tocopherol and selenium,
erythrocyte glutathione peroxidase (GSH-Px) activity, gastric tissue
selenium, tocopherol, GSH-Px and cyclooxygenase, and the incidence of
gastric lesions.

Supplementation of the BUD with E-Se did not affect growth rate in
either experiment. Blood data of experimental animals reflected the
dietary levels of E-Se such that serum tocopherol, serum selenium and
erythrOcyte GSH-Px values were significantly greater among supplemented
animals. LikewiSe, tocopheral-selenium-GSH-Px levels of gastric tissue
collected from several anatomic regions of the stomach.ref1ected (sig-
nificantly) dietary supplementation of the nutrients E-Sel' In particular,
the tocophero1 and selenium concentrations and GSH-Px activity of the

esophageal region appeared to be most sensitive to B-Se supplementation

David M. Bebiak

and depletion. Unlike the data concerning growth and blood values

whiCh are Consistent with previous work, data concerning the regional
distribution of these parameters within the stomach were not available.
Among all regions of the intragastric surface, and between animals of
both dietary groups, the distribution of cyclooxygenase among common
tissue layers was similar. Among the tissue layers of all anatomic
regions examined, a relative abundance of the enzyme was present in the
mucosal lamina propria, especially of the esophageal region. No previous
work of this type could be located for comparison.

Finally, the stomachs of (3S) unsupplemented animals in the two
trials were characterized morphologically as follows: (9) normal, (20)
preulcerous lesions and (6) ulcers. The stomachs of the (35) supplem~
ented animals in the two trials were characterized as: (13) normal,
(18) preulcerous lesions and (4) ulcers; The ulcerogenic effect of
the BUD utilized in one trial of this study was believed to be reduced

by maintaining the pigs on straw bedding.

Dedicated to my wife Kim

TABLE OF

CONTENTS

ABSTRACT
LIST OF TABLES iii
LIST OF FIGURES iv
INTRODUCTION 1
LITERATURE REVIEW 2
Introduction 2
Vitamin E and Selenium As Antioxidants 3
Vitamin E and Selenium Deficiency Signs 8
Feed Particle Size and the Incidence of Gastric Lesions 9
Prostaglandins 9
Gastric Ulcers in Swine 15
Summary 17
METHODS AND MATERIALS 18
Experimental Design 18
Tissue, Serum Analyses _20
Characterization of Ulcers and Tissue Sampling 20
RESULTS AND DISCUSSION 22
Perfbrmance 22
Blood Values 22
Gastric Tissue 23
Feed Values 25
Cyclooxygenase 25
Gastric Lesions 34
SUMMARY 38
Conclusions 38
BIBLIOGRAPHY 41
APPENDIX 44

VITA

Table 1.

Table 2.

Table 3.

Table 4.

LIST OF TABLES

Composition of Basal Ulcerogenic Diet.
Trial I.

Summary of Data. Trial I.
Summary of Data. Trial II.

Distribution of the PG-Forming Cyclooxygenase
in the Porcine Stomach

iii

19

35

36

37

LIST OF FIGURES

Figure 1. Role of vitamin E (Toc.) and selenium (GSH-Px)
in free radical pathology (Adapted from .Tappel,
1974). Superoxide anion (0'), hydrogen peroxide
(H 02) glutathione peroxidage (GSH—Px) lipid (RH)
hydroperoxide (ROOH) peroxy free radical (R02)

oxidized tocopherol (Toc-O). 5
Figure 2. Vitamin E and selenium function (Adapted from

Brady 2.2.1: 1979). 6
Figure 3. Prostaglandin metabolism. 11

Figure 4. Prostaglandin fbrming - cylooxygenase - catalyzed
reaction. - 12

iv

INTRODUCTION

Several researchers studying the vitamin E (E)-se1enium (Se)
deficiency syndrome in swine have noted a high incidence of gastric
ulcers among experimental animalss‘ In general, the pathogenesis of the
mucosal lesions has been attributed to several factors including genetic
selection of meat-type swine, confinement rearing and the feeding of
high energy finely ground rations for greater feed conversion (Muggenburg
"égpalg 1967). .Also, certain prostaglandins have been reported to protect
the gastric mucosa of several species against experimental ulcerogens
(Robert, 1973); The mechanism of protection is not understood and the
action is described as cytoprotective (Chaudhury, 1978). Interestingly,
ulcers in swine are unique in that they always occur in exactly the same
anatomic region of the gastric lumenal mucosa.

In swine, gastric ulcers present a practical production problem. A
conservative estimate of the incidence of esophagogastric lesions in
swine would be 5 to 20 percent (Muggenburg 93321., 1966, 1967). Determining
more precise pathogenic factors may be useful in assisting swine producers
in diminishing this problem. The curious association between E-Se
deficiency, prostaglandin biology and the predictable appearance of gast-
ric ulceration in swine inspired this research effbrt toward more

accurately defining the ulcerogenic process.

LITERATURE REVIEW

Introduction

 

Gastric ulcers are an important problem in swine production as well
as human health. A voluminous amount of research has been conducted on
the nature of the problem, especially the physiopathologic aspects
(Muggenburg, 1966). While considerable infbrmation is available on the
nature of the disease, few data are available on the specific cause(s) of
the problem. In swine, the problem has been associated with intensified
production and the use of finely ground high energy diets intended to
maximize growth and feed efficiency (Mahan,'g£;§g, 1966; Muggenburg, 1967;
Perry, gt_al,, 1963). Morbidity and mortality varies from herd to herd
and from year to year without specific relation to age, sex, climate or
breed (Muggenburg‘ gt a_1_, 1967).. '

An unusually high incidence of ulcers has been reported in herds
characterized as vitamin E (E) - selenium (Se) deficient and among E-Se
deficient experimental animals. The possible relationship between E-Se
status and ulcerogenesis is not defined, nor is the relationship between
E-Se status and prostaglandin biology. As a class of naturally occurring
compounds the prostaglandins elicit a wide range of biological effects
including antiulcerogenesis (Moncada, 1978; Chaudhmnn 1978). In particu-
lar, prostaglandin 12 is a potent cytoprotective agent (Chaudlury, 1978).

This research is important in providing information which may help
define the relationships mentioned above. The data and observations
-generated here haVe potential usefulness in biomedical scienCe as well

as in swine production.

VITAMIN E AND SELENIUM AS ANTIOXIDANTS

Basic to the theme of this review, and theSis, is an understanding
of the free radical pathology theory (Butterfield gt_al,, 1979). A free
radical is a chemical in which the outer electron orbital has an unpaired
electron whiCh spins unopposed. This situation creates an unstable
electronic distribution and henCe a free radical is quite reactive. In
biology, compounds are converted to free radicals by chemical agents
called initiators.

The molecular structure of oxygen (02) may be represented as having
a covalent double bond (0=0) or as being a diradical (0-0) and actually
oscillates between these two forms.

The simple theoretical biological membrane containing interdigitating
hydrophobic, hydrophyllic and amphipathic substances forms a lipid bilayer
with a hydrophobic midzone area in which 02 is very soluble. Hydrocarbon
chains of the polyunsaturated fatty acids also exist in this hydrophobic'
midzone area. Thus, in the typical bilayer membrane the highest concen-
tration of diradical oxygen exists in the hydrophobic area where it has
the most destructive potential. The allylic hydrogen bonds (those involv-
ed with carbons in the B-position relative to an unsaturated C=C) are
relatively weak and therefore susceptible to radical initiation reactions.
Demopoulos £3 31. (1977) suggest that cholesterol prevents the normal
plasma membrane from being destroyed by lipid peroxidation. Cholesterol
enters the hydrophobic midzone area perpendicularly to the surface of the
membrane. Hydrocarbon tails of the polyunsaturated fatty acids, as well
as those of other lipids, wrap themselVes (hydrophobic fortes) around the
nonpolar end of the amphipathic cholesterol. The SterOid serVes as a

physical barrier between radical oxidative components and the susceptible

allylic bonds of the fatty acids. Apparently then, one of the many
roles of cholesterol may be that of'a protective antioxidant in normal
plasma membranes.

The broad range Of lesions which has been observed in animals under
conditions of E-Se deficiency might be explained by this theory of "free
radical pathology". Indeed, the role 0f E as a deterrent to lipid per-
oxidation is similar to that proposed for cholesterol in the cell membrane.
Since oxygen is soluble in the membrane it may catalyze free radical
peroxidation reactions which could proceed unchecked in the absence of E,
ultimately disrupting membrane integrity. Normal membrane integrity is
critical to membrane functions of permeability, transport, and bioener-
getics. The result of free radical peroxidation would be ”free radical
pathology" i.e. cellular damage and necrosis. In Figure 1 the roles of
E and Se in the free radical pathology theory are diagrammed. In Figure
2 the antioxidant functions of E and Se (as a component of glutathione
peroxidase) are detailed.

A free radical generated in normal metabolism is capable of reacting
with an allylic bond of an unsaturated fatty acid (RH). The oxygen
molecule becomes stable at the expense of the lipid which itself becomes
a type of radical (R) burdened with electron disruption at the reaction
site. This lipid radical (R-), in the presence of molecular oxygen, forms
a lipid peroxy free radical (R02) which is capable of initiating auto-
catalytic peroxidation of allylic bonds of adjacent unsaturated lipids
(RH'). The products of this biochemical reaction (R02 + RH') are a lipid
radical (R-') which again, in the presence of molecular oxygen and an
adjacent unsaturated lipid, may perpetuate this cyclic autocatalytic

peroxidative process. The trail of lipid hydroperoxides (ROOH) left in

(prooxidant

free Hydrophobic
radical) midzone
area

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GSH - Px
ROOH R0

ROH noon 4.57091) L J
(non-toxic) ‘\I:::I:;;>p=:;,4;l l V-‘ -A ‘7‘

W0

 

FIGURE 1. Role of vitamin E (Toc.) and selenium (GSH-Px) in
free radical pathology (Adapted from Tappel, 01974).
Superoxide anion (O ), hydrogen peroxide (H
glutathione peroxidase (GSH-Px) lipid (RH) fiydro-
peroxide (ROOH) peroxy free radical (R02) oxidized
tocopherol (Toc-O).

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the wake of this peroxidative pathway presents a region of hydrophyllic
polar groups in the nonpolar hydrophobic midzone of the cellular membrane.
Clearly, this is disruptive to normal membrane function, and eventually
the attraction of theSe polar groups to the water phase of the exterior
surfaces of the membrane will lead to membrane-lipid decomposition.

When the propagating chain reaction meets a membrane bound molecule
of E the chain reaction is broken (Tappel, 1974). The tocopherol compound
accepts the oxidation reaction without generating a chain perpetuating
lipid radical.

The role of Se as a limiting agent in the mechanism of "free radical .
pathology" is quite different from that of ET Selenium is an essential
element of the enzyme glutathione peroxidase (GSH-Px). As a cytosolic
enzyme with hydrophobic character, GSH-Px reacts with both'cytosolic
hydrogen peroxide molecules (H202) and membrane bound lipid hydroperoxides
(ROOH). GSH-Px reduces the lipid hydroperoxides (ROOH) to relatively
harmless hydroxy acids (RDH). Similarly the toxic hydrogen peroxides are
reduced to molecules of water (Tappel, 1974).

At this point it is worthy of noting that both dietary polyunsat-
urated fatty acids (PuFA) and sulfur amino acids may influence E-Se
status. The role of these factors in relation to E-Se status may be
explained in the context of the theory of free radical pathology as
discussed above. Inclusion of PuFA in the diet increases substrate
(allylic bonds) involved in the initiation of the peroxidative process.

Of course, this statement assumes that the composition of dietary fat

influenCes the composition of total membrane lipids. With respect to
the Sulfur amino acids, their influenCe may be two-fold. First, thrbugh
the involvement of these amino acids in the production of glutathione

(GSH) a tripeptide (y-L-glutamyl-L-cysteiny1glycine) which is linked

through glutathione reductase (GSH Red) to the NADPH generating system of
the cell. Second, the selenoamino acids selenocysteine and selenomethio-
nine are significant in occurrence.‘ Therefore, organic sources of dietary
methione and cysteine may actually provide enough selenium to influence
E-Se status. (Neither the negative influence of PUFA nor the positive

influence of the sulfur amino acids were studied in this researdh).

Vitamin E and Selenium Deficiency‘Sign§_

The clinical signs which are characteristic of specific deficiencies
or marginal levels of E and Se include liver necrosis (Obel, 1953),
microangiopathy (Grant, 1966), yellowish brown discoloration of body fat
(Davis and Gorham, 1954), sudden death in young pigs (Michel SE.21: 1969),
exudative diathesis (Trapp‘étpél; 1970), muscular degeneration (Lannek
35 El: 1961) and erythrocyte hemolysis (Sondegaard, 1966). The clinical
signs of general dietary deficiencies in swine to which E and Se deficien-
cies are known to contribute are: slow or interrupted growth, reduced
appetite, lameness or stiffness and impaired reproduction (Trapp st 31.
1970). It is important to remember that a single mechanism (free
radical pathology) could be responsible for the necrotic lesions observed
in tissues (Butterfield, 1978).

Among experimental pigs fed E-Se deficient rations, and in field
cases of E-Se deficiency, the most frequently observed gross lesions
were liver necrosis and fibrosis, ulceration and preulcerous lesions of
the squamous epithelium of the nonglandular esophageal region of the
stomach GMichel g£_§1, 1969). Icterus, generalized edema, and transu-
dation were not especially evident in experimental pigs but were consis-

tently observed in field cases.

Feed PartiCle Size and the Incidence of GaStric Lesions

Mahan g£_gl, (1966) studied the effects of various physical proper-
ties of feed on the incidence of ulceration. Rations ground to a fine
particle size (.16 cm) were associated with an increased incidence of
ulcers.

Finely ground barley caused a strong increase in the frequency of
gastric lesions in growing pigs (Simonsson, 1978). Diets containing
coarsely ground barley or crushed oats caused very few lesions. Also,
in this study and others, the gastric contents of pigs consuming diets
of small particle size are more fluid and leave the stomach more quickly.
It may be that a fluid ingesta is more accessible to the physically
remote nonglandular esophageal region of the stomach. Increased exposure
of this region to the acidic fluid ingesta may be responsible for the
increased incidence of lesions. Gastric pH alone is not a likely causative

factor in ulcerogenesis (Kowalczyk and Muggenburg, 1963).

Prostaglandins

 

Approximately seventeen years have passed since the structures of

prostaglandin E and prostaglandin F1 (PGE1 and PGFl) were described

1
(Moncada and Vane, 1978). As the biological properties of the prostaglan-
dins were revealed, it became evident that these cyclical products of
arachidonic acid metabolism had diverse but symmetrical effects. That

is, PGE and PGF compounds were shown to have opposing actions in many
tissues; e.g. PGE dilates blood vessels and bronchi whereas PGF constricts
them. This concept of diversity and symmetry was extended by the later

identification of two other classes of prostaglandins, the thrOmboxanes

(Tx) and prostacyclin (P612). Thrbmboxane A2 is the principle prostaglandin

10

produced in the circulating blood platelets and induces platelet
aggregation as well as smooth muscle constriction. Conversely, prost-
acyclin (PGIZ) is produced in the endothelium of blood vessel walls
and is a potent antiaggrggant of platelets and a smooth muscle dilator
(Moncada and Vane, 1978).

With respect to ulcerogenesis, workers have shown the antiulcerogenic
affects of PGI2 (Robert, 1973; Chaudhury and Jacobson, 1978). At this
point the mechanism of action is not understood however, the term
"cytoprotective" (Chaudhury and Jacobson, 1978) seems appropriate.
Prostacyclin (PGIZ) protects the gastrointestinal mucosa against numerous
experimental ulcerogens. Among the agents which have demonstrated
ulcerogenic properties, and against which PGI2 offers protection, are
aspirin [acetyl salicylic acid (ASA)] and the nonsteroidal antiinflam-
matory compound indomethacin. Appropriately, both of these compounds
inhibit biosynthesis of endogenous prostaglandins in the mucosa (Chaudhury,
1978). Although the mechanism of inhibition of prostaglandin biosynthesis
by indomethacin is not understood, that of aspirin is defined. Also, the
site of action of both agents has been identified as the first unique

step in prostaglandin synthesis, that of arachidonic acid conversion to
PGGZ. Specifically, ASA acetylates the active site of prostaglandin
cyclooxygenase. The extent of acetylation correlates with the degree of
inhibition of cyclooxygenase activity.' The effect of acetylation seems
to be permanent as measured by its antiaggregant effect on platelets

(Marcus, 1978). The overall scheme of prostaglandin metabolism is depicted

in Figure 3. The reaction catalyzed by the cyclooxygenase enzyme is

depicted in Figure 4.

Bound

Arachidonic Acid

(phospholipase A2)

 

\/
Free

Arachidonic Acid

 

(02) (202)
(lipoxygenase) (cyclooxygenase)
HPETE - hydroperoxy metabolite PGG2
(peroxidase) (peroxidase)
J7
HETE GH

P 2
9612 TXAZ PGan 9652

J, PGD2
TxB2

MDA

 

6-Keto-PGF1
a

 

 

FIGURE 3. Prostaglandin metabolism

11

12

 

(5,8,11,14 — eicosatetraenoic
arachidonic acid acid (20:4)

cyclooxygenase

 

PGG (15—hydroxy-9a, 11a-

 

peroxido prosta -S, l3-dienoic acid)

a) Oxygenat1on at C11 and C15

b) Bond formation (cyclization) between C8 and C12
c) Oxygen molecule at C forms peroxide bridge with C

11 9

FIGURE 4. Prostaglandin fbrming - cyclooxygenase - catalyzed reaction.

13

The essential fatty acid arachidonic acid (20:4) is a component of
phospholipids found in cell membranes and subcellularly. Activation of
the enzyme phospholipase A2 initiates a hydrolytic process which releases
free fatty acids including arachidonic acid. Arachidonic acid is avail-
able to either lipoxygenase or cyclooxygenase enzymes and either oxy-
genated to form the hydroperoxide metabolite HPETE, or oxygenated and
cyclized to form the endoperoxide PGG2 respectively. Products of the
lipoxygenase pathway are bioactive nonprostaglandin metabolites which
will not be discussed here. All oxygenated and transformed products of
the cyclooxygenase pathway must pass through the endoperoxide (PGGZ,
PGHZ) stage. It should be noted that qualitative and quantitative
differences exist with respect to end products among tissues.

If P612 is to be implicated as a naturally occurring cytoprotective
agent in the stomach, it must be demonstrated that it does occur in that
tissue. Indeed, PGI2 biosynthesis from prostaglandin endoperoxide has
been demonstrated in microsomal fractions of gastric mucosa from the
following species: rat, mouse, guinea-pig, rabbit and monkey (Moncada,
1977; Sun, 1977). Whittle g£_al, (1978) investigated the role of PGI2
in the rat gastric mucosa and observed that intravenous infusion of P612
increased mucosal blood flow, inhibited gastric acid secretion and in-
hibited indomethacin-induced gastric erosions. Another possible action
of the prostaglandins in gastric tissue is that of stimulating the active
transport of sodium. Data presented by Chaudhury (1978) support the
hypothesis that active transport of sodium is inhibited by the ulcerogenic
(agent indomethacin and is stimulated by the cytoprotective agent PGEZ,

the latter appearing to act via increased accumulation of intracellular

CAMP. If the inhibition of active sodium transport is related to
ulcerogenesis, a possible mechanism might be that sodium accumulation
promotes osmotic swelling and disruption of epithelial cells.

The interrelationship between vitamin E and prostaglandin biology,
if any, is probably multifactorial and may be indirect. As noted before,
one Of the characteristic signs of E-Se deficiency is that of microangio-
pathy. Endothelial swelling and necrosis in blood vessel walls may
disrupt the PGI2 (endothelial antiaggregant) / TxA2 (platelet proaggre-
gant) functional symmetry which characterized prostaglandin biology.

That is, disruption of vascular endothelium (removal of P612 producing
capacity) is a proaggregant Stimulus in a "relative" proaggregant (TsAz)
environment. An episode of platelet aggregation in this microvasculature
could result in microthrombi capable of occlusion and focal necrosis.
[Three of 4 vitamin E deficient rabbits died from pulmonary thrombotic
episodes during an intravenous injection of arachidonic acid. The fourth
animal was sacrificed in a similar moribund condition. All four of the

E sufficient controls survived brief periods of respiratory distress
under similar conditions Nafsted (1978).] Another point of possible
interrelationship between E-Se status and the prostaglandins is one of

an indirect effect on prostaglandin metabolism. Lipid peroxides are
strong and selective inhibitors of P612 synthesis (Moncada and Vane,
1978). Lipid peroxides produced during the process of "free radical
pathology" such as might occur during E-Se deficiency again may promote

a situation which is predisposing to thrbmbus formation in vessels or
noncytoprotective in gastric tissue 0r both SimultaneouSly. Cornwell 33(21-
(1979) has presented data which Suggest that E inhibits HPETE production

thus making arachidonic acid more available for endoperoxide-PG synthesis,

15

P612 inclusive.

GaStric UlCers in Swine

 

The incidence of gastric ulcers in swine ranges from 5 to 20 percent
and the economics of the disease becomes significant when apparently
normal pigs die suddenly (Boenker, 1967); These may develop under varying
conditions of age, sex, diet, climate and feeding. In swine, lesions
occur only in the nonglandular esophageal region of the stomach and are
more appropriately termed esophagogastric lesions. The surface of the
normal swine stomach is white, smooth, and glistening. Lesions of gastric
tissue were described by Muggenburg §£_al, (1966) on the basis of gross
and histopathologic characteristics. In order of increasing severity,
Muggenburg differentiated between scars, epithelial changes, acute
erosions, subacute ulcers and chronic ulcers. A detailed description
of the characteristics of each class of lesion has been reviewed (Bebiak
gt_§l, 1977) with some discussion of the anatomy of the stomach.

Ulcers have been observed in the pig under a number of circumstances
and consequently a number of causes have been suggested. The relationship
between feed particle size and the incidence of ulcers was discussed
above. Our own previous work (Bebiak gt_§l, 1977) indicated that typical
swine diets, in which corn starch supplanted ground corn, were ulcerogenic
in nearly 100 percent of the animals examined. Though the mechanism is
not understood, mere awareness of the ulcerogenic property of finely
ground feeds is valuable in that it provides a model (negative control)
for studying this health problem.

Gastric hyperacidity has been discussed as an important causative

agent in ulcerOgenesis (Kowalczyk 1963). A direct relationship between

16

gastric hyperacidity and the incidence of ulcers has not been shown con-
sistently. In fact, most human gastric ulcer patients secrete less
gastric juice, and in many cases no acid is secreted at all, compared

to healthy controls (Dragstedt, 1978).. Young pigs having undergone
gastric vagotomy develop chronic gastric ulcers by 5 months of age. It
should be noted that such an operation effectively removes the nervous
phase of gastric secretion and reduces glandular response to gastrin

or histamine stimulation. The lesion produced in these vagotomized pigs
developed in the glandular tissue along the lesser curvature immediately
distal to the nonglandular esophageal region. Gastric lesions induced
through surgical manipulation of the acid environment are not representa-
tive of naturally occurring 1eSions. In fact, this evidence would suggest,
by default, that acidity is not a principal cousative factor in naturally
occurring esophagogastric ulcerogenesis;

The role of ischemia in the pathogenesis of gastric ulcers may be
important (Hottenrott g£_gl, 1978). Conscious piglets subjected to
hemorrhagic shock by injection of microspheres into the left atrium
developed mucosal lesions within 4 hours. The incidence and location
of lesions related directly to areas of reduced blood flow indicating
that local ischemia may render the mucosa susceptible to ulcerogenosis.
The possible roles of vitamin E, selenium and the prostaglandins in
regard to ulcerogenesis have been discussed. And in light of that
discussion one cannot disregard the possible association of E-Se def-

iciency, PGIz/Tx imbalance and ischemia as related pathogenic components.

17

Summary
In the literature reviewed here, it is clear that specific infor-
mation is available Concerning E-Se nutrition, prostaglandin biology and
ulcers, but little specific information relating these subjects. Since
the problem of gastric ulceration is important in biomedical science and
swine production, and since the theoretical relationship between E-Se
status-prOStaglandin biology and ulcerogenesis remains untested, a study
was designed with the following objectives:
1. Determine the effect of vitamin E-selenium supplementation
on the incidence of gastric ulcers in swine.
2. Provide original data on the regional distribution of
tocopherol, selenium, glutathione perOxidase, and cy-

looxygenase in the porcine stomach.

METHODS AND MATERIALS

Experimental Design_

 

In each experiment pigs were randomly assigned from 6 litters into
2 dietary groups. The pigs used in this experiment were born from sows
which had been fed a low E-Se diet thrOughout gestation and lactation.
These pigs were weaned to a low E-Se diet at 5 weeks of age. Two trials
were conducted using 36 pigs in the first experiment while the second
experiment employed 34 pigs:

The experimental diets conSiSted of a control ration (Table 1)
demonstrated to be ulcerogenic in pigs (Wesoloski gtpal,, 1974; Bebiak
g£_al,, 1977) and a test ration consisting of this basal ulcerogenic
diet (BUD) supplemented with 44 I.U. of B/kg of feed as d,£-alphatocopheryl
acetate and .2 ppm of Se from sodium selenite (BUD+E+Se)

In trial I pigs were maintained in confinement on slotted floors
until 10 weeks of age. After 10 weeks the pigs were maintained in con-
finement on straw bedding until they were killed at 20 weeks. In trial
II pigs were maintained on slotted floors throughout the 20 week study.
These animals were raised without the imposition of any determinable

environmental stress.

Periodically (at 5, 10 and 20 weeks of age), blood was obtained
from the anterior vena cava for serum E and Se analysis and for red blood
cell glutathione peroxidase (RBC GSH-Px) activity determinations. Also,
pigs were killed at 5, 10 and 20 weeks of age so that the Stomachs could
be collected for gastric tissue E, Se and GSH-Px analysis.or for cyclo-

oxygenase determination. Given certain analytical preconditions (time,'

19

Table 1. Composition of Basal Ulcerogenic Diet. Trial 1

 

Inggedient Percentage

Corn starCh - 70.70
Soybean meal (.49 protein) 20.00
Corn oil 3.00
Cellulose ' 3.00
Defluorinated phosphate 2.00
Vitamin-Trace mineral supplement .50
Salt .25
ASP-250 (antibiotic) .25
Lysine (.78) a .20
D,L-Methionine (.98) ' .10

100.00

* Composition of the Vitamin-Trace mineral supplement.

 

"Nutrient ‘Amount'in‘IO'kg_
Vitamin A as retinyl palmitate 6.6 x 106 IU
Vitamin D3 1.3 x 106m
Choline as choline chloride 220.0 g
Niacin as nicotinic acid 35.2 g
D-Pantothenic acid as calcium pantothenate 26.4 g
Riboflavin .6 g
Pyridoxine " .6 g
Thiamin .6 g
Vitamin K as menadione sodium bisulfate 4.4 g
Vitamin B12 39.6 mg
Zinc as zinc oxide 149.6 g
Iron as ferrous sulfate 118.8 g
Manganese as manganous oxide 74.8 g
Copper as copper sulfate 19.8 g
Iodine as potassium iodate 5.5 g
Antioxidant as BHT 99.0 g

Corn starch carrier' ' the balance of lO'kg_

 

20

sample size, sample freshness), not all parameters could be measured on
all pigs.

Samples of gastric tissue were taken from each of the anatomic
regions of the stomach and either analyzed as fresh tissue or frozen

for later analysis.

"TiSSue,'Serum Analyses

 

The procedure for determining tissue and serum selenium levels has
been described (Whetter and Ullrey, 1978) as a method modified from the
procedure developed by Olson'gtyal., (1975); The procedure for determin-
ing tissue and serum vitamin E levels has also been.described (Taylor,
1976).
The coupled assay for determination of RBC GSH-Px activity was
adapted from that described (Paglia and Valentine,-1967; Brady £3 21,, 1979).
The immunohistochemical assay fer the prostaglandin endoperoxide-
forming cyclooxygenase was developed by Smith and Wilkin (1977). Cryostat cross
sections of each anatomic region of the stomach were analyzed so that
specific tissue layers could be accounted for in regard to the presence

of the cylooxygenase enzyme.

‘CharacteriZation of Ulcers and TisSue Sampling_

The stomach was incised along the greater curvature, everted and
rinsed lightlywith water. The entire mucosal surface was examined
and the types of lesions were recorded. The classification of gastric

ulcers into five groups was based on the following gross characteristics

(Muggenburg 9."; fl. 1966):

epithelial changes: rough, corrugated, irregular shape,
yellow

21

subacute ulcer: rough, regular—oval to linear shape,
red, shallow-extending thr0ugh the
mucosa, mild keratinization

chronic ulcer: rough, regular-oval to round shape,
dark red or brown, deep-extending
to the muscle layer, extensive
keratinization

scar: stellate to linear shape, distorted,

tough

RESULTS AND DISCUSSION

PerfOrmance

 

The addition of E and Se to the basal diet did not affect performance
in either trial, as measured by body weight (Tables 2 and.3). Normal

growth Occurred among pigs in both dietary groups.

Blood Values

Serum levels of E and Se reflected dietary concentrations of those
nutrients (Tables 2 and 3). At ten and twenty weeks of age, supplemented
pigs in trial I had significantly higher levels of serum E (P<.0000).
Among pigs fed the basal diet in trial 1, serum tocopherol levels decreased
from 2.4 mg/ml at five weeks to 1:5 mg/ml at twenty weeks. Among pigs
supplemented with E-Se, serum.tocopherol levels increased from 2.6 mg/ml
at five weeks to 3.7 mg/ml at twenty weeks; At all ages, supplemented
pigs in trial 11 had significantly higher serum tocopherol levels, P<.0000;
than unsupplemented pigs. Serum tocopherol levels of unsupplemented pigs
decreased from 2.0 mg/ml to 1.3 mg/ml to 3.6 mg/ml at five and twenty
weeks of age, respectively. Serum selenium levels ranged from .07 ppm
at the beginning to .05 ppm at the end of trial I among unsupplemented
animals. Similarly, unsupplemented animals in trial 11 had initial and
final serum selenium levels of .07 ppm and .06 ppm respectively. Sup-
plemented animals in both trials had significantly higher serum selenium
levels than controls, P<0001:.ranging from .3 ppm to .19 ppm and from .11
ppm to .20 ppm at five and twenty weeks of age in trial I and trial II,

reSpectively. In light of previous studies, a deficient State might be

22

23

characterized by serum values below 1.0 In8/ml tocopherol and .05 ppm
selenium. Based on these figureska true E-Se deficient state may not
have been accomplished with the BUD usedl

At all ages, the addition of'E and Se to the basal diet elevated
erythrOcyte glutathione peroxidase Values significantly: Trial I, P<.02
and trial 11; P<.003 (Tables 2 and 3); Among pigs fed the basal diet,
RBC-GSH Px levels decreased from 7I0 U/mgHb at five weeks to 5.8 U/mgHb
at twenty weeks in trial I. Among pigs in the same trial that were
supplemented with E-Se, RBC-GSH Px levels increased from 7.6 U/mgHb
at five weeks to 9.3 U/mgHb at twenty weeks. Among animals in the
second trial the trends were the same: negative controls fed the basal
diet decreased from6.5 U/mgHb at five weeks to 6.0 U/mgHb at twenty
weeks; positive controls supplemented with E-Se increased from 7.4 .U/mgHb

to 9.1 U/mgHb from five to twenty weeks.

GaStriC‘TisSue

 

With regard to the Se content of the various anatomic regions of
the stomach, a significant response to dietary Se supplementation was
demonstrated for each tissue region, at all ages, in both trials. Again,
statistical analysis was used to determine if real intraregional differ-
ences existed among animals fed common diets. Among animals fed the
basal diet in trial I, the Se content of the nonglandular esophageal
(NGE) region tended to be less than that of all other regions combined;
.059 ppm y§_.066 ppm (P<.090.- Conversely, among animals fed the basal
diet supplemented with EeSe in trial I, the Se content of the NGE region
tended to be greater than that of all other regions combined; I155 ppm

' !§_.145 ppm (P<.228). In trial 11, the trend in intraregional selenium

differences continued. Among pigs receiVing the basal diet the NGE
region of stomachs sampled from these animals had significantly less
Se Content than all other regions; .057 ppm y§_.064 ppm (P<I048). Among
supplemented pigs; the Se content of the NGE region was significantly
greater than that of all other regions combined; .167 ppm‘y§_.l46 ppm
(P<.021; Tables 2 and.3)I

The E levels measured in the various tissue regions of the stomach
were significantly greater among supplemented pigs than controls at 10
and 20 weeks (P<.036) but not at 5 weeks of age in either trial.‘ When
the tocopherol level of the NGE region was compared with the tocopherol
levels of all other regions, no significant differences were observed
(P<. 29) among unsupplemented animals. When that same comparison was
made among supplemented pigs the NGE region had significantly greater
tocopherol content than the other regions as a group (P<.04). This was
the case in both trials (Tables 2 and 3); |

With regard to the GSH-Px activity levels of the various anatomic
regions of the stomach, a significant response to dietary Se supplementa-
tion was demonstrated for each tissue region, at all ages, in both trials
(P<.02). Statistical analysis (analysis of variance) was applied to these
data in order to determine if differences existed between anatomic regions
of stomach removed from pigs receiving common diets. In trial I, the
only significant intraregional difference occured among gastric tissue
from pigs fed the E-Se supplemented diet. The nonglandular esophageal
(NGE) region of supplemented pigs had significantly greater GSH-Px activity
than all other regions, 2.04 U/g vs 1.88 U/g (P<. 02) ' In a similar

comparison among pigs in trial 11 the only significant intraregional

25

difference occured among pigs fed the basal diet. Again, the NGE
region separated itself from all others; tending to have less GSH- Px

activity, 1.07 U/g vs 1.20 U/g (P<. ;10 Tables 2 and 3).

Feed Values

Vitamin E and selenium levels in the BUD ranged from 1.0 to 1.9 mg
tocopherol/g feed and from .04 to .09 ppm selenium. Vitamin E and
selenium levels in the E-Se supplemented ration ranged from 2:3 to 3.8

mg tocopherol/g feed and from .18 to .26 ppm selenium.

cyclooxygenase

As an indication of prostaglandin synthesis, the entire stomach
was surveyed for the prostaglandin—forming cyclooxygenase: Photographs
fer this semiquantitative assay were taken of each of the four tissue
layers which are common to the different anatomic regions (Photographs
l—8)I Fluorescence, in this assay, is an indication of the presence of
cyclooxygenase.

It should be noted that the epithelial layer of the cardiac, fundic
and pyloric regions consists of little more than a single layer of cells.
The submucosa, muscularis externa and serosa tissue layers of these
regions are essentially identical to the esophageal region with respect
to specific fluorescent staining and so those photographs are excluded.
The occurrence of cyclooxygenase-bearing cells among the various common
tissues of the various anatomic regions of the stomach is summarized in

Table 4.

Photograph 1 .

26

 

A low power photomicrograph of the mucosal layer of
the esophageal region showing the epithelial, lamina
propria and muscularis mucosa. There is an abundance
of positive staining cells in the lamina propria and
an absence of specific staining in the epithelium

and muscularis mucosa. The stratified squammous
epithelium of the esophageal region is unique among
the anatomic regions of the stomach.

Photograph 2.

27

 

A high power photomicrograph of the mucosal layer

(epithelium and lamina propria) of the esophageal

region. Note the perinuclear positive staining in
cells of the lamina propria which have been tenta-
tively identified as plasma cells.

Photograph 3.

28

 

A photomicrograph of the mucosal layer (lamina propria
and epithelium) of the esophageal region to which an
absorbed antisera was applied. This represents the
control used in this immunohistofluorescence assay.
Note that the nonspecific staining of the epithelium
remains constant (similar to that same tissue to which
immune antisera was applied) while cells of the lamina
propria do not contain the fluorescent indicator. Only
this one photomicrograph of a negative control is
included.

29

 

Photograph 4. A photomicrograph of the submucosa of the esophageal
region. This tissue layer contains a few positive
staining cells which appear to be similar to those
of the lamina propria.

Photograph 5.

30

 

A photomicrograph of the muscularis externa of the
esophageal region. This tissue layer, like the
muscularis mucosa did not appear to contain cyclo-
oxygenase-bearing cells. However, the vasculature
within those tissues contained a small population
of positive staining cells.

31

 

Photograph 6. This photomicrograph of the serosa of the esophageal
region depicts a tissue layer without specific positive
staining cells.

32

 

Photograph 7. A photomicrograph of the mucosa (lamina propria and

epithelium) of the fundic region of the porcine

stomach. Note the positive staining cells of the
lamina propria.

33

 

Photograph 8. A photomicrograph of the mucosa (epithelium and lamina
propria) of the pyloric region. Note the positive
Staining cells of the lamina propria.

34

Gastric LesiOns

 

The stomachs of all pigs were examined for ulcers. Tables 8a and
8b provide a compilation of the gross characterizations of those stomachs.
In summary the Stomachs of the (35) unsupplemented pigs in the two trials
were characterized as follows: 9 normal, 20 preulcerous lesions and 6
ulcers. The stomachs of the 35 supplemented pigs in the two trials were
characterized as: 13 normal, 18 preculcerOus lesions and 4 ulcers. Among
the 25 pigs in trial I maintained on straw bedding from 10 to 20 weeks of
age there were 7 normal, 12 preulcerous lesions and 5 ulcers. Among the
24 pigs in trial II maintained on slotted concrete floors from 10 to 20

weeks of age, there were 4 normal, 16 preulcerous lesions and 4 ulcers.

 

Table 2. Summary of Data. Trial I.
BUD
5 wk 10 wk 20 wk
Body wt (kg) 11.1 22.1 84.2
Serum tocopher61' 2.4 1.9 1.5
(ms/mu)
Serum Se (ppm) .07 .06 .05
RBC GSH-Px 7.0 5.7 5.8
(U 7mg Hb)
Gastric tissue
tocopherol (mg/g)
esophageal .15 .10 .12
gastric .15 .13 .12
fundic .16 w12 -11
pyloric .15 .11 .12
selenium,(ppm)
esophageal .06 -06 .06
gastric .08 .06 .06
fundic .09 .07 .06
pyloric .07 .05 .05
GSH-Px (U./g prot) »
esophageal 1.0 1.0 0.9
gastric 1.2 0.9 1.0
fundic 1.1 1.2 1.1
pyloric 1.1 1.1 1.0

 

BUD +‘E + Se
5 wk 10 wk 20 wk
10.0 19.7 89.3
2.6 3.4 3.7
.13 .17 .19
7.6 9.3 9.3
.18 .21 .21
.16 .20 .20
-15 .18 .19
.17 .18 .20
.13 .16 .18
.11 .14 .17
.11 .15 .16
.15 .15 .18
1.9 2.0 2.2
1.8 1.9 2.0
1.7 1.9 1.9
1.8 1.7 2.0

36

Table 3. Summary of Data. Trial 11.

 

 

 

'BUD .1 1f vvvvvvv ff BUD f E‘+ Se
5 wk 10 wk '20 wk ‘5 Wk 10 wk 20 wk
(Body wt (kg) 9.8 20.0 74.7 9.3 18.0 73.7
Serum tocopherol 2.0 1.5 1.3 3.1 3.7 3.6
(mg/m 1)
Serum Se (ppm) .07 (.07 .06 .11 .18 .20
RBC GSH-Px 6.5 6.2 6.0 7.4 9.0 9.1
( U ling HB)
Gastric tissue
tocopherOl m g/ g)
esophageal .10 .11 .10 .18 .21 .24
gastric .11 -.12 .ll .18 .20 .18
fundic .12 .11 .12 .17 .17 .19
pyloric .ll .12 .09 .18 .20 .21
selenium (ppm) - - n »
esophageal .07 .05 .04 .13 .19 .19
gastric .07 .06 .06 .12 .17 .19
fundic .08 .06 .06 .11 .15 .17
pyloric .06 .06 .05 .13 .17 18
GSH-Px ( UYg prot)~ ~ »
esophageal 1.3 0.8 1.0 1.7 2.2 2.1
gastric 1.3 1.2 1.2 1.7 2.0 2.0
fundic 1.3 1.1 1.1 1.8 1.8 2.0
pyloric 1.5 1.2 0.9 1.5 1.9 2.1

37

Table 4. Distribution of the PG-Forming Cyclooxygenase in the Porcine

 

Stomach.
Esophageal ‘Cardiac "Fundic Pleric

MucOsa

Epithelium ___ __ __ --

Lamina Propria *** ** ** **

Muscularis Mucosa * * * *
'Submucosa * * * *
'Muscularis Externa *- *- *- *.

 

SUMMARY .

Two experiments, involVing70 weanling pigs were conducted to evaluate
the effect of vitamin E and selenium on the incidence and severity of
gastric ulcers. At weaning (5 weeks of age) pigs were randomly assigned
from litters to two dietary groups, namely: a basal ulcerogenic diet (BUD)
composed of corn starch, soybean meal and corn oil adequately fortified
with minerals and vitamins without selenium and vitamin E; or, the BUD +
i2 ppm selenium from sodium selenite + 44 IU of vitamin E/kg from DL-alpha-
tocOpherylacetate.‘ The effect of E-Se supplementation was assessed on the
basis of growth rate, serum levels of tocopherol and selenium, erythrocyte
Vglutathione peroxidase (GSHQPx) activity, gastric tissue selenium, toco-
pherol, GSH-Px activity and cyclooxygenase, and the incidence of gastric
lesions. Data were collected from animals at 5, 10 and 20 weeks of age.
The regional diStribution of tocopherol, selenium and the enzymes GSH-Px

and cyclooxygenase in gastric tissue was determined.

ConclusiOns: l. Supplementation of the BUD with E-Se did not affect

 

growth rate in either experiment.

2. Blood data of experimental animals reflected the
dietary levels of E-Se such that serum tocopherol,
serum selenium and erythrOcyte GSH-Px values were
significantly greater among supplemented pigs than
non supplemented pigs. The conspicuous absence of‘
classical E-Se deficiency signs, accompanied by serum

tocopherol and selenium values whiCh are not indicative

38

39

of a deficient state, suggests that we failed to
induce a deficient negative control group: Serum
levels of leSS than 1.0 mg/ml tocopherol and 105

ppm selenium are indicative of deficiency. In
contrast, at 20 Weeks of age, serum levels of pigs
considered negative controls were 1.4 mg/ml tocopherol
and .06 ppm selenium:

Tocopherol, Se and GSH-Px analyses of gastric tissue
collected from each of the anatomic regions of the
stomach correlated significantly with dietary supplem-
tation of the nutrients E-SeI In particular, the
tocopherol, selenium and GSH-Px content of the eso-
phageal region appeared to be most sensitive to E-Se
"supplementation and depletion": However, since the
process of erosion in this area does alter the quality
of the tissue, and since it does not occur randomly
among anatomic regions, this effect may be artificial.
That is, analyzing that esophageal tissue which remains
after an erosive process amounts to an analysis of
serosa and keratin in contrast to the analysis of
normal cardiac, fundic and pyloric tissues.

Among all regions of the gastric lumen, and between
animals of both dietary groups, the distribution of
cyclooxygenase among common tissue layers was similar.
Among the tissue layers of all anatomic regions, a

relative abundance of the enzyme was present in the

40

mucosal lamina propria, especially of the esophageal
region. If a cyclooxygenase product is cytoprotective
of the gastric mucosa, and if the accessibility of

that product (likely P612) to the mucosa is a factor
contributing to Cytoprotection, then, the presence of

a greatly thiCkened esOphageal epithelium may be in-
directly reSponsible for regional (esophageal) gastric
ulcerogenesis by disallowing the lamina propriae-produced
cytoprotective agent acess to the site of effectiveness
(the gastric mucosa).

The presence of dietary vitamin E and selenium did
reduce the number and severity of gastric ulcers and
preulcerous leSions: Among the stomachs of 35 unsup-
plemented pigs in the two trials, there were: 9 normal,
20 preulcerous lesions and 6 ulcers. The stomachs of

35 supplemented pigs were characterized as: 13 normal,
18 preulcerous lesions,and 4 ulcers.

The ulcerogenic effect of the BUD utilized in this study
was reduced in trial I by maintaining the pigs on straw
bedding. Pigs in this trial did consume large amounts
of straw, however, this straw consumption did not appear
to affect E-Se status. This observation lends support
to the idea that the physical quality (fineness) of the
BUD is the principle ulcerogenic factor and that nutri-

tional status (E-Se).is secOndary.

BIBLIOGRAPHY

42

Marcus, A.J. 1978. The role of lipids in platelet function. J. Lip.
Res. 19(7): 793.

Michel, R.L., C.K. Whitehair and K.K. Keahey. 1969. Dietary hepatic
necrosis associated with Selenium-vitamin E deficiency in swine.
J. An. Vet Med. Assoc. 155:50.

Moncada, S., J.A. Salmon, J.R. Vane and B.J.R. Whittle. 1977. Formation
of prostacyclin-and its product~6-oxo-PGF1d by the gastric mucosa of
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Moncada, S. and J.R. Vane. 1978. The role of prostacyclin in vascular
tissue. New Dev. in PG and Tx Res. Symp. FASEB p. 63.

Muggenburg, B.A., S.H. McNutt and T.-Kowa1czykw 1966. Pathology of
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Muggenburg, B.A., T. Kowalczyk, W.G. Hoekstra and R.H. Grummer. 1967.

Effect of certain management variables on the incidence and severity
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Nafsted, I. .1978. Arachidonate-induced thrombosis in vitamin E-deficient
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Obel, A.L. 1953. 'Studies on the morphology and etiology of so-called
toxic liver dystrophy in swine. Acta Path et.‘Microb. Scand. 94:1.

Olson, O.E., J.S. Palmer and E.E. Carey. 1975. Fluorometry of selenium
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Paglia, D.E. and W.N. Valentine, 1967. Studies on the quantitative and
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Perry, T.W., A.A. Jimenez, J.E. Shively, T.M. Curtin, R.A. Pickett and
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Robert, A. 1973. Prostaglandins and the digestive system. Sem. Inserm.
Prostaglandins 297.

Simonsson, A. and N.E. Bjorklund. 1978. Some effects of the fineness of
_ ground barley on gastric lesions and gastric contents in growing pigs.
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Smith,'W.L. and G.P. Wilkin. 1977. Immunochemistry of prostaglandin
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Sondegaard, E. 1966. Symposium on selenium in biomedicine. p. 365.

Sun, F.F., J.P. Chapman and J.C. McGuire.‘ 1977. Metabolism of prostaglandin
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43

Tappel, A.L. 1974. Selenium—glutathiOne peroxidase and vitamin E.
Am. J. Clin. Nutr. 27:960.

Taylor, S.L. 1976. SenSitive fluorOmetric method for tissue tocopherol
analysis. Lip. 11(7):530.

Trapp, A.L., K.K. Keahey, D.L. Whitenack and C.K. Whitehair. 1979.
Vitamin E-selenium deficiency in swine. Differential diagnosis
and nature of field problem. J. Am. Vet. Med. Assoc. 157:289.

Wesoloski, G.D., A.H. Jensen, V.D. Ludwig and H. Gosser, 1974. Effects
of different concentrations of corn oil, magnesium sulfate and oat
hulls in a porcine ulcerogenic ration. Am. J. Vet. Res. 36(6):773.

Whetter, P.A. and D.E. Ullrey. 1978. Improved fluorometric method for
determining selenium. J. AOAC 61(4):927.

Whittle, B.J.R., N.K. Boughton-Smith, S. Moncada and J.R. Vane. 1978.
Actions of P61 and its product, 6-oxo-PGF ' on the rat gastric
mucosa in vivo and in vitro. Prostaglandins 15(6):955.

APPENDIX

44

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m.m m.~ m.H no-HAH A.H H.~ o.» ma-~em
¢.m m.~ ~.~ nfi-mo~ ~.H m._ ~.~ no-~e~
~.m ¢.m ~.m ¢~-no~ w.~ c.~ m.~ no-~o~
A.n O.” c.n Ho-noH . o.H ¢.~ m.~ ~c-~e~
x: cm x: o” x: m .oz mmm. x: on x: as x: m .ozxwmm.
om+m+n5m can

.H 3.29 Arias m 553.? 53$ .3 033.

47

 

.Hoaogmouou anyhow @0353 we: mum—5:8 amazon—3.33m .momm Sm 3?

 

Boo . X—
H.m«

.mqm mo-m¢~

A.” mo-¢v~

m.N ”H-M¢H

m.~ qc-se~

“.m. 0.4 n~-o¢~

.mam m.~ mo-¢e~

a.m o.m No-m¢~

a.» m.n No-m¢H

n.e n.~ n_-mvfi

“.4 a.~ “H-mefl

e.m. m.m ¢.~ no-04H
- A.n m.» so-aem
A.n n.e 4.4 ~H-¢¢H.
m.~ o.m N.» no-0?”
m.m o.n A.N «H-nvfi
.H.¢ n.n m.~ ec-¢¢a
A.m m.m m.» Nc-wvfl
:2 on x: cm x: m .02 «an
om + m + can

 

Hcoo.vm

ao..H c.~
.mqm ac-ovm
~.~ Ho-e¢H
- Ho-mq~
mooc.va o.~ oH-o4~
no..u m.H - mo-v¢H
.mqw m.~ oo-o¢~
m.H m.~ mc-aem
A.“ ~.~ No-44“
N.“ c.~ Ho-av~
“coo.va H.H m.~ NH-m¢~
may.“ n.~ V.“ a.” 4H-mvH
.«4m x.“ w.H vo-mv~
o.~ H.~ a.” ao-oem
v.“ a.“ H.~ HH-¢¢H
o.“ ~.H m.” mo-m¢_
~.H ¢.H ~.~ o~-nvfi
an” A”_ n." --mv~
x: cm x: on x: m .oz «ma
can
.HH Hanna .fi~e\mav m assaufi> aspom .nN ensue

48

Hooo.vm .mHo>oH sawcoHom Eduom vouw>ch we; mHmEficm popcoEonmam .momm HHa u<¢

 

 

Hooo.va

NH.. mac..u No.

.qu «H-moH mun oH-NoH

mH. eo-moH No. No-NoH

mH. Ho-moH No. HH-NeH

HH. Ho-HNH No. oH-NcH

NH. NH-NoH Hooo.va No. NH-NeH
NH.. 4H. HH-noH moo..u co. mo. . oo-ooH
M44. NH. oH-HNH .mmq oH. HH-moH
HN. NH. No-HNH No. co. oH-meH
NH. NH. eo-moH co. mo. Ho-NoH
NH. HH. no-moH co. co. Ho-NeH
mH. OH. No-moH Hoco.va No. mo. mc-NcH
0H. 4H. oH-noH 400..” me. No. mo. No-NoH
NH. oH. NH-HNH .mmq moJ co. HH-NcH
0H. NH. 4H-HNH co. No. . - oH-NeH
NH. . NH. mo-HNH 4o. . mo. No. NH-NeH
NH. HH. NH-moH mo. mo. No. No-NoH
NH. NH. ¢H-moH co. co. No. mo-NoH
NH. NH. Ho-noH mo. go. No” Ho-NcH
H: oH H: m .02 NHN N: ON H: oH H3 m .02 NHN

om.+ m + mam cam

.H Hmauh .neamu EzdcoHom Eduow .mm oHnee

Hooo.vm .mHo>o~ aswcoaom sshom vopm>oHo cm; nausfica voucosoHQQSN .momm HHN u<N

49

 

 

 

HNNN.VN

HH.. NNN..H NN.
.mmq NN-NNH . .mmq NN-NNH
NH. NN-N¢H NN. HN-NNH
NH. HH-NNH - HN-NNH
NH. NN-NNH HNNN.VN NN. NH-NNH
NH.. NH. NH-NNH NNN..H NN. - HN-¢NH
.mmq NH. NN-NNH .mmq NN. NN-NNH
NH. NN. NN-N¢H NN. NN. NN-NNH
NH. HH. NN.NNH NN. NH. NN-NNH
HN. NH. NH-NNH NN. NN. HN-NNH
NH. NH. HH-N¢H HNNN.VN NN. NN. NH-NNH
NN.. NH. NH. NN-NNH NNN..H NN. NN. NN. NH-NNH
- NH. NH. NH-NNH .mmq NN. NN. NN-NNH
NH. NH. NN. NH-N¢H NN. NN. NH. NN-NNH
HN. NH. HH. NN-NNH NN. NN. NN. HH-NNH
NN. NH. NH. NH-NNH NN. NN. NN. .HN-NNH
NH. NH. NH. NN-NNH NN. NN. NN. NH-NNH
NH. NH. NN. ANN-NNH NN. NN. NN. NH-NNH
NszN x: NH N2.N .oz NNN N: NN x3 NH x: N .NZINNM.
om + m + can can

HH Hmwuh .nemmv EsficoHom Esaom .nm oHneb

50

 

 

0.5:
.mqm NH-NNH
N.N NN-NNH
N.N HN-NNH
N.N HN-HNH
N.N NH-NNH
N.N. N.N HH-NNH
N.NH N.N NH-HNH
N.HH N.N NN-HNH
N.HH N.N «N-NNH
N.NH N.N NN-NNH
N.N N.N NN-NNH
N.N. N.N N.N NH-NNH
.mqm N.N N.N NH-HNH
N.NH - N.N HH-HNH
N.N N.N N.N NN-HNH
N.N N.NH - NH-NNH
N.N N.N N.N NH-NNH
N.N N.N N.N HN-NNH
N: NN N: NH x3 N .02 NHN
om + m + NNN

Hedge

amo.vav xmuzmo mo mHo>oH noum>oHo we: mHmEHcm voucosoammnm .momm HHN u<N

 

NNHN.VN
NH..H N.N
.mqm NH-NNH
- NN-NNH
N.N HH-NNH
- NH-NNH
HNNN.vN N.N «N-NNH
HN..“ N.N N.N NN-NNH
Nwmm N.N HH-NNH
N.N N.N NH-NNH
N.N N.N HN-NNH
N.N N.N HN-NNH
NNNN.VN N.N N.N NN-NNH
HN..“ N.N N.N N.N NN-NNH
, .mqm N.N N.N HH-NNH
N.N N.N N.N NH-NNH
N.N N.N N.N NH-NNH
N.N N.N N.N NN-NNH
-. N.N N.N NN-NNH
N.N N.N N.N HN-NNH
N: NN x: NH N: N .oz Nmm
NNN
.An: ma\=g ommvwxouom ocownumusHm HHoo vooHn pom .mv oHomh

$1

 

.moo.vm .xuw>fiuum xmnzmu mo mHo>oH woum>oHo was mHmawzu coucosoammzm .momm HHm u<«

 

¢.h«
.mqm NN-N¢H
N.N NN-NNH
N.N HH-N¢H
N.N «N-NNH
N.N“ N.N NH-NNH
N.N N.N NN-NNH
N.NH N.N NN-NNH
N.N N.N NN-NNH
N.NH N.N NH-NNH
N.NH N.N HH-NNH
H.N. N.N N.N NN-NNH
.mqm N.N N.N NN-NNH
N.N N.N N.N NH-¢¢H
N.N - N.N NN-NNH
N.NH - N.N NH-NNH
N.N N.N N.N NN-NNH
N.N - - NN-NNH
x: NN 4: NH x3 N .oz NNN
0N + m + NNN

 

NNNN.VN
NN..“ N.N

.mqm NN-NNH

N.N HN-NNH

N.N HN-NNH

HNNN.VN N.N NH-NNH

NN..“ N.N - HN-NNH

..Ju. N.N NN-NNH

N.N N.N NN-NNH

N.N N.N NN-NNH

N.N N.N HN-NNH

HNNN.VN N.N N.N NH-NNH

NN..“ N.N N.N N.N NH-NNH

.mqm N.N N.N NN-NNH

N.N N.N N.N «N-NNH

N.N N.N N.N HH-NNH

N.N N.N N.N HN-NNH

- N.N N.N NH-NNH

N.N N.N - NH-NNH

x: NN N: NH N: N .ozwmm

can

HH HNNNN .an NNNNN NNN>HNNN oNNNonNNN NNNHNNNNNHN HHoo NooHN NON .NN NHNNN

52

 

 

HNNN. NHNN. NNNN. HNNN. VN
HN. NN. NN. HN. NN
.www .mww .mww Nun.mw
NH. NH. NN. HH.
NN. NNNN. NNHN. NNNN. VN NH. HH. NH. NN.
HN. HN. HN. HN. NN NN. NN. NH. HH.
.mHl. F mm... .mHl.m HH. NH. HH. NH.
NH. NH. NH. NH.
NH. NN. NH. NH.
NH. NN. NH. NN.
NH. NH. NN. NH.
N N N NNz N .N N. .mNz
N: NN N: NH

HNN NNNNNN .HNN NHNNNNN .HNNzN HNNNNNNNNN NNHNNNNHNNNz

 

o.H o.H o.H mNmH. vm

He. no. Hc. No. mm
u u n u oHaon
.u. u. u u: NonhoH
0H. u 0H. NN. HHINcH
0H. NN. ow. NH. cHuNoH
9H. NH. mH. NH. vHuNoH
mH. 0H. mo. mo. ocuNoH
HHnmoH
oHuaoH
HeumoH
HounoH
mancH
NeuNoH
HHuNoH
oHuhoH
mHuNoH
nancH
menNcH
HouNoH
N N N NNz .oz NHN
92m

x3 m
.H HNHNH .mconoH may oHHonm cum

.Hm\mau m :NENNH> osmmwu :umsoum .Nm oHnNH

53

 

.HNN NNNNNN .HNN NHNNNNN .HNNzN HNoNNNNoNo NNHNNNNHNNNz

.pmw .mww Nmn. Nmnzm
NH. HN. NH. NH.
NN. NN. NN. NN.
NH. NH. HN. NN.
HN. NH. NH. NH.
N N N mNz
N: NN

mH.

cm.-

oH.

HN. NN.
mH. on.

NN.
HN.
HN..m NN.

HN.
mH.
no.

HH. HH.

 

 

m. u
x3 oH

muz m

.H HNHHH

u moz

eHuaoH
oeumoH
HOnmoH
HouHhH
NHumoH
HHumoH
oHanH
NOIHBH
voumoH
mcuaoH
NanmoH
oHnmcH
NHthH
HHIHBH
mouHhH
mHnaoH
vHunoH
HolmoH

oz «Hm

ow + m + 93m

x3 m

. .m:0HmoH may oNuonm can
.HM\MEV m :NEN9H> oammHu nomaoum .nm oHnmb

S4

 

HNN. HNN. HNN. HNN. VN
HN. HN. NN. HN. NN
NN. NH. HH. NH..m
NN. NN. NH. NH.
NN. HH. NH. NN.
NH. HH. NN. NH.
HH. NH. NH. NN.

N N N NNz
x: NN

.Hmv oncom .Hov ofiaummo .Hmon Hoomosmomo NNHsoanmcoz

 

NNN. NNNN. NHHN.
HN. HN. NN.
.NNN Nun. .wwu
NH. NN. NN.
NN. NN. NN.
NN. NH. NH.
NH. NH. NH.

N N NNz
N: NH
.HH

 

Nooo. Hmoo. HHoo. mNoo. VN
Ho. Ho. moo. No; mm
P NNN NH th
. I u u. NanoNH
:u: .t. s ,x; HouoNH
vm oH. NH. mH. NH. HoumNH
mm ac. . . No. we. oHINNH
m“ NH. NH. HH. NN. HN-NNH
NoncNH
monNNH
Nouva
HeuNNH
«HumNH
NHumNH
NoumNH
NouoNH
HHINNH
chmNH
oHumNH
«HumNH
N m u moz oz MNN
x: m can
HNNHH .mcofimon Hmv NNHonN vow
.wamsu m :NENNH> oommflu someoum .om oHoNN

55

 

Nmn. waw .mww .Nww

NH. NH. NH. NN.

NN. NH. NH. HN.

HN. NH. NH. NN.

HN. NN. NH. NN.

N N N NNz
N: NN

.NNN NNNNNN .NNN NNNNNNN .NNNzN HNNNNNNNNN NNHNNNNHNzoz

Ix

 

.pmw .Nww .wa .wa.m
NH. NH. NH. NH.
NN. NH. NN. NN.
NN. NN. NN. HN.
NH. NH. NN. NH.
N N N NNz
N: NH

 

 

x3 m

muz

neamvH
wolva
HHumvH
ecuan
ManvH
mauva
meaan
Nanm¢H
mHumvH
HHumVH
mesovH
scuth
NHIVVH
holovH
vHumvH
venevH
NanovH

.oz MHm

om + m + can

.mcoflmou HNV NNHoHNN vnm

.Hm\usv m :NsNuN> oommfiu someoum

.pm oHan

56

HNN. HHN. NNN. HNN. vN
NN. HN. HN. HNN. NN

NN.... EN g... .NINN N
- - - - NH-NNH
w-. 3-. -N _- NN-NNH
NN. NN. NN. NN. HH-NNH

HN. HNNN. HNNN. HNN. VN NN. NN. NN. NN. NH-NNH
HN. HN. HN. _HN. NN NN. NH. NN. NN. NH-NNH
ME. NH. .NIN... N.N... m NN. NN. NN. NN. NNNNH
- - - - HH-NNH

;-: _. - NH-NNH
NN. NN. NN. NN. HN-NNH

HN. HN. HN. NN. VN , NN. NN. NN. NN. HN-NNH
HN. HN. HN. HN. NN NN. NN. NN. NN. NN-NNH
NIN... MP N.NH En m NN. NN.. NN. NN. NN-NNH
- - _ -. -. HH-NNH

NN. NN. NN. NN. NH-NNH
NN. NN. NN. NN.. NN-NNH
NN. NN. NN. NN. . NN-NNH
NN. NN. NN. NN. HN-NNH

 

 

 

a m u muz m m o muz m m u muz .oz me
n=m

x3 on x3 oH x3 m

.H Hawks .mconon HNV ofiuoHNN Now
.HNV owccom .Hov ofiupmo .Hmuzv Hmowmgmomo HHmavomHmcoz .Hsmmv sowcoHoN oommHa goasoum .No oHnae

57

0H.
wH.

NH.
HH.
om.

NH.
vH.
HN.

NH. NH. HH.
H. m NH. NH. NH.

mH.
OH.
NH.
HN.

NH.
NH.
NH. m. NH.

oH. NH.
mo. HH.

 

 

 

x3 ON

.NNN NNNNNN .NNN NNNNNNN.HNN2N HNNNNNNoNo NNHNNNNHNNNz

muz m m u

x3 oH.

moz m

.H HoHHh

u muz

vHumoH
oouaoH
HonmoH
chHNH
NHunoH
HHumoH
oHuHNH
NQIHNH
venmoH
menmoH
NeumoH
oHunoH
NHnHNH
HHIHNH
neuHNH
mHnmoH
anmoH
chme

Noz mwm

om + m + can

N: N

.NconoH may NNHoHNN Now
.Hsmmv aoHcoHom comma» gouaoum

.NN NHNNN

58

HN. NNNN. NNNN. HNNN. VN
HN. NN. HN. HN. NN
.mpw. haw“ .Nww. waw .m
- - - - NN-NNH
HN. HN. HN. HN. VN NN. NN. NN. NN. HN-NNH
NN. HN. HN. HN. NN NN. NH. NN. NN. NH-NNH
N.NI. MP MN... NE“ on NN. NN. NN. NN. HN-NNH

- - - - NN-NNH

NN. NN. NN. NN. NN-NNH
HN. HN. HN. HN. VN NN. NN. NN. NN. HN-NNH
HN. HN. HN. HN. NN NN. NN. NN. NN. NH-NNH
.mmw .mpw .mpw .NNN. x NN. NN. NN. NN. NH-NNH

-... - - . - . . NN-NNH
NN. NN. NN. NN. HH-NNH
NN. NN. NN. NN. NH-NNH
NN. NN. NN. NN. NH-NNH
NN. NN. NN. NN. NH-NNH

 

 

 

N N N NNz N N N NNz N N N NNz .oz NHN
N: NN , N: NH x: N

w. _ .HH HNNNN .NNNNNNN NNN NNNNHNN NNN
.va N.N—NHHHHN £3 02930 .5qu Hmomaamomo HNHHHHNHHNHmcoz gammy 55:30.... 0333 528mm .3 038.

59

NH» mH.
”HO. 6.".

0H. NH. NH.
mH. mH. HN.
H..m NH. wH. 0H.

HN.
NH.
NN.
NH.

oH. co. NH.
m ..m NH. NH. HH.

HN.
mH.
wH.
NH.

mH.
OH..
OH.

 

 

 

N .N

#3 ON

.HNV ofivnsm .Huv uahummo .Hmozv Hmommnmomo NNHsvanucoz

muz m m u
x3 oH

muz m m u
x3 m

moz

mcumNH
mcuNNH
HHanH
NOINNH
mHnoNH
monNNH
mcumNH
NeumNH
mHumNH
HHumNH
neuoNH
NeuNNH
NHuNNH
NeuONH
NHumNH
NauNNH
NeuoNH

 

.oz NNN

om + m + 0::

.HH HNNNN .NNNNNNN NNN NNNNHNN NNN

.Hammv aofiooHoN comma» gomsoum

.vo oHnaN

60

NNNN. NNNN. NNNN. NNNN. VN
NN. _ NN. NN. NN. NN
H.H H.H N.H N.H .m
- - NH-NNH
N H N N HH-NNH
HNNN. HNN. HN. HN. vN N H N H NH-NNH
NN. NN. NN. NN. NN N.N N.N NN-NNH
H.H N.H N.N N.H N N H N H NN-NNH
- - - HH-NNH

HeumoH
Nooc. Ho. NNoo. Hooo. vm HeuNcH

H N H

N H H
NN. NN. NH. NN.. NN N. N.H NN-NNH
.NIH .HI..H. PM Mb m H .. . . N.N NNNNH

-.. . HH-NNH
;-; NH-NNH
N N NH-NNH
N H NN-NNH
N.H. NN-NNH
N N. HN-NNH

 

 

 

 

N N N NNz N N N NNz N N N NNz .oz NNN
NNN

.H HNNNN. .mzofiwon may oNHoHNN Nam
.HNV owvcsm .Huv ownummo .Hmozv Haowmgmomo uaqucmHmnoz .Hm\.=u xmuzmu oommwa somaoum .NN oHnNN

61

NH-NNH
NN-NNH
H HN-NNH
H HN-HNH
.N NH-NNH
H HH-NNH
NH-HNH
NN-HNH
N NN-NNH
H NN-NNH
.N NN-NNH
N NH-NNH
NH-HNH
HH-HNH
N H NN-HNH
H N NH-NNH
N.N . NH-NNH
N N HN-NNH

 

 

 

m u moz m m o mwz m m u mwz .oz wwm
. om + m + Gnu
x3 0N #3 oH x3 m

. .H HNHNN .moofimou HNV afiaoHNm vow
.HNV qunsN .Hov NNNNNNN .Hmozu Hmomunmomo NNHscanmooz .Hm\.=o xmummo oonNu :oNaoum .NN oHpNN

62

mme. nwoo. ano. mnHo. vm
HH. No. co. co. m
m.H MpH m.H m.H

lxco

, - -. NN-NNH
--. .-: - HN-NNH

NNNN. HNN. HN. HN. vN HN-NNH
NH. NN. NN. NN. NN NH-NNH
.NI..H. H.H N.H N.N m. 81.:

m.
m.
o

pq—cpq

o4‘0lfl
O

—1p4nd

- : u No-0NH
:u; :u; ,u; mo-NNH
N.N «N-NNH

N o

N .H I HenNNH
mc. we. mc. mH. mm m. H

n H

N.N NH-NNH
N.H .H..IH. NIH E m N.H NH-NNH

Hcoo. Hoco. Ncoo. Ncoo. vm

- . - . - NN-NNH
:u: :u; .u: NeucNH
o ~HI¢QH
H HOImVH
.H OHIMVH
O Nfilnvfi

 

 

 

 

N N N NNz N . N N NNz N N N .NNz N.Nz NNN
NNN
N3 NN x: NH , N: N

. . .HH HNNNN .NNNNNNN NNN NNNNHNN NNN
HNV ofionsN .HNV oHNpmNN .Hmqu HuomNnmoNo NNHoucmHmnoz .HM\=U xmuzmc comma» nomaoum .oN oHNNN

63

In
H
co
I-(
1‘
H
B
H
Ix

mcumNH
mcuNNH
HH-NNH
NanNH
NHnoNH
mo-NNH
m H moumNH
H N «N-NNH
o.N mHnmNH
H N HHnmNH
H.N N.N N.N H.N.m . nonoNH
NouNNH
NH-NNH
N H NN-NNH
n N NHumNH
N.H NouNNH
m N .

oomH
HHv—l
HaoN
NHH
some»
o—n-n-I
INNO
v-IHN

NancNH

 

 

 

 

N N N NNz N N N . . NNz N N. N NNz .oz NNN
. 0N + N + NNN
Na: NN NNNNH NN: N

. _ .HH HNNNN .NNNNNNN HNN NNNNHNN NNN
.HNN NNNNNN .HNN NNNNNNN .NNNzN HNoNNNNoNo NNHNNNNHNNNZ .NNNNN xN-=NN NNNNHN NNNNNNN .NN oHNNN

Table 8a.

BUD Pig No.

 

S'wks'

162-06
162-04
162-10
162-11
167-02
168-10

'10 Wks'

162-02
162-05
167-01
168-01
169-10
169-11

'ZO‘Wks

162-01
162-03
162-07
162-13
167-10

168-11.

Gross characterization

Trial I.

epithelial Change '

normal
normal .

epithelial change "

epithelial change
epithelial Change

subacute ulcer
epithelial change
acute erosion
acute erosion
subacute ulcer'
subacute ulcer‘

normal

acute erosion

subacute ulcer
normal

normal

acute erosion

64

of stomach surface (mucosa).

BUD + E + Se Pig No

 

'5'Wks
163-11
163-12
171-01
169-01
169-06
169-14

"lO'wks

163-10
169-02
169-03
169-04
171-02
171-10

"20'WRS

163-01
163-14
169-13
171-03
171-11
171-12

epithelial change
normal
epithelial change
epithelial change
normal
normal

acute erosion
subacute ulcer
acute erosion
epithelial change
acute erosion
normal

epithelial change
normal

normal

acute erosion
acute erosion
normal

65

Gross characterization of stomach Surface (mucosa).

 

Table 8b.
Trial 11.

BUD Pig No.

5 wks

144-01. normal

146-10 epithelial change

143-01 normal

146-01 normal

146-07‘ epithelial change '
"10'wks

145-14 epithelial Change

145-12 normal .

147-01 epithelial change '

144-02 subacute erosion

147-05 epithelial Change

146-06 acute erosion

ZO'Wks

143-12 acute erosion

143-10 epithelial change

145-01 acute erosion

144-11 subacute ulcer

146-04 epithelial change

145-01 chronic ulcer

BUD + B + Se Pig No.

 

"S'wks'

146-13
147-04
143-11
144-03
145-03

'lO'Wks

146-03
145-11
145-13
145-02
143-03
144-05

'ZO‘Wks

146—02
144-04
143-14
146-07
144-12
147-07

normal
acute erosion
subacute ulcer
normal
normal

acute erosion
acute erosion
normal

epithelial change
epithelial change
normal

normal

chronic ulcer
epithelial Change
chronic ulcer
acute erosion
epithelial change

Vita

The author was born in Lansing, Michigan on July 17, 1953.
Graduating from Lansing Waverly High School in 1971 the author enrolled
at Kalamazoo College, a private liberal arts college, in Kalamazoo,
Michigan. While at Kalamazoo College, he had the opportunity to spend
time at Michigan State UniverSity in East Lansing, Michigan, at the
Upjohn Company in Kalamazoo, Michigan and at L'universite' de Caen in
Caen, France. In 1975 the author graduated from Kalamazoo College and
returned to Michigan State University to enter graduate school in the
Department of Animal Husbandry. The author obtained the degree of Master
of Science from that department in 1977 and is currently pursuing the
degree of Doctor of PhiloSOphy from that department in conjunction with
the Michigan State University Institute of Nutrition.

The author married Kim Marie Green on August 22, 1975 and is
currently working as a nutritionist in the Department of Pet Nutrition

and Care Research for the Ralston Purina Company in St. Louis, Missouri.