ABSTRACT
THE ROLES OF VITAMIN E, SELENIUM, AND METHIONINE
IN DIETARY LIVER NECROSIS AND
NUTRITIONAL MUSCULAR DYSTROPHY IN THE PIG
by Robert L. Michel
Three experiments were conducted to study the pathology and patho-
genesis of dietary liver necrosis (DLN) in the pig and to clarify the
roles of vitamin E, selenium, the sulfur amino acids, and total dietary
protein in the prevention of this disease. A total of 48 pigs was useui
in the 3 trials. A basal 6% protein diet contained Torula yeast as a
source of protein and vitamin E-free lard as a source of fat. The
various supplements employed included vitamin E at 2 levels, selenium,
and methionine, each alone, and in several combinations. Other pigs
were fed a diet containing the antioxidant ethoxyquin, and 6 pigs were
fed rations containing sufficient Torula yeast to provide a 20% pro—
tein diet.
Dietary liver necrosis occurred consistently in pigs fed the basal
6% protein ration, or the basal 6% protein ration supplemented with
low levels of vitamin E or with methionine. Supplementation with
selenium, high levels of vitamin E, or additional protein completely
prevented dietary liver necrosis during the periods of these trials.
Lesions of DLN were observed in l of 4 pigs fed ethoxyquin.
Acute dietary liver necrosis appeared grossly as scattered red
spots on the surfaces and in the parenchyma of affected livers. Chronic
Robert L. Michel
changes were represented by irregularly shaped, roughened areas resulting
from the contraction of scar tissue.
Microsc0pically the most striking features were necrosis of hepato-
cytes, pooling of blood in the necrotic lobules, and a characteristic
limitation of the necrosis by the interlobular septa. Connective tissue
and bile duct proliferation occurred as a sequel to necrosis.
Microscopic lesions of nutritional muscular dystrOphy (NMD) were
observed in some pigs of all groups except those fed the higher level of
vitamin E or ethoxyquin. Muscle tissue of only 2 of 4 pigs fed ethoxy-
quin was examined for this lesion.
Ulcers or pre—ulcerous changes of the squamous epithelium of the
stomach were observed in virtually all the pigs in these experiments.
These lesions were somewhat less severe among pigs fed 20% protein
rations.
Hydropic degeneration of hepatocytes and extreme atrOphy of the
thymus occurred consistently in pigs fed 6% protein diets.
Serum ornithine carbamyl transferase activity of pigs with die-
tary liver necrosis was generally higher than that of pigs free of
DLN. However, none exceeded values reported as normal for pigs by
one author.
Nuclear abnormalities in erythroid cells in bone marrow smears
were relatively common among pigs maintained on vitamin E—deficient
diets, whereas such lesions were extremely rare among the group fed a
high level of vitamin E.
Growth rates were very poor in pigs fed 6% protein diets, but
tocopherol deficiency appeared to have little effect on growth.
THE ROLES OF VITAMIN E, SELENIUM, AND METHIONINE
IN DIETARY LIVER NECROSIS AND
NUTRITIONAL MUSCULAR DYSTROPHY IN THE PIG
By
.9
00
Robert L? Michel
A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Pathology
1967
(7’. {PW/OJ (,7
7â€}
ACKNOWLEDGEMENTS
The author wishes to express his sincere appreciation to Dr.
K. K. Keahey and Dr. C. K. Whitehair for counsel and guidance in the
conduct of this research and in the preparation of this manuScript.
The gratitude of the author is extended to all members of his
guidance committee and of the Department of Pathology at Michigan
State University for their generous assistance on many occasions.
The writer's appreciation is expressed to Dr. Elwyn R. Miller
of the Department of Animal Husbandry for assistance in formulating
the mineral and vitamin content of the basal ration.
Thanks are expressed to the National Institutes of Health for
the support granted the author in conducting this research.
Finally, the writer gratefully acknowledges the monumental debt
owed his wife, Marjorie, for her patience and assistance, and
all the Michels: Virginia, Janice, Andrea, and Richard, who also
sacrificed to make this study possible.
ii
TABLE OF CONTENTS
INTRODUCTION 0 O O O O O O O O O O O O O 0
LITERATURE REVIEW . . . . . . . . . . . .
Vitamin E Deficiency in Swine. . .
History of Dietary Liver Necrosis in Swine .
Field occurrences . . . . . . .
Experimental dietary liver necrosis in swine. . . . .
Experimental Dietary Liver Necrosis in the Rat . . . . . .
Serum Enzyme Activity in Dietary Liver Necrosis. . . . . .
Vitamin E. . . . . . . . . . . . . .
History . . . . . . . . . .
Metabolic role of vitamin E . .
Analytical Methods for Vitamin E Determination . . . . . .
selenium O O O O O O O O O O O O O O
Nutritional Muscular Dystrophy (NMD) in Pigs . . . . . . .
Hematologic Changes in Vitamin E-Deficient
Gastric Ulcers . . . . . . .-. . . .
MATERIALS AND METHODS . . . . . . . . . .
Experimental Animals . . . . . . . .
Housing. . . . . . . . . . . . . . .
Rations and Feeding Practices. . . .
Supplements. . . . . . . . . . . . .
Hematology . . . . . . . . . . . . .
Serum Ornithine Carbamyl Transferase
PathOIOgy O O O O O O O O O O O O O 0
iii
Pigs.
Determinations.
10
11
ll
12
18
20
23
24
24
28
28
29
29
30
34
35
35
Analyses of Tissues and Rations for Vitamin E Content.
Experimental Designs . . . . . . . . . . . . . . . .
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . .
Pathology of Dietary Liver Necrosis. . . . . . . . .
Gross lesions . . . . . . . . . . . . . . .
HistOpathology of dietary liver necrosis. . . .
Hydropic Degeneration of the Liver . . . . . . . . .
Nutritional Muscular Dystrophy . . . . . . . . . . .
Histopathology of NMD . . . . . . . . . . . . .
Degeneration and Ulceration of the Esophageal Region
the StomaCh O O O O O O O O O O O O O O O O O O O O 0
Gross lesions . . . . . . . . . . . . . . . .
HistOpathology of gastric lesions . . . . . .
Atr0phy of the Thymus. . . . . . . . . . . . . . .
Experiment I . . . . . . . . . . . . . . . . . .
Clinical signs and mortality. . . . . . . . . .
Feed consumption. . . . . . . . . . . . . . .
Growth. . . . . . . . . . . . . . . . . . . .
Liver weights . . . . . . . . . . . . . . . . .
Hematology. . . . . . . . . . . . . . . . . . .
OCT activity. . . . . . . . . . . . . . . .
Pathology . . . . . . . . . . . . . . . . . . .
Other gross lesions . . . . . . . . . . . . .
HistOpathology. . . . . . . . . . . . . . . . .
Vitamin E analysis of livers. . . . . . . . . .
Experiment II 0 O O O O O O O O O O O O 0 O O O O I 0
Clinical signs and mortality. . . . . . . . .‘.
iv
43
43
43
43
47
54
54
6O
60
60
65
65
65
65
68
68
68
68
75
75
75
79
79
79
Feed consumption. . . . . . . . . . . . . . . . . . . . 79
Growth. . . . . . . . . . . . . . . . . . . . . . . . . 79
Liver weights . . . . . . . . . . . . . . - . . . . . . 82
Hematology. . . . . . . . . . . . . . . . . . . . . . . 82
Serum ornithine carbamyl transferase activity . . . . . 82
Pathology . . . . . . . . . . . . . . . . . . . ... . . 82
Experiment III . . . . . . . . . . . . . . . . . . . . . . . 91
Clinical signs and mortality. . . . . . . . . . . . . . 91
Feed consumption - 6% protein groups. . . . . . . . . . 95
Feed consumption - 20% protein groups . . . . . . . . . 95
Feed consumption - pig fed standard grower. . . . . . . 95
Growth. . . . . . . . . . . . . . . . . . . . . . . . . 97
Liver weights . . . . . . . . . . . . . . . . . . . . . 97
Hematology. . . . . . . . . . . . . . . . . . . . . . . 97
Serum ornithine carbamyl transferase activity . . . . . 97
Pathology . . . . . . . . . . . . . . . . . . . . . . . 97
Bone marrow smears. . . . . . . . . . . . . . . . . . . 107
DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Clinical Signs and Mortality . . . . . . . . . . . . . . . . 109
Feed Consumption . . . . . . . . . . . . . . . . . . . . . . 111
Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . lll
Liver Weights. . . . . . . . . . . . . . . . . . . . . . . . 112
Hematology . . . . . . . . . . . . . . . . . . . . . . . . . 112
Serum Ornithine Carbamyl Transferase Activity. . . . . . . . 112
Pathology. . . . . . . . . . . . . . . . . . . . ... . . . . 113
Dietary liver necrosis. . . . . . . . . . . . .4. . . . 113
Atrophy of the thymus . . . . . . . . . . . . . . . . . 114
Hydropic degeneration of the liver. . . . . . . . . . . 114
v
Bone Marrow Smears .
Feed and Liver Analyses for Alpha-Toc0pherol .
SUMMARY .
Nutritional muscular dystrOphy. . . . . .
Lesions of the epithelium of the esophageal
of the stomach.
REFERENCES.
VITA.
vi
region
115
116
117
118
120
128
Table
10
11
12
13
14
15
-16
17
18
LIST OF TABLES
Basal ration, Experiments I, II, and III. .
20% protein ration, Experiment III. . . .
Standard Michigan State University pig grower ration.
Alpha-t0c0pherol cOntent of feeds, Experiment III .
Number of days each pig was maintained on experimental
regimen, Experiment I . . . . . . . . . . .
Number of days each pig was maintained on experimental
regimen, Experiment II. . . . . . . . . . .
Number of days each pig was maintained on experimental
regimen, Experiment III . . . . . . . . . .
Experimental design, Experiment I . . . .
Experimental design, Experiment II. . .
Experimental design, Experiment III . . .
Weights of pigs, Experiment I (Gm.) . . . .
Total weight gains and average daily gains,
1 (Gm.) o o o o o o o o o o o o o o o o o 0
Liver weights and ratios of body weights to
Experiment I O O O O O O O O O O I O I O O I
Experiment
liver
Hematocrit values (packed cell volume per cent),
Experiment I. o o o o o o o o o o o o ‘0 o o
Hemoglobin values (Gm./100 m1.), Experiment
I O 0
Serum ornithine carbamyl transferase activity,
Experiment I. . . . . . . . . . . . . . . .
Gross evidence of liver disease and extreme atrophy
thymus, Experiment I. . . . . . . . . . . .
Incidence of microscopic lesions, Experiment I. .
vii
weights,
32
33
36
37
38
40
40
42
69
70
71
72
73
74
76
78
my;
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Alpha-tocopherol content of livers, Experiment I. .
Feed consumption, Experiment II . . . . . . . . . .
Weights of pigs, Experiment II. . . . . . . . . . .
Total weight gains and average daily gains, Experi-
ment II (Gm.) o o o o o o o o o o o o o o o o o 0
Liver weights and ratios of body weights to liver
weights, Experiment II. . . . . . . . . . . . .
Hematocrit values (packed cell volume per cent),
Experiment II o o o o o o o o o ’0 o o o o o o o o o
Hemoglobin values (Gm./100 m1.), Experiment II. . .
Serum ornithine carbamyl transferase activity,
Experiment II I O O O O O I O O O I O O O O O O I 0
Gross evidence of liver disease and extreme-atrophy
of thymus, Experiment II. . . . . . . . . . . . .
Incidence of microscopic lesions, Experiment II .
Feed consumption, 6% protein group, Experiment III.
Weights of pigs, Experiment III . . . . . . . . . .
Total weight:gains and average daily gains, Experi-
ment III (Gm.) O O O O O O O O C O O O O O O O O O 0
Liver weights and ratios of body weights to liver
weights, Experiment III . . . . . . . . . . . . . .
Hematocrit values (packed cell volume per cent),
Experiment III. 0 O O O O O O O O C C O O O O O O O
Hemoglobin (Gm./100 m1.), Experiment III. . . . . .
Serum ornithine carbamyl transferase activity, Experi-
ment III 0 O O O O O O I O O O O O I O O O O O O O 0
Gross evidence of liver disease and extreme atrophy
of thymus, Experiment III . . . . . . . . . . . . .
Incidence of microscopic lesions, Experiment III. .
viii
83
84
86
87
88
89
9O
92
96
98
99
100
101
102
103
104
106
Figure
10
11
12
13
14
15
16
LIST OF FIGURES
Acute dietary liver necrosis. Basal ration plus
methionine. O O O O I O O O O O O I O O O O I I O O 0
Acute dietary liver necrosis. Basal ration plus
low level Vitamin E . . . . . . . . . . . . . . . . .
Chronic dietary liver necrosis. Basal ration . . . .
Chronic dietary liver necrosis. Basal ration plus
low level vitamin E . . . . . . . . . . . . . . . . .
Liver with acute necrosis and fibrosis. Basal ration
plus methionine plus low level vitamin E. . . . . . .
Dietary liver necrosis. Basal ration . . . . . . . .
Dietary liver necrosis. Basal ration . . . . . . . .
Dietary liver necrosis. Polymorphonuclear leukocytic
infiltration. Basal ration plus low level vitamin E.
Dietary liver necrosis. Mineralization. Von Kossa's
Stain O O O O O O O O O O O O O O I O O O I O I O O 0
Focal intralobular necrosis. Basal ration plus
methionine and low level vitamin E. . . . . . . . . .
Dietary liver necrosis with fibrosis. Basal ration 1
plus methionine O O I O O O I O O O O O O O O O O O 0
Chronic dietary liver necrosis. Basal ration . . . .
Acute necrosis and fibrosis. Basal ration plus low
level Vitamin E O O O O I I O O O O O O O O O O O O 0
Massive dietary liver necrosis. Unaffected bile
duct. Basal ration plus methionine . . . . . . . . .
Hydropic degeneration of the liver. Basal ration plus
low level vitamin E . . . . . . . . . . . . . . . . .
Hydropic degeneration of the liver. Basal ration plus
selenium plus low level vitamin E . . . . . . . . . .
ix
44
45
45
46
46
48
48
49
49
50
50
51
51
52
52
Figure
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Hydropic degeneration. Oil-red-O stain . . . . . . . .
HydrOpic degeneration of the liver. Best's carmine
Stain O O O O O O O O O O O O O O O O O O O ...-O O 0
Normally staining hepatocytes. 20% protein ration. .
Nutritional muscular dystrophy. Basal ration plus
Selenium. O O O O O O O O I O O O O O O O O O I O I O O
Nutritional muscular dystrOphy. Basal ration plus
methionine I O O O O I O O O O C O O O O O O O. O O O O
Nutritional muscular dystr0phy. 20% protein ration . .
Fragmentation. Nutritional muscular dystrophy. Basal
ration plus selenium plus low level vitamin E . . . . .
Fragmentation in nutritional muscular dystrOphy. Basal
ration plus methionine. . . . . . . . . . . . . . . . .
Increased number of.nucléi in nutritional muscular
dystrophy. 20% protein ration. . . . . . . . . . . . .
Mineral deposits in nutritional muscular dystrOphy.
Basal ration plus methionine. . . . . . . . . . . . .
Same muscle as Figure 26. Von Kossa's stain. . . . . .
Centrally located nuclei in nutritional muscular
dystrophy. Basal ration plus selenium plus low level
Vitamin E O O O I O O O I O O O O O O O O O O O O O O
Mitotic division in dystrophic muscle. Basal ration
Plus methionine O O O I O O I O O O O O O O O O O O O O
Hyperkeratosis and parakeratosis in the stomach. Basal
ration. O O O O O O O O O O O O O O O O O O O O O O O C
Hyperkeratosis and parakeratosis in the stomach. 20%
protein ration. . . . . . . . . . . . . . . . . . . .
Gomori's methenamine silver stain of g, albicans in
the stomach. Basal ration. . . . . . . . . . . . . . .
Vacuolation of gastric epithelium. 20% protein ration.
Intra-epithelial phstule in the stomach. 20% protein
ration. O O I O I O O O O . O O O O O I. O O O C O O O O 0
g, albicans in gastric ulcer. Gomori's methenamine
silver stain. Basal ration . . . . . . . . . . . . . .
55
55
56
56
57
57
58
58
59
59
61
61
62
62
63
63
64
Figure
36
37
38
39
4O
41
42
43
44
45
46
47
48
49
50
Mucinous degeneration in the stomach. Basal ration . .
Intimal degeneration in gastric blood vessel. Basal
ration plus high lava]. Vitamin E. o o o o I o I. o o o o
Degenerative changes in wall of gastric-b1ood vessel.
Basal ration plus low level vitamin E . . . . . . . . .
Intimal swelling in the wall of a gastric blood vessel.
Basal ration. . . . . . . . . . . . . . . . . . . . . .
Appearance of some of the pigs of Experiment I. . . . .
Perforated ulcer in the stomach of pig A—ll . . . . . .
Liver of pig A-ll. Dietary liver necrosis. . . . . . .
Appearance of some of the pigs of Experiment II . . .
Appearance of some of the pigs of Experiment II .
Degeneration in the myocardium. Basal ration plus
methionine O O O O O O O O I O O I O I O O O O I O O O O
Mineralization in the myocardium. Von Kossa's stain. .
Extracellular vacuolation in the liyer. Basal ration
plus methionine . . . . . . . . . . . . . . . . . . .
Greater magnification of lesion illustrated in Figure 47.
Nuclear abnormalities in bone marrow. Basal ration . .
Nuclear abnormalities in bone marrow. Basal ration
plus high level vitamin E . . . . . . . . . . . . . . .
xi
66
67
67
77
77
85
85
93
93
94
94
108
108
INTRODUCTION
Dietary liver necrosis (DLN) in the rat has been described by
Gyorgy and Goldblatt (1939) and Schwarz (1951). Obel (1953) described
a similar condition in the pig. The disease constitutes a signifi-
cant clinical problem in commercial swine operations in some parts of
the world, notably the Scandinavian countries, Germany, and New
Zealand. A morphologically indistinguishable condition occurs in
the Pacific Northwest of the United States and has been observed by
veterinarians at Washington State University. There is a considerable
volume of literature dealing with these field occurrences and with the
experimental studies undertaken as a consequence of them. The
research described herein was undertaken in order to study the patho-
genesis and pathology of this disease, and to clarify the roles of
vitamin E, selenium, methionine, and other dietary factors in its
prevention.
LITERATURE REVIEW
Vitamin E Deficiency in Swine
The lesions of vitamin E deficiency in the pig, as in other species,
may assume several forms and often overlap. So-called "yellow fat" may
occur under conditions of high intake of unsaturated fats (Davis and
Gorham, 1954). Other manifestations of vitamin E deficiency which
have been reported include: anemia (Nafstad, 1965), muscular degenera-
tion, female infertility (Adamstone.g£â€al., 1949), and necrosis of the
liver (Obel, 1953). It is with necrosis of the liver that this
treatise is mainly concerned.
History of Dietary Liver Necrosis in Swine
Field occurrences. Hutyra and Marek (1913), in Special Pathology and
Therapeutics of the Disease of the Domestichnimals (3rd ed.) cite
several early reports of necrotic liver conditions in swine. Among
those mentioned were Semmer in Russia, who described the livers as
enlarged, nodular, and marked with red blotches. Nonewitch isolated
cocci from such livers and injected them into pigs. After 7 to 8
weeks the pigs died with liver lesions similar to those described
by Semmer. Bradel made a histologic study of a porcine liver necro-
sis of unknown cause. He associated the condition with swine fever
(hog cholera). Quin and Shoeman (1933) described field losses in
Iowa from a condition which they termed "idiopathic hemorrhagic
hepatitis of swine". Whether this was identical to dietary liver
2
3
necrosis is impossible to determine.
Obel (1953), in an extensive study of dietary liver necrosis
("hepatosis diaetetica") in swine, described the condition as encountered
in commercial swine operations in Sweden. Up to 10% of the pigs pre-
sented for necropsy to the State Veterinary Medical Institute at
Stockholm were affected. The clinical course was characteristically
acute, with depression, vomiting, diarrhea, and bloody feces. Icterus
was rare. Usually several in a litter were affected, most typically
between 3 and 15 weeks of age.
Gross lesions included yellow fat, waxy degeneration of the skele-
tal muscles, ulceration of the esophageal area of the stomach, mucous
colitis, and patchy, hemorrhage—like areas and fibrosis of the liver.
Microsc0pically the principal lesion was necrosis of the liver.
The peculiar lobular distribution of the liver lesions was explained
on the basis of the unique vasculature of that organ.
Dodd and Newling (1960) described a field outbreak of dietary
liver necrosis and nutritional muscular dystrophy in 40 of a herd of
200 pigs in New Zealand. The diet included fish liver oil supplements.
There was necrosis and a notable lack of affinity for the ordinary
tissue stains in the parenchymal cells of the liver. There were also
degenerative changes in the intima of the hepatic arteries and in the
biliary epithelium. Accompanying these changes were lesions of
nutritional myopathy which were observed in both skeletal and heart
muscle in many cases. The trouble ceased when the fish liver oil was
deleted from the ration.
In the Annual Reports of the Ruakura and Wallaceville Animal
Research Stations and the Whatawhata Hill Country Station (Anon.,
4
1963-1964) it was reported that sudden deaths among pigs occurred
frequently as a result of diseases of the "mulberry heart disease -
hepatosis diaetetica complex". Such outbreaks were commonly associated
with barley feeding. On 2 occasions when barley meal was analyzed, it
was found to be low in selenium (0.008 and 0.011 p.p.m.). Peroxide
values were not excessive (less than 15 meq./100 Gm. of fat extractures).
Oral administration of selenium appeared to be the best method of
controlling the problem. Intestinal emphysema was reported as a
common accompanying lesion. A histologically identical condition has
been observed at the College of Veterinary Medicine, Washington State
University, Pullman, Washington. These occurrences were also fre-
quently associated with the feeding of barley (G. R. Spencer, unpublished
data).
Experimental dietary liver necrosis in swine. Adamstone e£_§1, (1949),
in an investigation of the effects of vitamin E deficiency in swine,
fed marine liver oils and rancid lard to 70-lb. gilts. They noted
pool-like accumulations of blood and degeneration of the liver cords
in one animal.
The Swedish worker Obel (1953) reported producing "hepatosis
diaetetica" experimentally in pigs fed rations which included dried
brewer's yeast (18%) as a source of protein and either cod liver oil
(6%) or lard (6%) as a source of fat. The lesion was produced more
consistently when the diet included cod liver oil. Alpha toc0phery1
acetate administered at the rate of 150 mg. twice weekly prevented
liver lesions in pigs on the lard diet but not in those fed cod liver
oil. The liver lesions were also prevented by a 15% casein diet or
5
supplementation of the necrogenic diet with cystine (0.5%) or methio-
nine (0.5%).
Hove and Siebold (1955) were among the early investigators of the
effects of vitamin E—deficient diets in swine. They produced dietary
liver necrosis in weanling pigs fed a 6% protein (soybean meal) ration
with 2% cod liver oil. In addition to the typical changes of necrosis,
they observed calcium deposits in the liver, ceroid pigment in the
adipose tissue, and incipient cirrhosis in some of those which sur-
vived the necrotic process. There were no lesions suggestive of
nutritional muscular dystrophy. They also described a failure of the
cytoplasm of the hepatocytes to stain, and ascribed this to protein
depletion. They considered cod liver oil essential to the pathogenesis
of dietary liver necrosis.
The roles of vitamin E and selenium in swine nutrition were further
investigated by Eggert and co-workers (1957). They fed a basal ration
which included 40% Torula yeast, 5% vitamin E-free lard, 0.4% methionine,
and 0.27% cystine. Four of 6 pigs on the basal ration died of dietary
liver necrosis within 53 days. Two of the 4 pigs also had discolored
fat. The addition of 40 p.p.m. of tocopheryl acetate or 1 p.p.m. of
selenium as selenite completely controlled the disease.
Grant and Thafvelin (1958) fed pigs a hepatonecrogenic ration
based on soya meal. The addition of 0.2 mg. sodium selenite per kg.
to the deficient ration prevented liver necrosis. However, skeletal
muscle degeneration and deposition of ceroid in adipose tissue were not
prevented by the selenium.
Stowe (1962), in a study of the lesions of toc0pherol deficiency
in swine and mink, observed liver lesions which he described as
6
centrolobular hemorrhages. Alpha-toc0pherol supplementation of the basal
ration at the rate of 100 p.p.m. was adequate to prevent all lesions of
the deficiency.
Stowe (1962) also cited Pellegrini (1958) as having produced fatal
dietary liver necrosis and nutritional myopathy in swine by feeding a
diet of 32 to 45% Torula yeast. Pellegrini found that cystine prevented
the liver necrosis but not the muscle changes, whereas 50 mg. alpha-
tocopheryl acetate and 0.45 p.p.m. sodium selenite prevented both con—
ditions. It is interesting to note that the rates of gain were satis-
factory in all groups.
Swahn and Thafvelin (1962) produced dietary liver necrosis in pigs
experimentally by adding cod liver oil or heated maize or cottonseed
oils to their diets. These rations also resulted in muscular lesions
and microangiOpathy. Yellow fat occurred in pigs fed cod liver oil.
Pigs fed heated vegetable oils had a rise of serum glutamic—oxala-
cetic transaminase from a normal of approximately 22.5 i 7.7 Kramer-
Ordel units to values as high as 300 in 30 days. Supplementation of
the vegetable oil diets with vitamin E or selenium prevented these
changes except for a slight myopathy. Supplementation of the cod liver
oil diet with selenium prevented all lesions of the deficiency except
pigmentation of the fat. An interesting sidelight to this study was
the fact that the livers of vitamin E-supplemented pigs had adequate
levels of vitamin A despite the feeding of heated maize oil. Those on
the basal ration or basal plus selenium had no detectable vitamin A in
their livers.
Stokstad ggnal. (1958) fed pigs a vitamin E-deficient diet which
included Torula yeast as a source of protein and vitamin E-free lard.
7
The pigs grew normally, but died suddenly with dietary liver necrosis.
Some pigs also had edema, discolored fat, or hemorrhages of the gastro-
intestinal tract. They observed no changes in the hemoglobin values or
red blood cell counts. Vitamin E or selenite supplementation prevented
the liver necrosis.
Keahey (1963), in the course of a study on the relationships
between protein malnutrition and infection, observed dietary liver
necrosis in pigs fed a 6% crude casein diet which included commercial
lard and 11.7 mg. dl-alpha-toc0pherol per kg. of feed. The lesion did
not appear in pigs fed higher levels of casein.
EXperimental Dietary Liver Necrosis in the Rat
In 1935 Weichselbaum published the first report on dietary liver
necrosis in the rat. He fed diets deficient in cystine and methionine
and added cod liver oil as a source of vitamins A and D. Supplementa-
tion with cystine or methionine prevented the lesion. Cystine, and to
a lesser extent methionine administration would also cause regression
of the early signs of the disease. Subsequently, Gyorgy and Goldblatt
(1939) observed the condition, again in the course of an experiment in
which cod liver oil was a part of the ration. They published the first
clear description of the liver necrosis lesions.
Glynn ethgl, (1945) employed a diet in which pure amino acid mix-
tures replaced other protein sources. They also concluded that dietary
liver necrosis is a manifestation of cystine deficiency.
Himsworth and Linden (1949) were among the first to demonstrate
the protective effect of alpha—tocopherol against dietary liver necrosis.
They saw no necrosis in 8 rats fed 15 to 20 mg. alpha-tocopherol twice
8
weekly. Necrosis occurred in 8 of 11 controls on the deficient ration.
During the early period of investigation into the nature of this
liver necrosis, there was confusion between liver necrosis as the result
of nutritional deficiencies and an entirely distinct nutritonal problem
characterized by fatty changes and fibrosis (Gyorgy and Goldblatt, 1942).
In 1942 Daft e; 31. reported proof that these were separate entities
and that the latter condition was primarily the result of choline
deficiency. Methionine, a source of methyl groups in the biosynthesis
of choline, could also prevent fatty degeneration and fibrosis. By
contrast, dietary liver necrosis was preventable with tocopherol or
the sulfur amino acids, methionine or cystine.
Gyorgy and Goldblatt (1949) were among the first to call attention
to the fact that toc0pherol deficiency was enhanced by the inclusion
of highly unsaturated fats (e.g., cod liver oil) in the diet. Abell
and Beveridge (1951) noted that all the reliably necrogenic experimental
rations had contained cod liver oil as a source of vitamins A and D.
Fite (1954) published a pathologic study of dietary liver necrosis.
He observed that the structural changes appeared suddenly and that
there was little or no gross or microsc0pic evidence of disease until
the acute necrosis occurred.
Gyorgy e£_§l, (1951) demonstrated that some antimicrobial agents,
such as chlortetracycline, when administered pe£_g§_ to rats on necro-
genic diets, significantly delayed, but did not prevent, the onset of
dietary liver necrosis. They interpreted this as evidence that the
necrosis occurred as the result of the absorption and transport to the
livercï¬ftoxic substances elaborated by intestinal microorganisms. The
deficient liver was less able to carry out its normal function of
9
detoxification, and necrosis was the result. They theorized that the
antibiotic temporarily suppressed the intestinal microflora but resistant
strains eventually developed.
Schwarz (l958b)demonstrated that such antioxidants as ethoxyquin
and diphenyl-p-phenylenediamine (DPPD), when included in the diet at
adequate levels, would completely protect rats from dietary liver necrosis.
Schwarz and Foltz (1957), aware that in addition to cystine and
tocopherol there was a third factor which would prevent necrosis of the
liver in rats, reported that the essential component of this poorly
understood "factor 3" was selenium. Two years later Schwarz _£_§l,
(1959) reported that cystine, available from commercial sources, was
contaminated with traces of selenium. It was their contention that
the protective effect of cystine against dietary liver necrosis was
due solely to its selenium content. Chemically pure cystine would
delay but would not prevent the occurrence of dietary liver necrosis in
the rat.
Schwarz (1965) found that liver slices from vitamin E-deficient
rats decline in oxygen consumption after 30 to 60 minutes of incubation.
This was characteristic of the latent (prenecrotic) stage of dietary
liver necrosis. This occurred in whole homogenates as well as in liver
slices, but not in isolated mitochondria. Also at this stage, degenera-
tive changes of the mitochondria could be demonstrated by electron;
microscopy. Simon's metabolites (p.15 ) but not alpha-tocopherol, when
added to such preparations 23.32559, prevented respiratory decline.
This phenomenon bore no relationship to the rate of peroxide formation
in the preparation. Schwarz concluded that trace element atoms, present
in whole homogenates or liver slices, blocked active sulfhydryl groups
10
of essential enzymes. In support of this he pointed to the protective
effect of such chelating agents as ethylenediaminetetraacetate (EDTA).
Schwarz speculated that the toc0pherols functioned in the body as
quinones, identical to, or closely resembling Simon's metabolites. In
this form they might oxidize the vulnerable sulfhydryl groups to less
labile disulfides, and thus prevent the respiratory decline of dietary
liver necrosis.
Olson and Dinning (1954) also found abnormalities of some of the
enzymes of the citric acid cycle in rats with dietary liver necrosis.
McLean (1963) noted that liver slices from vitamin E-deficient rats
were unable to reaccumulate potassium removed by leaching in cold
saline. Such slices also had a reduced rate of oxygen uptake. He
speculated that the deficient tissues were unable to stand the stress
of cooling, and the result was a disturbance of certain mechanisms
involved in ion movement. McLean suggested that in the bodies of
deficient animals other stresses may produce effects on these mechanisms
similar to the effects of cooling lE;Xl£IQn thus precipitating de-
ficiency signs. He pointed out that there may be a high intracellular
sodium and low potassium in dystrophic muscles of vitamin E-deficient
animals.
Serum Enzyme Activity in Dietary Liver Necrosis
Musser 33331. (1966), studied the changes in serum enzyme activity
in human beings with various liver diseases. They recommended a combi-
nation of glutamic-oxalacetic transaminase (GOT), alkaline phosphatase
(AP) and ornithine carbamyl transferase (OCT) determinations as diag-
nostic aids in hepatic disease.
11
Orstadius egngl. (1959) noted that serum glutamic—oxalacetic
transaminase, serum glutamic-pyruvic transaminase (GPT), and serum
ornithine carbamyl transferase were all elevated in dietary liver ne-
crosis of swine. By contrast, only GOT and GPT activities increased
in nutritional muscular dystrophy. They pointed out that muscle tissue
has little OCT, and therefore recommended this determination as highly
Specific for liver disease. The bromsulfalein excretion test and
icteric index determinations were unreliable indicators of dietary
liver necrosis.
Wretlind e£_§l, (1959), employing Reichard's technic (Reichard,
1957), determined the plasma ornithine carbamyl transferase activity
in 19 normal pigs. They reported a mean normal value of 5.93 units
with a standard deviation of i 2.42.
Vitamin E
History. The essential nature of vitamin E in nutrition was reported by
Evans and BishOp (1922) of the University of California. They noted
reproductive failure in female rats as a result of fetal resorption.
The condition could be prevented by the feeding of fresh lettuce, dried
alfalfa, or additional butterfat. Neither orange juice (vitamin C) nor
cod liver oil (vitamins A and D) were effective in the prevention of
this disorder.
The name "toc0pherol" was suggested to Evans by George M. Calhoun,
a professor of Greek at Berkeley. The term is derived from the Greek
words tocos, child birth, and phero, to bear or carry. The suffix
"-01" indicates the alcoholic nature of the compound.
12
To date, numerous toc0pherols have been isolated, differing in the
number and placement of methyl groups on the basic chromane ring
(Emerson g£_§l,, 1937). Of these, the one designated as alpha-
tocopherol is the most abundant in nature and the most active biologi—
cally (Dam, 1957).
Since the original description of infertility in the rat by Evans
and Bishop, a great variety of vitamin E deficiency manifestations have
been noted in many different species. Among the better known of these
are myopathies in the pig (Lannek £3 31" 1960), in the lamb (Metzger
and Hagan, 1927), in the calf (Schofield, 1953), and in the guinea pig
and the rabbit (Goettsch, 1931). Other well known diseases associated
with vitamin E deficiency include: exudative diathesis in the chick
(Dam and Glavind, 1939), fat pigmentation in the mink (Hartsough and
Gorham, 1949) and the rat (Mason _£._l., 1946), degeneration of the
testes in the rat (Mason, 1940), and necrosis of the liver in the rat
(Gyorgy and Goldblatt, 1949) and in the pig (Obel, 1953). This is by
no means a complete list. Vitamin E deficiency has never been char—
acterized as a clear—cut clinical or experimental entity in man. Whether
human beings can indeed suffer such a deficiency is questionable.
Metabolic role of vitamin E. The precise metabolic role or roles of
vitamin E are not known. The diverse manifestations of vitamin E
deficiency render improbable the finding of a simple, single biochemical
explanation of its function. However, it has been possible to demon—
strate, by in yi££g_studies of the chemical behavior of the toc0pherols,
and by ig;yigg_trials using experimental rations, some of the more
likely mechanisms of action of this unique nutrient.
13
Much of the research to elucidate the function of vitamin E has
related to the well known ability of the toc0pherols to undergo
oxidation-reduction reactions i§;yi££g_to form the corresponding qui-
nones. This fact suggested that vitamin E might serve as a biochemical
antioxidant to protect essential labile compounds in the body,such as
fatty acids, vitamins, and enzymes, from oxidation by hydrogen peroxide
or molecular oxygen.
Among the leading pr0ponents of this theory are the Danish worker
Dam and Tappel of the University of California. Dam (1957), in a com-
prehensive review of the subject, concluded that the principal effect
of an antioxidant in protecting fats is the neutralization of free
radicals which represent the initial stage in an autoxidative chain
reaction. The succeeding stages are the formation of peroxidic free
radicals, hydroperoxides, keto- and hydroxy- compounds, and polymeri-
zation products. In the reaction with the free radical, the antioxidant
itself is converted to a free radical form. In this form it is un-
stable and is thus irreversibly destroyed. Although alpha-toc0pherol
is actually the least active iggyggrg_antioxidant of the known toc0pherols,
its greater activity in 3139 may be due to its greater tendency to
accumulate in the tissues. Although commercially the toc0pherols are
often converted to the acetate ester to increase their stability in
storage, only the free phenolic forms are active as antioxidants.
Therefore, the esters must first be hydrolyzed in the body. The anti-
oxidant effect of the toc0pherols in giggg is enhanced by the presence
of ascorbic and phosphoric acids (Dam, 1957). The latter observation
is interesting in light of apparent successes in the treatment of nutri-
tional myOpathies of lambs and calves with phosphoric acid (Schofield,
1953).
14
Olcott and Emerson (1937) did not agree that the free hydroxyl group
is necessary for antioxidant activity. They found that the allophonates
of the toc0pherols also act as antioxidants.
Tappel (1962) stated that vitamin E is the principal lipid anti-
oxidant in the animal body. He espoused the theory that damage to the
lipid components of the mitochondrial, microsomal, and lysosomal mem-
branes was the fundamental disorder in vitamin E deficiency. This
occurred as the result of lipid peroxidation which was in turn initiated
by free radical chain reactions. Tappel and Zalkin (1959a) found that
tocopherol added to ignxi££g_preparations of isolated mitochondria
markedly reduced peroxidation and at the same time increased the sta-
bility of DPNH-cytochrome C reductase. In a companion paper (Tappel
and Zalkin, 1959b) they reported that mitochondria contained about 25%
lipids, mainly unsaturated, and that they were subject to hematin-
catalyzed oxidative deterioration in the presence of molecular oxygen.
They suggested that vitamin E, normally present in mitochondria, sta-
bilized the unsaturated lipids.
Pollard and Bieri (1959), of the National Institutes of Health,
found no difference in DPHN-cytochrome C reductase activity in heart
muscle preparations from normal and vitamin E-deficient chicks. Alpha-
toc0pherol could not be demonstrated in the preparations from the
deficient birds in contrast to those from the controls which did con-
tain alpha-toc0pherol.
Tappel's views are supported by the fact that many of the condi-
tions associated with vitamin E deficiency occur only if the diet includes
significant amounts of easily oxidizable, i.e., unsaturated fats.
Indeed, most of the manifestations of vitamin E deficiency in all
15
species are more readily produced experimentally if such fats are
included in the diet. Deficiency states may occur under such condi-
tions even when the diet contains relatively high levels of vitamin E
(Evans and Burr, 1927). Examples of diseases which require the presence
of greater amounts of dietary polyunsaturated fats for their occurrence
are encephalomalacia of chicks (Century and Horwitt, 1958; Kummerow,
1964), and "yellow fat" disease of mink (Mason and Hartsough, 1951).
An example of a deficiency state which is produced more readily when
relatively high levels of polyunsaturated fats are fed is nutritional
muscular dystrophy (NMD) of pigs (Lannek etugl., 1961). Cod liver oil
is often employed in experimental rations because of its high content
of polyunsaturated fats. McCoy g£_§l, (1938) demonstrated that if cod
liver oil were first hydrogenated, it did not enhance vitamin E
deficiency in the experimental production of nutritional muscular
dystrOphy of rabbits.
Csallany and Draper (1962) lent further support to the antioxidant
theory of vitamin E function. They demonstrated the conversion of
labeled alpha-tocopherol to a quinone in the body. Similarly, Simon
e£_§l, (1956) reported the isolation of a quinone from urine of sub-
jects following oral administration of large doses of dl-alpha-
tocopherol. The quinone in the urine was conjugated as a glucuronide.
The isoprenoid side chain of alpha-toc0pherol had been shortened by
13 carbons and the terminal methyl group had been oxidized to a car-
boxyl group. A gamma lactone of this compound was also isolated.
These two molecules have come to be known as "Simon's metabolites".
16
The antioxidant concept of vitamin E function is supported by
numerous reports wherein structurally unrelated compounds with anti-
oxidant activity have prevented deficiency signs in animals fed vitamin
E-deficient diets. Crider e; 31. (1961), using diphenyl-p-phenylene-
diamine (DPPD) or ethoxyquin (EQN) maintained rats on vitamin E-
deficient diets through normal reproduction. These compounds did not
prevent the disappearance of toc0pherol from the tissues. They con-
cluded, therefore, that the action of DPPD or ethoxyquin was not due
simply to a sparing or protective effect on tocopherol, but that they
actually replaced the vitamin biologically. Singsen e£_gl, (1955)
prevented encephalomalacia in chicks fed 2% cod liver oil by supple—
menting the ration with DPPD (0.025%).
Schwarz (l958b)maintained rats on a hepatonecrogenic diet by
supplementing with ethoxyquin (0.25%) or DPPD (0.02%). He did not
believe the antioxidants prevent liver necrosis simply by stabilizing
dietary fats because liver necrosis was readily produced with fat-free
rations and his basal ration was stable against rancidity. This sta-
bility was due to antioxidants in Torula yeast which he employed as a
protein source.
Bieri and Anderson (1960) found that peroxide formation in incu-
bated tissue homogenates was prevented when adequate supplements of
tocopherol were fed to chicks and rats on vitamin E-deficient diets.
There was considerable peroxide formation in the tissues of unsupple—
mented animals.
Bunyan e£_§1, (1963) used the thiobarbituric acid test (Wilbur gt
31,, 1949) as an index of lipid peroxidation in tissue homogenates.
This is a colorimetric test which depends for color development on the
17
breakdown of peroxides to malonaldehyde. They found little correlation
between the inhibition of lipid peroxidation and protection from die-
tary liver necrosis. They concluded that either lipid peroxides were
not increased in necrotic livers or that the peroxides were not broken
down to malonaldehyde.
Green (1962) likewise concluded that the antioxidant activity of
the toc0pherols in the reduction of lipid peroxidation could not alone
explain their functions in prevention of the many manifestations of
vitamin E deficiency.
Bouman and Slater (1957), using horse heart muscle preparations,
presented evidence that alpha-toc0pherol was intimately involved with
the respiratory chain. They speculated that the molecule might act
catalytically to transfer phosphate groups from phosphoric acid to
adenosine diphOSphate. Thus, the essential effect of vitamin E
deficiency was an uncoupling of oxidative phosphorylation. It is known
that this effect increases respiratory chain activity by reducing the
inhibition ordinarily exerted by the accumulation of adenosine tri-
phosphate. They supported this theory by pointing out that oxygen
consumption is increased in dystrOphic muscle.
An increasing number of scientists believe that, although vitamin
E is unquestionably active as a biological antioxidant, it has other,
specific functions which characterize it as a true vitamin. Scott
(1962), at Cornell University,in a review of vitamin E in poultry nu-
trition, pointed out that while this vitamin is the most effective,
but not the only, antioxidant for the prevention of encephalomalacia,
it serves also in a specific role, interrelated with selenium, to pre—
vent exudative diathesis as reported by Patterson 35 31. (1957).
18
‘Analytical Methods for Vitamin E Determination
Emmerie and Engel (1938) reported a colorimetric method for the
quantitative determination of alpha-tocopherol in certain foodstuffs,
such as wheat germ oil and cottonseed oil. The technic was based on
the ability of tocopherol to reduce ferric to ferrous iron. In the
presence of alpha, alpha' dipyridyl, the ferrous ion formed a colored
salt which could be measured colorimetrically. In a subsequent paper
(Emmerie and Engel, 1939) they reported wider adaptation of the method
for the analysis of biologic materials which contained significant levels
of interfering substances, such as vitamin A and carotenoids. They
separated these from toc0pherol by adsorption on Floridin XS earth,
eluting the tocopherol with benzene. The basic principle of the
Emmerie—Engel colorimetric determination using ferric chloride and
alpha, alpha' dipyridyl is still widely employed for vitamin E deter-
mination in feeds and tissues.
A micro-technic for the determination of total toc0pherols in
blood serum or plasma was described by Quaife _£Hgl. (1949). The
procedure was based on the Emmerie-Engel reaction. Light absorption
due to carotenoids was measured at 4601nfx , and a correlation was made
for the contribution of the carotene to the Emmerie-Engel reaction color
at 520 m/u.
Bro-Rasmussen and Hjarde (1957) reported a method for separating
alpha-toc0pherol from other toc0pherols and vitamin A. They eluted
alpha-toc0pherol from a column of secondary magnesium phosphate using
petroleum ether containing 2% ethyl ether. The other compounds could
then be eluted by increasing the percentage of ethyl ether in the eluant.
19
This purification procedure was suitable when quantitative colorimetric
determinations were to be done using the Emmerie-Engel technic.
Dicks and Matterson (1961) reported a procedure for the determina-
tion of alpha-toc0pherol in tissues. They employed the Bro-Rasmussen-
Hjarde technic of secondary magnesium phosphate column chromatography.
However, they modified the method with respect to the saponification
step and the colorimetric determination with ferric-chloride-dipyridyl.
Duggan (1959) described a technic for the determination of plasma
toc0pherols based on the fact that toc0pherols exhibit ultraviolet
fluorescence. He reported good correlation with chemical methods.
Edwin and co-workers (1960) described a procedure for the determi-
nation of toc0pherols in tissues. After extraction and separation from
the saponifiable lipids, toc0pherols were further purified by Floridin
earth column chromatography and finally isolated by 2-dimensional paper
chromatography. It was their contention that this technic avoided the
problem of false high values to the Emmerie-Engel colorimetric determi-
nation which may occur due to other reducing substances in the tissues.
They reported a 90% recovery of added toc0pherols.
Mason (1942) studied the distribution of vitamin E in the tissues
of the rat. He concluded that the amount of tocopherol in the liver
was the best index of the dietary level and body stores of the vitamin.
The total amount of stored vitamin E in all the tissues of the rat,
however, represented only a small fraction of that ingested. From this
Mason inferred that the tocopherol was either poorly absorbed or rapidly
degraded. Quaife and Harris (1944), using a method for tocopherol
determination based on the Emmerie-Engel reaction, reported the normal
plasma range for man as 0.90 to 1.59 mg.% with 1.20 mg.% as the average.
20
Selenium
Prior to the 19505 the element selenium was thought to have bio—
logical significance only as a poison. Forage crOps with high selenium
content were responsible for numerous losses among livestock in the
central plains and Rocky Mountain states (Draize and Beath, 1935; Moxon
and Rhian, 1943). In 1954, however, Pinsent (1954) reported that cer-
tain bacteria of the coli-aerogenes group required selenite for the
biosynthesis of formic dehydrogenase. The organisms did not have an
absolute requirement for selenite, however, in that they were capable
of carrying out all essential oxidations without this enzyme.
Numerous subsequent reports were concerned with the use of selenium
compounds in treatment of diseases associated with vitamin E deficiency.
Among the conditions which were reported to be preventable or treatable
with selenium were dietary liver necrosis in swine (Eggert e£_§1,,
1957) and rats (Schwarz and Foltz, 1957), exudative diathesis in chicks
(Patterson 23 31., 1957), nutritional muscular dystrophy (white muscle
disease) of lambs and calves (Muth e£_§l,, 1959), and the corresponding
condition in swine (Lannek e£_§l,, 1961).
Despite these examples of the interrrelationship of selenium and
vitamin E, no one has been able to explain in biochemical terms the
exact mechanism of this relationship. Bieri (1961) suggested that
selenium in some form serves as an antioxidant and can substitute for
vitamin E in this capacity. In a later paper, however (Bieri, 1959),
he pointed out that although lipid oxidation is reduced in homogenates
of liver and muscle from selenium supplemented chicks, there was no
reduction when selenium was added directly to the preparation i§_vitro.
21
This was interpreted as evidence that selenium compounds do not act
directly as lipid antioxidants.
Welch'ggnal. (1960) produced nutritional myopathy in lambs by
feeding fish liver oils to the dams. The plasma tocopherol levels of
the dams were reduced. The condition could be prevented if the dams
were fed supplementary vitamin E. The addition of sodium selenite to
the rations of the dams was much less effective in controlling NMD in
the young.
Oldfield £5 31. (1960), at Oregon State University, noted that
white muscle disease in the lamb could be prevented by giving selenium
or large doses of vitamin E at birth, but the growth rate was better
in the selenium-supplemented group. The growth-stimulating effect
of selenium was even more pronounced if it were given prenatally to
the dam. They concluded that selenium is essential for normal growth
in lambs.
In discussing the role of selenium in nutrition, Schwarz (1960)
stated that although it frequently serves to prevent the occurrence
of the signs of vitamin E deficiency, it is also an essential micro-
nutrient in its own right. As evidence for this view he mentioned poor
growth in rats and chicks on diets adequate with respect to vitamin E
but deficient in selenium. Schwarz (1958a) propounded the theory that
vitamin E and selenium were each essential to the proper function of
two different but equivalent metabolic pathways. Either one of these
pathways was sufficient to maintain the animal's health. Thus, such
conditions as dietary liver necrosis represented a simultaneous de-
ficiency of vitamin E and selenium.
22
Green etngl. (1961) reported that both selenium and vitamin E
supplementation increased tissue ubiquinone. In tocopherol—deficient
rats ubiquinone levels in the uterus were reduced. Administration of
vitamin E resulted in increased uterine ubiquinone and correction of
the accompanying resorption sterility. Selenium supplementation did not
affect ubiquinone levels in uterine tissue and did not prevent resorp—
tion sterility.
Tappel (1962), in discussing the relationship of selenium to vitamin
E, speculated that dietary inorganic selenium compounds are incorporated
by the body into unknown organic antioxidants. Such selenium antioxi-
dants are about 500 to 2000 times as effective as alpha-tocopherol on
a molar basis for the prevention of lipid peroxidation and these com—
pounds react with free radical intermediates to break chain reactions.
Olcott e£_§l, (1961) reported that selenomethionine had strong
antioxidant properties ignyi££g_as indicated by its ability to control
lipid oxidation and rancidification. They suggested that the failure
of selenium supplements to prevent some of the diseases due to vitamin
E deficiency could be explained on the basis of different degrees of.
availability in different tissues.
Bunyan.e£_§1, (1963) found that reduction in lipid peroxidation is
not the essential mechanism by which selenium protects rats from necrosis
of the liver. The lesion could be prevented with dietary levels of
selenium lower than that required to control peroxidation in tissues.
Sondergaard-egual. (1958) studied the role of selenium in the diet
of the tocopherol-deficient rat. They concluded that selenium would
not prevent the following manifestations of vitamin E deficiency in the
rat:
23
l. Decreased liver storage of vitamin A.
2. Depigmentation of the incisors.
3. Peroxidation and discoloration of body fat.
4. Increased erythrocyte fragility.
Nutritional Muscular DystrOphy (NMD) in Pigs
Adamstone e£_al, (1949), at Illinois, were among the early investi—
gators of the importance of vitamin E in swine rations. Feeding natural
diets which contained rancid lard and marine liver oils, they observed
degenerative lesions of the muscles in addition to liver necrosis and
reproductive failures. Forbes and Draper (1957), at the same institu-
tion, also observed muscle degeneration in pigs fed low vitamin E
rations with 5 to 10% cod liver oil. Dodd and Newling (1960), in New
Zealand, reported nutritional muscular dystrophy in pigs affected with
dietary liver necrosis. The ration contained fish liver oil. Losses
ceased‘when the addition of oil to the feed was discontinued. Thafvelin
(1960) demonstrated that nutritional muscular dystrophy could occur in
pigs on rations containing no fats other than vegetable fats.
Lannek gtpal, (1961) fed a vitamin E-deficient diet containing
stripped lard, casein, and brewer's yeast to pigs. No NMD was observed.
If, however, the lard were replaced with cod liver oil, muscle degenera—
tion did occur. Sodium selenite was said to have a curative effect.
However, Swahn and Thafvelin (1962) reported that selenium provided pigs
only partial protection from NMD when diets contained fish liver oils
or oxidized vegetable oils.
24
Grant and Thafvelin (1958) fed a known hepatonecrogenic diet to
newly weaned pigs to determine the effect of selenium supplementation
under such conditions. Although liver necrosis was completely prevented,
ceroid deposition in adipose tissue and degeneration of skeletal muscle
did occur. Thus, selenium apparently exerted a preventive action on
some but not all of the manifestations of vitamin E deficiency in the
pig. Selenium has also been found ineffective or only partially effective
in preventing NMD in rabbits (Hove e£_§l,, 1958).
Hematologic Changes in Vitamin E-Deficient Pigs
Nafstad (1965) studied the blood and bone marrow of vitamin E-
deprived pigs. She fed a ration which included 10% casein and either
4 or 5% cod liver oil. After 4 weeks, deficient pigs had reduced hemo—
globin values and packed cell volumes. There was also an increased
total leukocyte count due principally to a neutrophilia. There was
hypercellularity of the bone marrow involving mainly the erythroid
elements. Multinucleated cells representing all stages of the erythro-
poietic series were observed. Vitamin E supplementation effectively
prevented these changes. Supplementation with 0.25 p.p.m. sodium selenite
or with a combination of methionine, lysine, and threonine did not in-
fluence the occurrence of these changes in the blood and blood-forming
tissues.
Gastric Ulcers
The problem of gastric ulcers in swine has received a great deal
of attention in recent years. Although the so-called "peptic ulcer" of
the glandular epithelium occurs in swine, lesions of the esophageal por-
tion of the stomach are far more common (Muggenberg g5 11., 1964).
25
Reese and co-workers (1966a) reviewed several studies on the incidence
of this condition. They cited reports indicating that the incidence
of ulcers and preulcerous lesions may exceed 50%. The changes apparently
begin as a hypertrophic parakeratosis of the squamous epithelium which
progresses to erosion, ulceration, and finally to chronic inflammatory
changes. Severe, even fatal, hemorrhages may occur (Muggenberg g; 31.,
1964).
Many attempts haVe been made to relate the incidence of gastric
ulcers in pigs to various dietary or environmental influences. Nafstad
(l967a),in Norway, studied the effects of various dietary factors on
ulcer develOpment. She concluded that the incidence of lesions was not
influenced by the level of protein or fat, thiamine supplementation of
the ration, or addition of tryptophan, lysine, and methionine. Vitamin
E appeared to have a mildly mitigating effect. Nafstad 35 31. (1967b)
reported that rations containing high levels of rash or oxidized unsatu-
rated fats were consistently ulcerogenic. Vitamin E had a slight pro-
tective effect. Increasing the level of protein, amino-acids, or selenium
did not alter the incidence of lesions. In a study of the effects of
different proteins and fats on the occurrence of ulcers, Nafstad e£_§l,
(1967c) found that a diet containing saturated fat (hydrogenated coco-
nut oil) was far less ulcerogenic than one containing unsaturated fat
(cod liver oil). Similarly, the incidence of ulcers was markedly reduced
when soybean oil meal was substituted for part of the casein used as a
source of protein. If the casein were eliminated and replaced with soy-
bean meal (36%), ulcers were completely prevented. The authors speculated
that soybean oil meal may possess buffering properties which casein does
not.
26
Jensen (1946) studied the effects of toc0pherols on the incidence
of gastric ulcers in rats maintained on a diet deficient in vitamin A.
Ulcers deveIOped in a large percentage of vitamin A-deprived rats.
Vitamin E supplementation substantially reduced ulcer formation. How-
ever, if large doses of alcohol were included in the rations, the
apparent protective effect of vitamin E was overcome.
Reese £3 al. (1966a) studied the effects of various nutritional and
environmental factors on the occurrence of gastric ulcers in swine.
They fed antibiotics, arsenilic acid, dry skim milk, fluid milk, pre—
viously heated ground soybeans, 15% soybean oil, the water soluble B
group of vitamins, and the fat soluble vitamins A, D, E, and K. They
observed no change in the over-all incidence of ulcers as a result of
adding any of these to the ration. In a companion study (Reese e£_§l,,
1966b) they found that oats had a marked protective effect when added
to corn or wheat rations. If the diet consisted of 85% oats, no lesions
were observed. By contrast, all pigs fed a 76% corn ration had lesions
of the esophageal portions of their stomachs, and, in 53% of these
animals, the changes had progressed to the erosion or ulcer stage.
Nuwer 25.31, (1965) fed pigs a highly ulcerogenic diet containing
gelatinized corn. They found no effect on the incidence of ulcers from
feeding a number of different additives, including oxytetracycline,
oxytetracycline plus copper sulfate, vitamin A, tocopherol, tocopheryl
acetate, menadione, methionine, and an antihistamine.
Rothenbacher etngl. (1963) surveyed the problem of gastric ulcers
among swine in Michigan. They reported that both bacteria and fungi
could commonly be demonstrated in the "fibrino-necrotic membranes" often
associated with gastric ulcers. These organisms were regarded as
27
secondary invaders, since they were not observed in the deeper structures
of the affected areas.
***
Thus, it is_c1ear that although there has been a considerable amount
of investigation of the problem of dietary liver disease, there is as
yet an incomplete understanding of the roles of various dietary factors
in combatting these diseases, and of the interrelationships among these
factors.
MATERIALS AND METHODS
This dissertation describes 3 experiments which were done at
Michigan State University during the period from February 1966 through
the Spring of 1967. Although the 3 trials were similar in many
respects, there were some important differences which are explained
in the following general description of materials and methods.
Experimental Animals
Young Yorkshire-cross pigs were used as the experimental animals
in all experiments. Except for pigs numbered C-l through C-18, which
were bought from a local farmer, all pigs were.farrowed at the Veteri-
nary Research Farm at Michigan State University. They were identified
by ear notching and assigned particular experimental rations by random
selection. Supplemental iron was administered before beginning each
experiment. Pigs were observed at least twice daily for obvious clini—
cal signs. They were weighed at the beginning and end of each experi—
ment and at intervals during the course of each experiment. Final weight
was determined at the time of euthanasia or, in the cases of pigs which
died for other reasons, as soon after death as practicable.‘
A single litter consisting of 7 males and 5 females was used for
Experiment I. They were started on the experimental regimen when 16 days
of age.
A single litter of 6 male and 6 female pigs was used in Experiment
II. They were 29 days of age when confined in individual cages and
started on the experimental rations.
28
29
Pigs from 3 different litters were used in Experiment III. One
litter of 7 males and 3 females (pigs C-1, C—3, C-5, C-7, C-10, C—12,
C-l4, C-l6, C-17, C-18) were 20 days of age at the start of the experi-
ment. Another litter of 4 males and 4 females (pigs C-2, C-4, C-6,
C-8, C-9, C-ll, C-l3, C-15) were 18 days of age at the start of the
experiment. A 3rd litter of 2 males and 4 females (pigs C-19 through
C-24) were 25 days of age at the start of the experiment. These last
6 were all fed a basal ration containing 20% protein.
Housing
Excepting C-l9 through C-24, all pigs were maintained in individual
metal metabolism cages in a heated, isolated room. Throughout Experi-
ment III pigs C-l9 through C-24 were maintained in ordinary pens with
a concrete floor. At first pigs C419 and C-20 were kept in one pen
while C-21 through C-24 were kept in an adjoining pen. In hosing down
the floors some of the excreta were washed into the adjoining pens and
the pigs thus had access to the wastes from animals on other experi-
mental regimens. It was thought that the feces and urine of C-19 and
C-20 might contain enough selenium to jeepardize the experiment. There-
fore, on the 12th day of the trial, C-19 and C-20 were moved to a dif-
ferent room, and pigs C-21 and C-22 were separated from C-23 and C-24
and placed in the pen vacated by C-19 and C-20.
Rations and Feedingigractices
All pigs were fed twice daily. Those in individual cages were fed
and watered in separate bowls. An effort was made to keep water
available at all times,but some of the caged pigs consistently emptied
their bowls between waterings, particularly during the night. The pigs
30
which were penned on the floor were fed using one pan for each 2 pigs.
Note was made of pigs which failed to eat all of the feed provided, and
the amount was adjusted accordingly.
During Experiments I and II, feed was prepared in quantities suf-
ficient to last 1 to 2 weeks. Mixing was accomplished with a Hobart
mixer in 7 kg. batches. The mixed preparation during these 2 trials was
stored in galvanized containers at room temperature. Unused stripped*
lard in opened cans was refrigerated until used the next time feed was
mixed.
During Experiment III feed was mixed at intervals of one week or
less except at the beginning of the trial, when sufficient feed was
prepared for 17 days. Feed was kept at refrigerator temperatures in
polyethylene bags. The standard Michigan State University pig grower
which was fed to pigs C-17 and C-18 was kept at room temperature.
The basal ration is given in TABLE 1.
The 20% protein ration is given in TABLE 2.
The standard Michigan State University pig grower ration is given
in TABLE 3.
The diets fed in Experiment III were analyzed for alpha-tocopherol
content and the results are given in TABLE 4.
Supplements
The experimental supplements employed in these trials included vita—
min E, selenium, methionine and ethoxyquin. Vitamin E was supplied as
dl-alpha-toc0phery1 acetate.* It was administered at the rate of
*Vitamin E-free. - -
**Rovimix E-lOOW, Hoffman La Roche.
31
TABLE 1. Basal ration, Experiments I, II, and III.
Torula yeast 12%
Cerelose 72%
Stripped lard 5%
Mineral mix* 6%
Cellulose 5%
Vitamin mix** 25 ml./kg.
* Mineral mix:
COCO3 1.00
KI 0.02
C0304 1.00
Mnso4~H20 1.00
FeSO4'2H20 7.00
Zn804°H20 4.00
MgCO3 20.00
KCl 100.00
CaCO3 125.00
Cerelose 131.00
NaHCO3 250.00
CaHPO4:2H20 360.00
1000.00 Gm.
** 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 B12 (0.1% triturated) 10.000 Gm.
Vitamin D2# 0.044 Gm.
Menadione sodium bisulfite 0.400 Gm.
Water soluble vitamin A## 1.000 Gm.
Absolute ethyl alcohol 250 m1.
Distilled water q.s. 2500 ml.
# Water dispersible vitamin D2 (500,000 I.U./Gm.)
## Rovimix A—250-W, 294,000 U.S.P. units vitamin A/Gm., Hoffman
La Roche, Nutley, N. J.
32
TABLE 2. 20% protein ration, Experiment III
Torula yeast 40%
Cerelose 44%
Stripped lard 5%
Mineral mix* 6%
Cellulose 5%
Vitamin mix** 25 m1./kg.
* See TABLE 1.
** See TABLE 1.
TABLE 3. Standard Michigan State University pig grower ration.
S._;‘ p— _
Ground shelled corn 1554 lb.
Soybean oil meal (50% protein) 300 lb.
Meat and bone scraps 60 lb.
Alfalfa meal (17% protein) 50 1b.
Limestone 12 1b.
Dicalcium phosphate 4 1b.
High Zn trace mineral salts 10 1b.
Vitamin, antibiotic, trace mineral premix* 10 1b.
2000 lb.
* Vitamin A and D premix# 6
Merck 1231 Mixture (vitamin
B complex) 3
Dawes B12 10
Prostrep 20 ## 20
Zn oxide (74% Zn) 1
Ground shell corn 60
‘ 100
# Vitamin A and D premix: ## Procaine penicillin and
Vitamin A - 3,628,720 I.U.llb. streptomycin
33
TABLE 4. Alpha-toc0pherol content of feeds, Experiment III.
Ration Alpha-tocopherol (mg.%)
Basal (6% protein) 0.38
Basal (6% protein) + ethoxyquin 0.43
Basal (20% protein) 0.24
Standard pig grower ration 1.13
34
21.8 I.U., 3 times weekly (hereinafter designated "low level E") in
Experiments I, II, and III and at the rate of 150 I.U., 2 times weekly
(hereinafter designated "high level E") in Experiment III.
Selenium was furnished as sodium selenite, 0.2 mg., 3 times weekly.
At the beginning of Experiment I the vitamin E and selenium supple-
ments were both added to the ration at feeding time. After 29 days,
when feed consumption by some animals began to decline, these supple-
ments were administered to such animals orally with a syringe. This
means of administration was employed exclusively for vitamin E and
selenium in Experiments II and III.
Supplemental methionine was furnished as DL methionine. This was
mixed with the basal ration at the rate of 1.0 Gm. per 1000 Gm. of feed.
This seemingly low level of supplementation was used in order to keep
the sulfur-amino acids in prOper relation to the other amino acids in
this low protein ration. Thus, the ration contained 0.21% total sulfur-
amino acids after the addition of methionine. The basal ration (6%
protein) contained 0.12% sulfur-amino acids.
For those pigs whose basal ration was sUpplemented with ethoxyquin,
the antioxidant was added to the ration at the level of 0.1% (1 Gm.
per 1000 Gm. feed) at the time of mixing.
Hematology
Blood samples were collected from the anterior vena cava. Blood
was collected from the brachial artery after electrocution at the time
of necropsy. The disodium salt of ethylenediaminetetraacetic acid (EDTA)
was used as the anticoagulant. Hemoglobin determinations were made by
the cyanmethemoglobin method. Packed cell volumes were determined using
microhematocrit tubes.
35
Serum Ornithine Carbamyl Transferase (OCT) Determinations
Blood for serum enzyme activity studies was routinely collected from
the anterior vena cava. Blood was collected from the brachial artery
at the time of euthanasia. Samples were allowed to clot and were then
centrifuged. Serum was removed with a pipette and promptly frozen and
stored at -70° C until tested for OCT activity. The determination was
made using the technic of Reichard (1957).#
Pathology
The number of days that each pig was maintained on an experimental
regimen is given in TABLES 5, 6 and 7. The pigs were necropsied at the
ends of the indicated periods.
Pigs in all experiments were killed by electrocution. The car-
casses were immediately weighed and the brachial arteries severed for
blood collection for laboratory procedures. The livers were removed at
necropsy, trimmed of the lymphoid tissue and extrahepatic biliary struc-
tures, and weighed on a Mettler balance. Tissues collected for histo-
pathologic studies included sections from the: esophagus, esophageal
region of the stomach, glandular region of the stomach, duodenum, jejunum,
ileum, colon, mesenteric lymph node, right and left kidneys and adrenals,
right and left lateral lobes of the liver, left central lobe of the
liver, thymus, pancreas, right quadriceps femoris, skin from the medial
aspect of the right thigh, spleen, left ventricle, aorta, cardiac lobe
of the right lung, and others where indicated.
* Sigma kit 108AC, Sigma Chemical Co., St. Louis, Mo.
36
TABLE 5. Number of days each pig was maintained on experimental
regimen, Experiment I.
Number of days on experiment
Pig before necrOpsy
A-l 53
A—2 53
A—3 53
A-4 53
A—5 43*
A—6 59
A77 53
A—8 36**
A-9 53
A—10 53
A-ll 36*
A-12 53
* Died during course of experiment.
** Killed
37
TABLE 6. Number of days each pig was maintained on experimental
regimen, Experiment II.
.4_‘
Number of days on experiment
Pig before necropsy
B-l 43
B-2 36
B-3 36
B-4 43
B-5 43
B-6 36
B—7 43
B—8 43
B—9 10*
B-lO 43
B-ll 36
B-12 36
* Killed when found comatose.
38
TABLE 7. Number of days each pig was maintained on experimental
regimen, Experiment III.
Number of days on experiment
before necropsy
"U
H
00
I
HI—‘OCDVO‘UIbUJNH
bWNHOVOmVO‘m-bWNl-‘O
000000000000
I I I I I I I I I l
cataracaraqaqucaracacac:
Nah)NJNDNJP‘P‘P‘P'P‘P*P‘P'
47
46
46
47
52
47
46
47
47
47
52
46
43*
46
52
52
52
10 **
38
38
38
38
52
52
* Died during course of experiment.
**Killed early to provide examples of tissues at beginning of experiment.
39
Tissue sections were routinely fixed in 10% buffered formaldehyde
solution. They were processed in an automatic processing machine,*
sectioned at 6 microns, and stained with hematoxylin and eosin. Sepa-
rate sections of the livers were fixed with Carnoy's solution and
stained with Best's carmine for glycogen content. Formalin-fixed sec-
tions were frozen and stained with oil-red-O for fat content. Where
indicated,other special stains were employed.
Smears were made of bone marrow from the 5th left rib immediately
after euthanasia. They were fixed in methyl alcohol and stained with
the May-Granwald-Giemsa stain.
Analyses of Tissues and Rations for Vitamin E Content
Because of the difficulties inherent in analytical technics for
vitamin E, the Specimens for analysis were sent to the laboratories
of Hoffman La Roche where these procedures are conducted routinely.
The technic of Dicks and Matterson (1961) was used for liver analysis.
Vitamin E content of rations in Experiment III was determined by a
procedure employing solvent extraction, saponification, Florex and
secondary magnesium phosphate column chromatography, and colorimetric
determination with ferric chloride and alpha, alpha’ dipyridyl.
Experimental Desiggg
1. Experiment I. The various experimental diets for Experiment I
and the pigs assigned to each are given in TABLE 8.
2. Experiment 11. In TABLE 9 the experimental design of Experiment
II is outlined.
* Autotechnicon,'Technicon Co., Chauncey, New York.
40
TABLE 8. Experimental design, Experiment I
~v
Ration Pig Numbers
Basal A-3, A-6, A-8, A-9
Basal + low level E A-l, A-lO
Basal + selenium A-7, A-12
Basal + methionine A—5, A-ll
Basal + low level E + selenium A-2, A-4
+ methionine
TABLE 9. Experimental design, EXperiment II
"A A _
-:_
f
7-—
Ration Pig Numbers
Basal + low level E B—3, B—7
Basal + selenium B-5, B-6
Basal + methionine B-9, B-lO
Basal + low level E + selenium B—l, B-ll
Basal + low level E + methionine B-2, B-8
Basal + selenium + methionine B-4, B-12
41
3. Experiment III. In TABLE 10 the experimental design of Experi-
ment III is outlined.
42
TABLE 10. Experimental design, Experiment III.
A
Ration Pig Numbers
Basal C-1, C-2
Basal + low level E C-3, C-4
Basal + high level E C-9, C-lO, C-ll, C-12
Basal + selenium C-5, C-6
Basal + ethoxyquin C—l3, C-14, C—lS, C-16
Basal + low level E + selenium C—7, C-8
Basal with 20% protein C-23, C-24
Basal with 20% protein.+ C-21-C-22
low level E
Basal with 20% protein + C-19, C-20
selenium
Michigan State University standard C-l7, C-l8
grower ration
RESULTS
The lesions observed with greatest frequency in the pigs of these
3 experiments were dietary liver necrosis, hydropic degeneration of
the hepatocytes, nutritional muscular dystrophy, ulceration and pre-
ulcerous lesions of the squamous epithelium of the stomach, and atrophy
of the thymus. The pathologic changes were Similar in all 3 experi-
ments; therefore a general description is given for each lesion. In
addition,there is a section on pathology included with the results of
the individual experiments.
Pathology of Dietary Liver Necrosis
Gross lesions. Some of the livers were swollen, and over the surfaces
of the organs there were numerous dark red, punctate or slightly larger
lesions suggestive of hemorfhage. The livers of pigs A-5 and B-3 are
typical of this change (Figures 1 and 2). The surfaces of other livers
had irregularly Shaped, depressed, roughened areas. This is illustrated
in Figures 3 and 4, in photographs of the livers of pigs A-8 and B-7.
The livers of many pigs had both lesions (Figure 5).
Histopathologygof dietary liver necrosis. The hemorrhage-like lesions
observed grossly were sharply demarcated areas of necrosis. This process
affected lobules individually or in groups, while the immediately adjoin-
ing or surrounding lobules were often not visibly affected (Figure 6).
The most distinctive feature of this necrosis was the limitation of
43
44
Figure 1. Acute dietary liver necrosis. Pig fed
basal ration plus methionine.
Figure 2. Acute dietary liver necrosis. Pig fed
basal ration plus low level vitamin E.
45
Figure 3. Chronic dietary liver.necrosis with
fibrosis. Pig fed basa1.ration.
Figure 4. Chronic.dietaryultver_necrosis.,.Pig fed
basal ration plus low level vitamin E.
46
Figure 5. Liver with both acute necrosis and fibrosis.
Pig fed basal ration plus methionine and low level vitamin E.
7 . ‘ If" ,1 w 7 7—vle__
Figure 6. Dietary liver necrosis illustrating char-
acteristic pattern of necrosis. Pig fed basal ration.
Hematoxylin and eosin. x 30.
47
the necrosis by the interlobular connective tissue. There was an absence
of hepatic cells in most necrotic lobules. In many cases, karyorrhectic
or pyknotic nuclei were the only remaining traces of the necrotic cells
(Figure 7). Infiltration of.necrotic lobules by polymorphonuclear leuko-
cytes was observed in some sections (Figure 8). In a few instances
there was dystrophic mineralization of the degenerative cells (Figure 9).
A typical necrotic hepatic lobule consisted of a blood-filled
reticulum with few surviving hepatocytes. However, occasionally foci
of necrosis occurred within lobules in which most of the cells were not
visibly affected (Figure 10). These foci were located in any region of
the lobule. In some sections there were collapsed lobules with an increase
of interstitial connective tissue and bile ducts (Figure 11). Often a
few surviving parenchymal cells formed gland-like patterns. These cells
were frequently very large (Figure 12). Most sections contained combi-
nations of the above changes (Figure 13).
The structures of the portal triads were usually spared during the
acute necrotic process (Figure 14).
_Hydropic Degeneration of the Liver
A common observation in all 3 experiments was an almost complete
failure of the cytoplasm of the hepatocytes in many of the livers to
stain with hematoxylin and eosin (Figures 15 and 16). The lesion was not
due to fat content, as may be seen in Figure 17, a photomicrograph of a
section stained with oil-red-O. Best's carmine stains for glycogen
(Figure 18) also failed to explain this change. TherefOre, it was con-
cluded that this cytoplasmic change was hydropic degeneration. It was
not apparent by gross examination. In such livers the sinusoids were
48
Figure 7. Dietary liver necrosis. Pig fed basal
Hematoxylin and eosin..x 120.
ration.
necrotic lobule by polymorphonuclear.leukocytes.
basal ration plus low level vitamin E. Hematoxylin and
eosin. x 480.
f
Figure 8. Dietary liver.necrosis.,.Infiltration o
Pig fed
49
Figure 9. Dietary liver necrosis. Von Kossa-
positive material in necrotic area. Pig fed basal ration
plus methionine. Hematoxylin and eosin. x 480.
Figure 10. Focal intralobular necrosis. Pig fed
basal ration plus methionine and low level vitamin E.
Hematoxylin and eosin. x 120.
50
\
. u ' 1 - '
'o ' . ‘7 ' ‘3, fl .
I? . w . -
'n . . ‘ , .
9.- ...". by“ ‘tll ." < '
Figure 11. Dietary liver necrosis with desmoplasia
and bile duct proliferation. Pig fed basal ration plus
methionine. Hematoxylin and eosin. x 120.
Figure 12. Chronic dietary liver necrosis with in—
creased connective tissue and liver cells forming gland-
like nests. Pig fed basal ration. Hematoxylin and eosin.
x 120.
51
Figure 13. Acute necrosis and fibrosis. Pig fed
basal ration plus low level vitamin E. Hematoxylin and
eosin. x 30.
Figure 14. Massive dietary liver necrosis. Bile duct
adjacent to necrotic tissue unaffected. Pig fed basal
ration plus methionine. Hematoxylin and eosin. x 480.
52
7
Figure 15. Hydropic degeneration of the liver.
Pig fed basal ration plus low level vitamin E. Hematoxylin
and eosin. x 480.
-17.,,_‘, ._ , *7
Figure 16. Hydropic degeneration. Pig fed basal
ration plus selenium plus low level vitamin E. Hema-
toxylin and eosin. x 300.
53
Figure 17. Hydropic degeneration. Same liver as
Figure 16. Fat Stain which is essentially negative.
Oil-red—O stain. x 300.
..l .__ ._
Figure 18. Hydropic degeneration. Same liver as
Figure 16. Glycogen stain which is essentially negative.
Best's carmine stain. x 300.
54
compressed to the degree that their lumina often appeared obliterated
by the bordering hepatocytes..
The parenchymal cells of the livers of pigs fed 20% protein rations
stained normally (Figure 19).
Nutritional Muscular DystrOphy
Nutritional muscular dystrophy (NMD) was observed in some pigs in
all 3 experiments (Figures 20, 21, and 22). This disease may occur in
varying degrees of severity and is often not detected during gross
examination deSpite fairly extensive microsc0pic lesions. No NMD was
observed grossly in any of the pigs in these 3 trials.
J
Histopathology of NMD. Nutritional muscular dystrOphy was characterized
by swelling of the muscle fibers with loss of cross striations. Often
there was fragmentation of the fibers (Figures 23 and 24) and the appear-
ance of increased numbers of mesenchymal nuclei in affected areas
(Figure 25). These cells frequently formed elongated groupings in and
around degenerating muscle fibers. Their exact identity is not clear.
They are commonly termed "sarcolemmal cells", but this term is now regarded
as a misnomer. Many of them are probably scavengeretype reticuloendo-
thelial phagocytes which have infiltrated in response to the destruction
of tissue. These groups of nuclei, often arranged in linear patterns,
were the most Striking microscopic feature in many cases.
Mineralization was observed in one case of NMD (Figure 26). This
material stained well by von Kossa's method (Figure 27). Fibers in an
advanced State of degeneration and fragmentation usually had an increased
affinity for eosin. Occasionally, centrally located nuclei were observed
in degenerated fibers which had been cut in cross section (Figure 28).
55
Figure 19. Normally staining hepatocytes in the
liver of a pig fed 20% protein ration. Hematoxylin and
eosin. x 480.
Figure 20. Nutritional muscular dystrophy in a pig
fed the basal ration plus selenium. Hematoxylin and eosin.
x 120.
56
Figure 21. Nutritional muscular dystrophy in a pig
fed the basal ration plus methionine. Hematoxylin and
eosin. x 120.
Figure 22. Nutritional muscular dystrophy in a pig
fed a 20% protein ration. Hematoxylin and eosin. x 120.
57
Figure 23. Fragmentation and loss of cross striation
in the quadriceps femoris muscle of a pig fed the basal
ration plus selenium and low level vitamin E. Hematoxylin
and eosin. x 300.
\
Figure 24. Fragmentation of fibers and loss of cross
striations in the quadriceps.femoris muscle of a-pig fed
the basal ration plus methionine. Hematoxylin and eosin.
x 480.
Figure 25. Increased numbers of nuclei in the quadri—
ceps femoris muscle of a pig fed a 20% protein ration.
Hematoxylin and eosin. x 480.
Figure 26. Mineral deposits in the quadriceps femoris
muscle of a pig fed the basal ration plus methionine.
Hematoxylin and eosin. x 480.
59
Figure 27. Section from the same muscle as Figure 26.
Von Kossa's Stain. x 480.
Figure 28. Cross section of a focal area of nutritional
muscular dystrophy illustrating fibers with centrally located
nuclei. Pig fed basal ration plus selenium plus low level
vitamin E. Hematoxylin and eosin. x 480.
60
Mitotic figures occurred rarely (Figure 29).
Degeneration and Ulceration of the Esophageal Region of the Stomach
Gross lesions. There was a yellowish-white, granular, pseudomembranous
material on the squamous epithelium of the stomachs of many of the pigs
in these 3 experiments, irreSpective of diet. This was often present
in the esophagus also. Ulcers and erosions of various sizes were also
observed in the esophageal region of the stomach of a large percentage
of the animals in all trials. These usually began near the junction with
the glandular epithelium. At times the ulcer involved the entire eSOpha-
geal region of the stomach.
Histopathology onggStric lesions. The yellowish pseudomembranes observed
in the eSOphagi and stomachs during gross examinations represented
degenerated, hyperkeratotic and parakeratotic epithelium (Figures 30 and
31) which usually harbored a profuse growth of a fungus. The organism
in question occurred as hyphae and as budding yeast forms and stained
well with Gomori's methenamine Silver (Figure 32). It was identified as
Candida albicans. The epithelial cells in these areas were tremendously
swollen and vacuolated (Figure 33) and frequently there were intrai
epithelial'pustules (Figure 34). The underlying papillary layer was
edematous.' '
Ulcerations and/or erosions often occurred alone or in association
with the degenerative changes of the squamous epithelium. The denuded
areas and underlying structure of these ulcers were often invaded by
large numbers of Candida organisms and bacteria (Figure 35). There were
extensive inflammatory changes, including polymorphonuclear infiltration
61
4,
Figure 29. Mitotic division in a cell in an area of
nutritional muscular dystrophy. Pig fed basal ration plus
methionine. Hematoxylin and eosin. x 480.
Figure 30. 'Hyperkeratosis and parakeratosis of squa-
mous epithelium of the stomach of a pig fed the basal
ration. Hematoxylin and eosin. x 120.
62
Figure 31. Hyperkeratosis and parakeratosis in esopha-
geal region of stomach of a pig fed a 20% protein ration.
Hematoxylin and eosin. x 30.
Figure 32. Fungus stain of section from the esopha-
geal region of the stomach of a pig fed the basal ration.
Note the budding yeast forms and hyphae. Gomori's methena—
mine silver stain. x 480.
63
Figure 33. Vacuolation and ballooning degeneration
of the squamous epithelium in the stomach of a pig fed a
20% protein ration. Hematoxylin and eosin. x 120.
Figure 34. Intra-epithelial pustule in the esophageal
region of the stomach of a pig fed a 20% protein ration.
Hematoxylin and eosin. x 480.
64
Figure 35. Candida albicans in ulcerated area in the
esophageal region of the stomach of a pig fed the basal
ration. Gomori's methenamine silver stain. x 120.
Figure 36. Mucinous degeneration in glandular epi-
thelium adjacent to an ulcer of the squamous epithelium in
the stomach of a pig fed the basal ration. Hematoxylin
and eosin. x 120.
65
and fibroplasia. Mucinous degeneration of nearby glandular epithelium
was common (Figure 36). Occasionally degenerative changes were observed
in vessels of the muscularis or serosa near ulcerated areas (Figures 37,
38, and 39). It is to be re-emphasized that these lesions occurred to
some degree in most of the animals in the 3 experiments, regardless of
diet.
Atrophy of the Thymus
There was extreme atrophy of the thymus among many of the pigs fed
6% protein diets. In some cases, no thymic tissue was detected at
necropsy.
Experiment I
Clinical Signs and mortaligy. There was relatively little clinical evi-
dence of disease referable to the diet among the experimental animals,
other than general unthriftiness and poor appetite. Pig A-ll had a
fever and vomited blood before dying as a result of a perforated gastric
ulcer. Pig A-5 appeared normal the night before it was found in a coma
and dying apparently from liver necrosis.
Feed consumption. The 2 pigs (Ar2 and A54) which were fed all 3 supple-
ments had better appetites than the others. They were each eating
approximately 380 Gm. of ration daily by the end of the trial, and
undoubtedly would have eaten more. The group which ate next best
included pigs A-7 and A-12, whose rations were supplemented with selenite.
They were receiving 200 to 240 Gm. daily and often left feed in their
bowls. All other surviving pigs were eating considerably less than
200 Gm. daily at the conclusion of Experiment I.
66
Figure 37. Thickening and vacuolation of the intima of
a blood vessel in the serosa of an ulcerated area of the
(stomachn Pig fed basal ration plus high level vitamin E.
Hematoxylin and eosin. x 120.
Figure 38. Degenerative changes in the wall of a blood
vessel in the serosa near a gastric ulcer. Pig fed basal
ration plus low level vitamin E. Hematoxylin and eosin.
x 120.
67
Figure 39. Intimal swelling in the wall of a vein in
the serosa near an ulcer of the esophageal region of the
stomach. Pig fed basal ration. Hematoxylin and eosin. x
300.
Figure 40. Appearance of some of the pigs of Experiment
I: pig 4 fed basal ration plus selenium, low level E, and
methionine; pig 7 fed basal ration plus selenium; pig 10 fed
basal ration plus low level vitamin E; pig 6 fed basal ration.
68
Growth. The weights of pigs during the course of Experiment I are
presented in TABLE 11. Total weight gain, number of days on experiment,
and average daily gain are given in TABLE 12. In Figure 40 it is possi-
ble to compare the physical appearance of pigs on the complete supple-
ment (pig 4), selenium (pig 7), low level E (pig 10), and the basal
ration (pig 6). There was a great difference between pig 6 and the
others, but relatively little difference among the other 3 pigs.
All pigs made poor gains on the 6% protein ration. Those receiving
methionine, alone or in combination with other supplements, grew Sig:
nificantly better than the others. Pig A-ll, affected with severe
gastric ulceration, was an exception.
Liver weights. The weights of the livers of the pigs of Experiment I
at necropsy and the ratio of this weight to body weight are presented
in TABLE 13. Although there was great variation among these figures,
there was no Significant pattern of variation.
Hematology. Packed cell volume and hemoglobin values for the pigs of
Experiment I are presented in TABLES 14 and 15. There was a fairly
uniform decline in both values among all groups throughout the course
of the experiment. Hemoglobin values are omitted for day 54 because of
technical problems with the determination.
OCT activity. Serum ornithine carbamyl transferase activities for the
pigs of Experiment I are presented in TABLE 16. Although the highest
values occurred in pigs with lesions of dietary liver necrosis'(A-3 and
A-6), none exceeded the values reported by Wretlind ggnal. (l959)as normal
for pigs.
69
TABLE 11. Weights of pigs, Experiment I (Gm.)
Pig Supple- Days of Age
No. ment 16 22 28 35 42 49 52 56 59 63 69 74 75
A-l Low E 2580 2835 3204 3034 3402 3686 3742 3714 3680
A-2 Se + 2743 3232 3686 4167 4876 5670 6322 6549 6010
meth +
low E
Ar3 Basal 2892 3147 3374 3572 3856 3997 4338 4224 3970
A-4 Se + 2963 3430 3572 4168 5018 5557 5925 6095 5950
meth +
low E
A-5 Meth 2991 3289 3657 4309 4990 5840 6662 6500
A-6 Basal 2849 3147 3402 3572 3600 4054 3997 3856 3969 3770
A-7 Se 3041 3402 3657 3941 4309 4848 5188 5301 5315
A-8 Basal 2892 3147 3600 3629 3827 4026 4000
A-9 Basal 2821 3204 3487 3714 3827 4139 4253 4366 4470
A-lO Low E 2800 3119 3289 3827 3856 4253 4167 4309 4220
A—ll Meth 3377 3799 4167 4678 4933 5188 4600
A-12 Se 3055 3289 3742 3969 4196 4848 5075 5012
4593
70
TABLE 12. Total weight gains and average daily gains, Experiment I (Gm.)
Pig Supple- No. days on Approximate average
No. ment Total weight gain experiment, daily gain
A-l Low E 1100 53 21
A-2 Se + 3267 53 62
meth +
low E
A-3 Basal 1078 53 20
A-4 Se + 2987 53 56
meth +
low E
A-5 Meth 3509 43* 82
A-6 Basal 921 59 16
A-7 Se 2274 53 43
A-8 Basal 1108 36** 31
A—9 Basal 1649 53 31
A-10 Low E 1420 53 27
A—ll Meth - 1223 36* 34
A-12 Se 1957 53 37
* Died
** Killed
71
TABLE 13. Liver weights and ratios of body weights to liver weights,
Experiment I
J. V 1.14.—
Ll
Pig Supple- Age at ne- No. days on Liver Ratio; of body
No. ment cropsy (days), experiment wt. (Gm.) wt. to liver wt.
A-l Low E 69 53 145.0 25.0
A-2 Se + 69 53 191.0 31.4
low E +
meth
A—3 Basal 69 53 111.5 36.0
A-4 Se + 69 53 138.0 43.1
low E +
meth
A-5 Meth 59 43 320.0 20.0
A-6 Basal 75 59 73.0 52.0
A—7 Se 69 53 132.2 33.0
A—8 Basal 52 36 130.0 31.0
A-9 Basal 69 53 105.0 43.0
A-lO Low E 69 53 --- ---
A-ll Meth 52 36 200.0 23.0
A-12 Se 69 53 155.0 32.0
72
TABLE 14. Hematocrit values (packed cell volume per cent), Experiment I
Pig Supple- Day of Experiment
No. ment 8 15 24 30 ‘37 _ 44 52 _ 54 58
A-l Low E 38 32 30 32 29 29 --— 30- ---
A-2 Low E + 35 31 clot- 30 28 28 --- 33 ~--
se + ted
meth
A-3 Basal 36 28 32 33 27 20 --- 28 ---
A-4 Low E + 36 34 28 30 26 26 --- 29 ---
se +
meth
A-S Meth 35 28 31 34 29 --- -—— ——- ———
A-6 Basal clot- 30 38 38 ‘ 37 33 32 --- 30
ted
A-7 Se 33 30 33 28 29 30 --- 31 —--
A-8 Basal 37 32 37 36 34 -—— ——— —-- ---
A-9 Basal 34 32 33 29 29 27 --- 28 ---
A—lO Low E 37 32 34 34 36 33 --- 35 —--
A-ll Meth 35 37 33 28 --- --- --- --- --—
A-12 Se. 37 32 .32 30 29 29 --- 34- ---
73
TABLE 15. Hemoglobin values (Gm./100 m1.), Experiment I
Pig Supple- Day of Experiment
No. ment 8 15 24 30 '37 44 52 54 58
se + ted
meth
A-4 Low E + 11.8 11.2 . 9.5 9.7 8.1 8.5
se '1'
meth
A-6 Basal clot- 10.0 12.5 12.2 12.2 11.0 9.6 --- 9.5
ted
A-7 Se 10.6 10.0 10.5 9.2 9.2 9.8
A-8 Basal 12.0 10.4 12.5 11.7 11.2
A-9 Basal 10.9 10.6 11.0 9.8 9.5 9.0
A-lO Low E 12.2 10.3 11.5 11.0 11.5 11.2
A-ll Meth 11.2 11.8 11.0 9.2
74
TABLE 16. Serum ornithine carbamyl transferase activity, Experiment I
J.
5 - Serum OCT activity on day of experiment indicated
Pig Supple- (Reichard units)
No. gygment Day 44 A Day 52 . Day 59
A—l Low E 0.29 0.23
A-2 Se + 0.31 0.27
low E +
meth
A-3 Basal 0.29 0.43
A-4 Se + 30.35 0.31
low E +
meth
A-6 Basal 0.39 , 0.45 0.50
A-7 Se 0.29 0.30
A-8 Basal L 0.35 ‘ ---
A-9 Basal 0.23 0.29
A-lO Low E 0.26 0.29
A-ll ' Meth 0.31 ---
A-12 Se .25 0.29
75
Pathology. The incidences of grossly evident liver disease and extreme
atrOphy of the thymus among the pigs of Experiment I are summarized in
TABLE 17. "Extreme atrOphy" in this case indicates that no thymic tissue
was detected at necrOpsy.
Otherggrpss lesions. Subcutaneous edema of the hindquarters was observed
in pigs A-3, A-6, A-8, and A-9, all of which had extensively fibrosed
livers. A mild fibrinous peritonitis occurred in pigs A-5 and Ae9. Pig
A—5 also had subserous edema of the intestine and a considerable amount
of fluid in the peritoneal cavity. The heart of A-8 was collapsed and
flabby. Pig A-ll died following perforation of an ulcer of the glandular
region of the stomach near the greater curvature (Figure 41). This pig
also had a rent in the left Side of the diaphragm through which part of
the liver had herniated. There were about 100 m1. of dark brown, watery
fluid in the pleural and peritoneal cavities. The liver was a mottled
yellowish brown with many small, scattered, dark, depressed areas of
irregular shape (Figure 42).
Histopathology, The incidences of microscopic lesions of dietary liver
necrosis, hydrOpic degeneration of the liver, nutritional muscular
dystrophy, and gastric epithelial changes are summarized in TABLE 18.
Lesions of acute necrosis which had not been observed at necropsy were
detected microscopically. 'Lesions of the squamous epithelium of the
stomach occurred in all but one animal of those examined. Selenium did
not protect against nutritional muscular dystrophy.
76
TABLE 17. Gross evidence of liver disease and extreme atrophy of
thymus, Experiment I
Dietary Liver Necrosis
Pig Supple- Acute Extreme atrOphy
No. ment necrosis Fibrosis of thymus
A-l Low E 0 0 +
A-2 Se + 0 0 +
low E +
meth
A-3 Basal 0 + 0
A-4 Se + 0 0 not examined
low E
meth
A-5 Meth + 0 0
A-6 Basal 0 + +
A-7 Se 0 0 +
A-8 Basal 0 + +
A-9 Basal 0 + +
A-10 Low E 0 + +
A-ll Meth + 0 0 '
A-12 Se 0 0 +
77
I. II
4 s
IIIIIIIIIIIIIIIIIIIIIIl|"" """IIlllllllllllll
1-, . o' . ..‘v. .1___-,.1 —
Figure 41. Perforated ulcer of the glandular epi-
thelium of pig A—ll. Pig was fed basal ration plus
methionine.
Figure 42. Liver of pig A—ll. Swelling typical of
acute dietary liver necrosis.
78
TABLE 18. Incidence of microscOpic lesions, Experiment I
4
DLN* Hydropic Lesions of epithelium of
Acute degenera- eSOphageal region of stomach
Pig Supple- anecro- Fibro- tion of Epithelial Ero- Ulcera-
No. ment sis sis liver NMD** degeneration Sion tion
A-l Low E 0 0 + 0 +
A-2 Se + 0 0 + 0 0 0 0
low E +
meth
A-3 Basal + 0 + 0 +
A-4 Se + 0 0 0 0 not examined
low E +
meth
A-5 Meth + 0 + 0 +
A-6 Basal + + + 0 +
A-7 Se 0 0 + + + 0
A-8 Basal + + + 0 +
A—9 Basal 0 + + 0 +
A-10 Low E 0 0 + 0 + 0 0
A-ll Meth + + 0 0 perforated ulcer of glandu-
lar epithelium
A-12 Se 0 0 + 0 +
* Dietary liver necrosis
** Nutritional muscular dystrOphy
79
Vitamin E analysis of livers. The livers of the pigs of Experiment I
were analyzed for alpha-tocOpherol content. The results are given in
TABLE 19.
Experiment II
Clinical Signs and mortality. AS was the case in Experiment I, there
was little clinical evidence of nutritional disease other than general
unthriftiness and poor.hair coat. On the 10th day of the experiment
pig B—9 was found comatose, although it had appeared normal the previous
evening. It was electrocuted, and massive necrosis of the liver was
observed at necropsy. On the 25th day of the experiment pig B-7 was
depressed, weak in the hindquarters, and vomiting.
Feed consumption. In general, pigs of Experiment II had better appetites
than those of Experiment I. At the conclusion of the trial, pigs B-2
and B-8, receiving both methionine and low vitamin E, and pig B-4,
receiving methionine and selenite, and pig B-l, receiving selenite and
low level vitamin E, were each consuming approximately 700 Gm. of feed
daily. This was considerably more than the amount eaten by any of the
pigs receiving single supplements. Approximate daily feed consumption
Shortly before termination of the experiment is given for each animal in
TABLE 20. Such figures are somewhat inexact due to spilling and the
difficulties of measuring leftovers accurately. Also, there was often
wide variation in the daily feed consumption by individual animals.
Growth. Weight gains were considerably better than those of Experiment
I. With one exception (pig A-12), the methionine-supplemented pigs grew
80
TABLE 19. Alpha-tocopherol content of livers, Experiment I
L
L
Pig Alpha—tpcOpherol
No. Supplement (mg.%)
A-l Low E 0.26
A-2 Se + low E + meth 0.17
A-3 Basal 0.06
A—4 Se + low E + meth 0.26
A-5 Meth 0.18
A-6 Basal 0.12
A-7 Se 0.24
A-8 Basal 0.08
A-9 Basal 0.14
A~10 Low E 0.23
A-ll Meth 0.61
A-12 Se 0.04
81
TABLE 20. Feed consumption, Experiment II
No. days Approximate daily feed consumption
Pig Supple— on experi- at conclusion of experiment
No. ment ment (Gm.)
B-l Se + 43 700
low E
B-2 Meth + 36 700
low E
B-3 Low E 36 360
B-4 Se + 43 700
meth
B—S Se 43 400
3‘6 Se 36 450
B-7 Low E 43 280
B-8 Meth + 43 700
low E
B-9 Meth 10 died on 10th day
B-lO Meth 43 380
B-ll Se + 36 340
low E
B-12 Se + 36 470
meth
82
at a rate well above the average rates of growth of all other groups.
Weights of pigs throughout Experiment II are presented in TABLE 21.
Total weight gains and average daily gains are given in TABLE 22. In
Figures 43 and 44 it is possible to compare the appearances of the pigs
fed doubly supplemented rations (Figure 43, pigs B-l, B-4, and B-8)
with the appearances of pigs fed single supplements (Figure 44, pigs
B-S, B-7, and B-lO).
Liver weights. Weights of livers and.ratios of body weights to liver
weights are presented in TABLE 23. There was no apparent relationship
between dietary liver necrosis and ratio of body weight to liver Size.
Hematology. .Hemoglobin and packed cell volume values through day 27
of Experiment II are presented in TABLES 24 and 25. These values
declined generally throughout the experiment. The few exceptions to
this trend were not of experimental significance.
Serum ornithine carbamyl transferase activity. Serum ornithine carbamyl~
transferase activity values on the days indicated are presented in
TABLE 26. As in Experiment I, the highest values occurred in pigs with
lesions of dietary liver necrosis (B-3, B-7, B-8, B-lO). However, all
fell within the range of normal values reported by Wretlind g£_§l, (1959).
Pathology
Gross lesions. The occurrence of grossly visible lesions of dietary
liver necrosis and extreme atrophy of the thymus are summarized in TABLE
27. Liver lesions occurred in all pigs which had not received selenium.
83
TABLE 21. Weights of pigs, Experiment II
Pig Supple— Days of Age
No. ment 29 35 39 42. 49 4561 64 65 70' ,72
B-l Se + 6308 6464 7088 7938 9044 10660 11737 11895
low E
B-2 Meth + 6291 6435 7286 8647 10348 12559 12902
low E
B-3 Low E 5794 5897 6606 7371 8165 8562 8810
B-4 Se + 5672 5868 6747 7598 8930 11057 12219 12640
meth
B-5 Se 4750 4933 5387 6124 6861 7711 8222 8310
B-6 Se 4053 4224 4423 4791 5415 5585 5550
B-7 Low E 6246 6350 6747 7569 8023 8987 8363 8947
B-8 Meth + 5869 6039 6861 8136 9441 11567 12928 13488
low E
B-9 Meth 4811 5046 4590
B—10 Meth 4296 4479 5018 5925 6917 7995 9072 8995
B-ll Se + 4161 4423 4593 5160 5727 6294 6215
low E
B-12 Se + 3055 3147 3657 4196 4848 5185 5180
meth
84
TABLE 22. Total weight gains and average daily gains, Experiment II (Gm.)
Pig Supple- No. days on Approximate average
No. ment Total weightggain5~ experiment 1 daily gain
B-l Se + 5587 43 130
low E
B-2 Meth + 6611 36 184
low E
B—3 Low E 3016 36 84
B-4 Se + meth 6968 43 162
B—S Se 3560 43 83
B-6 Se 1497 36 42
B-7 Low E 2701 43 63
B-8 Meth + 7620 43 177
low E
B—9 Meth -221 10* -22
B—lO Meth 4699 43 109
B-ll Se + low E 2054 36 57
B-12 Se + meth 2125 36 59
4__
* Pig B-9 was in a coma on the morning of day 10., It was killed and
necropsied at that time.
85
Figure 43. Appearances of some of the pigs fed double
supplements, Experiment II: pig B-l fed basal ration plus
selenium and low level vitamin E; pig B—4 fed basal ration
plus selenium and methionine; pig B-8 fed basal ration plus
methionine and low level vitamin E.
Figure 44. Appearances of some of the pigs fed single
supplements, Experiment 11: pig B-10 fed basal ration plus
methionine; pig B-7 fed basal ration plus low level vitamin E;
pig B-5 fed basal ration plus selenium.
86
TABLE 23. Liver weights and ratios of body weights to liver weights,
Experiment II
l.
Pig Supple‘ Age at ne- No. days on Liver Ratio of body
No. mentgï¬ cropsy (days) experiment wt. (Gm.) wt. to liver wt.
B-l Se + 72 43 446.0 27
low E
B-2 Meth + 65 36 366.0 35
low E
B-3 Low E 65 36 342.0 26
B-4 Se + meth 72 43 357.8 35
B-5 Se 72 43 281.8 30
B-6 Se 65 36 262.1 21
B-7 Low E 72 43 355.8 25
B-8 Meth + 72 43 411.5 33
low E
B-9 Meth 39 10 103.0 45
B-lO Meth 72 43 300.5 30
B-ll Se + low E 65 36 192.8 32
B-12 Se + meth 65 36 132.5 39
87
TABLE 24. Hematocrit values (packed cell volume per cent), Experiment II
.—
Pig Supple- Day of Experiment
No. ment -2 6 12 13 20 27
B-l Se + 37 33 36 36 35
low E
B-2 Meth + 36 35 36 33 32
low E
B-3 Low E 36 31 34 33 35
B-4 Se + 35 33 35 33 32
meth
B-S Se 39 37 36 40 36
B-6 Se 37 30 39 33 35
B-7 Low E 36 36 39 33 39
B-8 Meth + 36 34 35 35 33
low E
B-9 Meth 38 30 40
B-lO Meth 31 32 33 32 31
B-ll Se + 34 29 31 30 30
low E
B-12 Se + 31 30 30 29 26
meth
88
TABLE 25. Hemoglobin values (Gm./100 m1.), Experiment II
Pig Supple- Day of Experiment
No. ment +2 6 12 13 20 27
B-l Se + 11.2 10.7 11.5. 11.8 11.0
meth
B-2 Meth + 11.2 11.5 11.8 11.0 10.3
low E
B—3 Low E 10.8 10.0 10.9 10.8 11.0
B-4 Se + 10.6 10.5 10.8 10.6 10.3
meth
B-S Se 12.4 11.8 11.8 12.5 11.5
B-6 Se 11.2 9.7 12.5 10.5 11.0
B-7 Low E 11.7 11.5 12.3 11.0 12.2
B-8 Meth + 11.2 11.2 11.2 11.3 10.9
low E
B-9 Meth 12.0 9.7 11.2
B-lO Meth 9.7 10.5 11.0 10.3 9.7
B-ll Se + 10.5 9.2 10.0 9.5 9.2
low E
B-12 Se + .9.5 9.5 9.5 9.2 8.0
meth
89
TABLE 26. Serum ornithine carbamyl transferase activity, Experiment II
A
Serum OCT activity on day of experiment indicated
Pig Supple- (Reichard units)i
No. ment Day 6 Day 10' Day 27 Day 35
B-l Se + low E 0.30 0.28 0.24
B-2 Low E + 0.33 0.31 0.26
meth
B-3 Low E 0.28 1.43 0.41
B-4 Se + meth 0.79 0.50 0.23
8+5 Se 0.35 0.35 0.23
B-6 Se 0.90 0.33 0.21
B-7 Low E 0.26 0.69 0.53
B-8 Low E + meth 0.34 1.18 0.20
B-9 Meth --- 2.55 --— ---
B-lO Meth 0.29 0.88 0.46
B-ll Se + low E 0.46 0.26 0.21
B-12 Se + meth 0.26 0.25 0.21
90
TABLE 27. Cross evidence of liver disease and extreme atrophy of
thymus, Experiment II
Pig Supple— Dietary liver necrosis 7 Extreme atrophy
No. ment Acute necrosis Fibrosis . of thymus
B—l Se + 0 0 0
low E
B-2 Meth + 0 + +
low E
B-3 Low E + 0 0
B-4 Se + 0 0 0
meth
B-5 Se 0 0 +
B-6 Se 0 0 +
B-7 Low E 0 + +
B-8 Meth + + + +
low E
B-9 Meth + 0 +
B-10 Meth + 0 +
B-ll Se + 0 0 0
low E
B-12 Se + 0 0 +
meth
91
The lesions observed in pig B-9 were unusual. This pig was electro-
cuted when apparently near death. In addition to typical acute necrosis,
the liver had a mottled appearance due to the contrast of the dark
necrotic areas with a yellowish, granular fibrinous membrane which
covered the organ in a patchy pattern.
Histopathology, -The occurrence of microsc0pic lesions of dietary
liver necrosis, hydrOpic degeneration of the liver, nutritional muscular
dystrOphy, and gastric ulcers among pigs of Experiment II is summarized
in TABLE 28.
In addition to the lesions indicated in TABLE 28, there was a focal
area of degeneration containing von Kossa-positive material in the
myocardium of the left ventricle of pig B-lO (Figures 45 and 46). An
unusual change was observed in the liver of pig B-9. This was one of
the few protein-deficient animals which did not have hydrOpic degenera-
tion of the liver. However, there were numerous vacuoles in the few
lobules which had not undergone necrosis. In contrast to hydropic
degeneration, this vacuolation appeared to be extracellular (Figures
47 and 48). It is thought that this lesion.represented edema of the
Space of Disse as described by Obel (1953).
Experiment III
Clinical.sigps and-mortality...Pig,C-l3 was the only animal to die during
.the course of the.experiment. The carcass was found bloated and in a‘
poor State of preservation on.the.morning of day 43. C-13 had appeared
normal the previous‘evening and.had consumed its feed and water.
92
TABLE 28. Incidence of microscopic lesions, Experiment II
4..—
DLN* HydrOpiC' Lesions of epithelium of
A
'
Acute degenera- espphageal region of Stomach
Pig Supple- necro- Fibro— tion of Epithelial Ero- Ulcera-
No. ment sis sis liver NMD** degeneration Sion tion
B-l Se + 0 0 + + + 0 0
low E
B-2 Meth + + + + 0 + 0 0
low E
B-3 Low E + + + 0 +
B—4 Se + 0 0 + 0 + 0 0
meth
B-S Se 0 0 + + + 0 0
B-6 Se 0 0 +' 0 + 0 0
B-7 Low E + + + 0 +
B-8 Meth + + + + O +
low E
B-9 Meth + 0 0 0 + 0
B-10 Meth + + + + + 0
B-ll Se + 0 0 + 0 +
low E
B—12 Se + 0 0 0 0 + 0 0
meth
* Dietary liver necrosis
** Nutritional muscular dystrophy
93
Figure 45. Degeneration in the myocardium of a pig
fed basal ration plus methionine. Hematoxylin and eosin.
x 300.
Figure 46. Mineralization in the same area shown in
Figure 45. Von Kossa's Stain. x 480.
94
Figure 47. Extracellular vacuolation in the liver.
Pig fed basal diet plus methionine.~ Hematoxylin and
eosin. x 300.
1' . ' ..... . . . . . .71-â€.-.—
’,
Figure 48. Greater magnification of lesion illustrated
in Figure 47. Hematoxylin and eosin. x 480.
95
Pigs C-l9 through C—24, all on 20% protein rations, had frequent
attacks of diarrhea,.but their appetites were not affected at these
times.
Pigs C-19 and C-20 had a febrile illness during the period from day
22 through day 24. During this time they ate poorly,and C—20 was
observed vomiting blood.
Pig C-l6 was very unthrifty throughout the experiment.
On day 45, C-3 was very depressed and passed dark feces. The next
day its eyelids were swollen,and the pig appeared weak. LOn the same day
C-4 was ataxic but ate as usual.
Feed consumption - 6% proteinggroups. Feed consumption by pigs being
fed the 6% protein ration varied considerably even within groups receiv-
ing the same supplements. This was at times due to illnesses probably
unrelated to diet. The approximate daily feed consumption by pigs on
the 6% protein ration at the end of Experiment III is presented in
TABLE 29.
Feed consumption - 20% proteinpgroups. Except for a brief period as
mentioned under the diScuSSion of clinical Signs, pigs in this group ate
all their feed and undoubtedly would have eaten more. Pigs C-l9 through
C-22 were eating 2200 Gm. of feed per pair daily by day 38 when they
were killed. Pigs C-23 and C-24 together were eating 3000 Gm. of feed
daily when killed on day 52.
Feed consumption -ppig fed standardpgrower. Pig C-l7, fed a standard
pig ration, was eating approximately 850 Gm. daily when killed on day
52. It would have eaten more, but this amount represented the approxi-.
mate capacity of the feed bowl.
96
TABLE 29. Feed consumption, 6% protein group, Experiment III
t.. - No. days Approximate daily feed con-
Pig Supple- on experi- sumption at conclusion of
No. ment ment experiment (Gm.)
C-l Basal 47 330
C-2 Basal 46 250
C-3 Low E 46 210
C-4 Low E 47 500
C-5 Se 52 650
C-6 Se 47 560
C-7 Se + 46 500
low E
C-8 Se + 47 500
low E
C-9 High E 47 450
C-lO High E 47 450
C-ll High E 52 650
C-12 High E 46 560
C-13 EQN* 43** 560
C-14 EQN 46 240
C-15 EQN 52 550
C—16 EQN 52 200
* Ethoxyquin
** Died
97
Growth. The weights of pigs throughout Experiment III are given in
TABLE 30. The total gain and average daily gain are given in TABLE 31.
Those pigs receiving 20% protein rations grew at a much better rate
than those fed 6% rations. Pig C-l7, fed a standard grower ration,
also made much better gains than those on the 6% protein ration.
Liver weights. Weights of livers and ratios of body weights to liver
weights are given in TABLE 32. There is no apparent relationship be-
tween this ratio and dietary deficiency or supplementation with any of
the various supplements.
Hematology. In TABLES 33 and 34 the packed cell volume and hemoglobin
values for the pigs of Experiment III are given. These data do not
suggest a relationship between any of the diets and a Specific effect
on the hemogram.
Serum ornithine carbamyl transferase activity. Serum OCT activities on
the days indicated are Shown in TABLE 35. Values were somewhat higher
among the pigs with lesions of dietary liver necrosis. Unfortunately,
some samples were unsatisfactory for analysis.
Pathology
Gross lesions. The incidences of gross lesions of dietary liver
necrosis and extreme atrOphy of the thymus are summarized in TABLE 36.
Neither of these lesions was observed in any of the pigs fed the higher
levels of vitamin E or protein. Selenium prevented necrosis of the liver.
98
aflov%xonum «
:ku
ONNNN NONON NOOON ONOON OOOON ONOO NNNN -oua NON NN-O
Gummy
.NONOO ONONO ONNNN ONNNN NNNON NNNNN ONOO -OOO NON ON-O
O 30N
+ ONOO
ONNNN NNONN OOOON ONONN OOOON ONOO -ONO NON NN-O
O soN
+ ONOO
NNNON ONNNN OOOON NONNN OONON NNNN -ouO NON NN-O
mm + GHmu
ONNNN NONNN ONONN NNNON NONNN NNOO -ona NON ON-O
mm + Gï¬mu
ONONN OOONN OOOON OOONN NONNN ONOO -oua NON ONuo
ONNN --- --- “OzouO Om: ONno
OONNN. ONOON NOOO NOOO NOON NNON OOOO Omaoum Om: NN-O
OOON OOOO OOOO OONN OOOO NNOO ONOO zOO ON-O
OOON ONNN NOOO NNNO OOON NNON OOON zOO ONuu
ONNO OONO OOON OOON NOON OOOO NNOO zOO NNuO
OONON ONNO OONO NOOO OONO NNNO «zOO ONIO
OOOO OOOO NOON NNNN NOOO OOOO OONO O OONO NN-O
ONOO NNON ONNN NNOO OOOO ONNO NNON O OONO NNuo
OONO NNOO ONOO OONO NOON NNNN ONNN O NONO ON-O
OOOO OOOO NNNO NNNN OOOO OONO OONN O OONO O-O
ONON OOON NOOO NNNO NNON NNNN NNOO O BON + OO O-O
OOON NOON NNOO ONOO OOON OOON NNNN O 30N + OO N-O
ONOO OOOO NONN NNOO OOOO ONON ONNN mO Ouu
ONNO OONO OONN NNNO ONNO OONN NNON mO Ono
OOOO OONO NONO ONNN NNOO ONNO OOON O SON Nuo
ONNO NOOO ONOO ONON OONN NNNN NNOO O SON Ouo
OOOO NNOO NONO OONO NOOO ONOO ONON NOOOO NIO
OOOO OONO NONN NOONN NOOO NONO OONO NOOOO N-O
NO NN ON NN ON OO NO OO ON ON O N Same .oz
unmawummxm mo hmn ImHQQSm wNm
NNN uaOaNumaxO .OONO No OOOONms .OO ONOON
99
TABLE 31. Total weight gains and average daily gains, Experiment III (Gm.)
4-_
:—
Pig Supple- No. days on Average daily
No. ment Total weight gain experiment gain
C-l Basal 2867 47 61
C-2 Basal 1782 46 39
C—3 Low E 2318 46 50
C—4 Low E 4147 47 88‘
C-5 Se 4489 52 86
C-6 Se 4141 47 88
C—7 Se + low E 3736 46 81
C-8 Se + low E 4034 47 86
C-9 High E 3837 47 82
Cth High E 2327 47 50
C-11 High E 3399 52 65
C-12 High E 3247 46 71
C-13 EQN* 4683 43 109
C-14 EQN 1313 46 29
C-15 EQN 3484 52 67
C-16 EQN 565 52 11
C-17 MSU grower 8788 ' 52 169
C-18 MSU grower --- 7-- ----
C-19 20% protein 11794 38 310
+ Se
C-20 20% protein 12701 38 334
+ Se
C-21 20% protein 12701 38 334
+ Se
C-22 20% protein 12247 38 322
+ low E
C-23 20% protein 28123 52 541
C-24 20% protein 19505 52 375
* Ethoxyquin
100
TABLE 32. Liver weights and ratios of body weights to liver weights,
Experiment III
Pig Supple- Age at ne- Days on Liver wt. Ratio b.wa
No. ment crepsy (days) experiment (Gm.) to liver wt.
C-l Basal 67 47 --- ---
C42 Basal 64 46 238 28
C-3 Low E 66 46 231 25
C-4 Low E 65 47 216 42
C-5 Se 72 52 308 30
C-6 Se 65 47 275 31
C-7 Se + low E 66 46 306 26
C-8 Se + low E 65 47 228 35
C-9 High E 65 47 234 37
C-10 High E 67 47 246 27
C-11 High Ev 70 52 345 23
C-12 High E 66 46 311 27
C—13 EQN* 61 43 280 36
C-14 ‘EQN 66 46 238 22
C-15 EQN 70 52 238 33
C-16 EQN 72 52 115 36
C-17 MSU grower 72 52 277 . 43
C-18 MSU grower 30 10 80.2 31
C-19 20% protein + Se 63 38. 559 38
C-20 20% protein + Se 63 38 740 29
C-21 20% protein + 63 38 648 32
low E
C-22 20% protein + 63 38 717 30
low E
C-23 20% protein 77 52 1132 34
C-24 20% protein 77 52 959 28
* Ethoxyquin
101
TABLE 33. Hematocrit values (packed cell volume per cent), Experiment III
Pig Supple- Day of Experiment
No. ment _ , 3_ . ll , . 30 . 37 44
C-1 Basal 34.0 --- 36.0 39.0 40.0
C-2 Basal 41.5 --- 42.0 40.0 41.0
C-3 Low E 23.5 --- 39.0 --- 29.0
C-4 Low E 42.0 --- 40.0 43.0 40.0
C-S Se 41.0 --- 36.0 --- 39.0
C-6 Se 35.5 --- 37.0 --- 37.0
C-7 Se + low E 40.0 -—- 36.0 41.0 37.0
C-8 Se + low E 38.0 --- 36.0 41.0 41.0
C-9 High E~ 35.0 --- 44.0 43.0 42.0
C-lO High E 41.0 --- 35.0 27.5 39.0
C-ll High E 42.0 --- 35.0 36.0 34.0
C-12 High E 40.0 --- 35.5 39.0 38.0
C-13 EQN* 36.0 --- 37.0 36.0 ---
C-l4 EQN 39.0 --- 33.0 36.0 37.5
C-15 EQN 34.5 --- 34.0 32.0 34.0
C-l6 EQN 41.0 -—— 39.0 33.0 33.0
C—17 MSU grower~ 41.0 —-— 34.0 35.0 35.0
C-18 MSU grower 36.0 33.5 --— --— ---
C-19 20% protein 36.5 --- 38.5 37.0 ---
+ Se
C-20 20% protein 40.0 --- 37.0 42.0 --—
+ Se
Ce21 20% protein 41.0 --- 40.0 40.0 ---
+ low E
C-22 20% protein 37.0 —-- 40.5 38.0 ---
+ low E
C-23 20% protein 34.0 —-- 46.5 42.0 42.5
C-24 20% protein 37.0 --- 38.0 3940 139.5
* Ethoxyquin
TABLE 34. Hemoglobin (Gm./100 m1.), Experiment III
102
LL
Pig Supple- Day of Experiment
No. ment 3 ll 30 37 44
C-1 Basal 11.0 --- 11.9 12.6 12.4
C-2 Basal 12.4 --- 13.1 13.1 12.8
C-3 Low E 7.7 --- 12.9 --- 9.2
CL4 Low E 13.1 --- 12.7 14.2 12.7
C—5 Se 13.1 --- 12.0 --- 12.8
C-6 Se 10.5 --- 11.4 ——- 11.7
C—7 Se + 12.7 --- 12.0 13.1 12.2
low E
C-8 Se + 12.0 --- -1.4 13.1 13.1
low E
C-9 High E 10.5 --- 13.4 14.2 13.1
C-lO High E 12.6 --- 11.7 9.4 12.2
C-ll High E 12.7 --- 11.9 12.0 10.8
C-12 High E 13.1 --- 11.9 12.4 12.0
C-l3 EQN* 11.1 -- 12.0 11.7 ---
C-l4 EQN 12.2 -—- 11.1 11.7 11.8
C-15 EQN 10.8 -—- 10.8 10.5 10.6
C-l6 EQN 12.6 --- 12.5 10.8 10.9
c-17 MSU grower 13.1 --- 11.1 11.1 11. 3
C-18 MSU grower 11.4 10.0 --- --- ---
C-19 20% protein 11.4 --- 12.4 12.4 ---
+ Se
C-20 20% protein 12.7 --- 12.0 13.4 -—-
+ Se
C-21 20% protein 12.9 --- 12.7 13.0 ---
+ low E
C-22 20% protein 11.4 --- 12.7 12.6 —--
+ low E
C-23 20% protein 10.2 --- 14.6 13.8 13.6
C-24’ 20% protein 11.4 --- 12.4 12.7 12.6
* Ethoxyquin
103
TABLE 35. Serum ornithine carbamyl transferase activity, Experiment III
Pig Supple- Day of Experiment
No. ment 17 44
C-1 Basal ~-- 0.51
C-2 Basal --— 0.56
C-3 Low E --- 0.48
C-4 Low E --- 0.23
C—5 Se 0.21 0.23
C-6 Se 0.23 0.23
C-7 Se + low E 0.20 0.21
C~8 Se + low E 0.21 0.20
C-9 High El --— 0.24
C~10 ‘High E 0.23 --
C-ll High E 0.28 0.23
C-12 High E --- 0.19
C-l3 EQN* 0;30 ---
C-l4 EQN --- 0.24
C-15 EQN 0.17 0.19
C—l6 EQN 0.13 0.16
C-l7 Grower ration 0.21 0.19
C—18 Grower ration --- ---
C~19 20% protein + Se 0.25 ---
C-20 20% protein + Se --- ---
C-21 20% protein + low E 0.23 ---
C-22 20% protein + low E 0.20 ---
C-23 20% protein --- 0.19
C424 20% protein 0.33 0.30
* Ethoxyquin
104
TABLE 36. Gross evidence of liver disease and extreme atrophy of
thymus, Experiment III
Pig Supple- Dietary liver necrosis Extreme atrophy
No. ment . Acute necrosis, Fibrosis of thymus
C-l Basal + + 0
C-2 Basal + + +
C-3 Low E 0 + 0
C-4 Low E 0 0 0
C-5 Se 0 0 0
C-6 Se 0 0 0
C-7 Se + low E 0 0 +
C-8 Se + low E 0 0 0
C-9 High E 0 0 0
C-lO. High E 0 0 0
C-ll High E 0 0 0
C-12 High E 0 0 0
C-13 EQN* + 0 O
C-14 EQN 0 0 +
C-15 EQN O 0 +
C-16 EQN 0 0 +
C-17 Grower ration 0 0 not examined
C-l8 Grower ration 0 0 0
C-19 20% prOtein 0 0 0
+ Se
C-20 20% protein 0 0 0
+ Se
C-21 20% protein 0 0 0
+ low E
C—22 20% protein 0 0 0
+ low E
C-23 20% protein 0 0 0
c-24 20% protein 0 0 o
* Ethoxyquin
-105
One of 4 pigs (C-13) fed the antioxidant EQN was affected with
dietary liver necrosis. This pig died suddenly, although.the.extent
of the liver changes was very limited by gross examination. Other
observations made during the necrOpsy of C-13 included a pronounced
gaseous distention of the gastrointestinal tract, a crepitating subcu-
taneous edema of the inguinal and scrotal regions, and congestion of
the brain, thymus, lungs, and skin of the anterior 1/2 of the body.
The gross evidence of liVer necrosis in pig C-13 consisted of a few
superficial reddened spots representing individual lobules or lobules
in small groups. Bacteriologic cultures of the edematous subcutaneous
tissues yielded Clostridium perfringens..
Pig C-3 was observed at necropsy to have edema of the eyelids, and
the carcass was pale. Pig C-l6 had severe ulceration of the squamous
epithelium of the stomach. The stomach contents were watery and the
color of digested blood.
Histopathology. The incidence of microsc0pic lesions of dietary
liver necrosis, hydropic degeneration of the liver cells, nutritional
muscular dystrophy, and gastric epithelial defects among the pigs of
Experiment III is summarized in TABLE 37. No additional cases of liver
necrosis were discovered by microscOpic examination. The higher level
of vitamin E and the ration containing 202 protein appeared to be fully
protective against dietary liver necrosis. There was no hydropic
degeneration of the hepatocytes in pigs which were fed 20% protein
*diets. High level vitamin E apparently completely controlled nutritional
muscular dystrophy. Lesions of the gastric squamous epithelium were
not as severe among pigs fed the higher protein diets and standard
106
TABLE 37. Incidence of microscopic lesions, Experiment III
DLN* Hydropic Lesions of epithelium of
Acute degenera- esophageal region of stomach
Pig .Supple— necro- Fibro- tion of Epithelial Ero- -Uléeta-
No. ment sis sis liver NMD** degeneration sion tion
C-l Basal + + + 0 +
C-2 Basal + + + + +
C-3 Low E + + + 0 +
C—4 Low E 0 0 O O +
C-S Se 0 O + + +
C-6 Se 0 0 + + +
C-7 Se + 0 O + + + 0 0
low E
C-8 Se + 0 0 O 0 not examined
low E
C-9 High E 0 O O 0 not examined
C-lO High E 0 0 O 0 +
C-ll High E O O + 0 + 0
C-12 High E 0 0 + 0 +
C-l3 EQN*** + 0 0 not not examined
examined
C-14 EQN 0 0 + 0 +
C-lS EQN 0 0i + 0 + 0
C-16 EQN 0 0 not +
examined
C—l7 Grower 0 0 0 not not examined
ration examined
Cr18 Grower 0 0 0 0 + 0 0
ration
C-19 20% pro- 0 0 0 0 + O 0
tein +
se
C-20 20% pro- 0 ' O 0 0 + O
tein +
se
C-21 20% pro- 0 O 0 0 + 0 0
tein +
low E
C-22 20% pro- 0 0 0 0 + 0 0
tein + '
low E
C-23 20% pro- 0 0 0 + + 0 0
tein
C-24 20% pro- 0 0 0 + + 0 0
tein
* Dietary liver necrosis
** Nutritional muscular dystrophy
*** Ethoxyquin
107
grower ration. However, the incidence of gastric lesions was similar
to the incidence among pigs on 6% protein diets. Hemorrhages were
observed in the thymus of pig C-l3.
Bone marrow smears. In the bone marrow smears of many of the pigs fed
vitamin E-deficient diets, including those fed low level vitamin E,
there were numerous cells of the erythroid series which contained 2
nuclei or a single, lobed nucleus (Figure 49). This abnormality was
observed in prorubricytes, rubricytes, and metarubricytes. Such forms
also occurred in the marrow of pig C-17, which was fed the standard pig
ration. Among the 4 pigs fed high level vitamin E, only C-lO had an
occasional erythroid cell with these nuclear abnormalities (Figure 50).
108
, VAV-_V ..r ..
Figure 49. Binucleate cells and lobed nuclei of cells
of the erythroid series in bone marrow smear from a pig
fed basal ration. May-Grunwald-Giemsa stain. x 1200.
Figure 50. Binucleate erythroid cell in bone marrow
smear of a pig fed basal ration supplemented with high
level vitamin E. May-Grï¬nwald-Giemsa stain. x 1200.
DISCUSSION
Clinical Signs and Mortality
These experiments support Obel‘S’statement (l953).that clinical
signs~are not a prominent feature of dietary liver necrosis in swine..
Pigs, which died with lesions suggestive of dietary liver necrosis as_
the cause of death, were sometimes found dead in the morning although
they showed no signs of serious illness the previous evening. In one
case (B-9), a pig was found comatose in the morning after having
refused its meal the night before., Other signs noted occasionally or
in individual animals included vomiting, edema of the eyelids, posterior
weakness, and dark feces which appeared to contain digested blood.
The last mentioned sign was observed in a pig which had a severe hemor-
rhagic gastric ulcer. Obel mentions the appearance of blood in the4
feces as one of the signs of hepatosis diaetetica.
It is probable that some of the pigs with nutritional muscular
dystrophy had disturbances of locomotion, but no such signs were
noticed. This may have been due in part to the close confinement of
most of the pigs in small cages. However, no abnormalities of.gait
were.observed in pigs C-23 and C-24, both of which were maintained in
a large pen and had microsoppic lesions of-NMD.
The brief, acute illness of C-19 and.C-20_was not thought to be
related to the experiment.
Severe, hemorrhagic gastric ulceration was-the most likely reason
for the poor condition of pig C-16 throughout most of Experiment III.
109'
110
The watery, dark stomach contents, observed in this pig at necropsy,
are in agreement with the observations of Reese ggyal, (1966a).
The over-all mortality rate among the pigs of the 3 experiments
was relatively low, deSpite the fact that many animals had extensive,
chronic lesions of dietary liver necrosis. One may conclude, therefore,
that DLN in.pigs has a greater tendency toward a chronic course than
the same disease in rats. In the latter species, early death: is
the rule (Schwarz, 1958). S
The circumstances under which pig C-l3 died and the unusual necropsy
findings bear several similarities to some of the features reported by
Stowe (1962) in connection with sudden deaths among tocopherol-
deficient pigs. Stowe reported several instances in which deaths were
associated with rapid, extreme postdmortem tympdhites, crepitating
effusions in the posterior parts of the body, and the isolation of
clostridial organisms. He Speculated that deaths in such cases were
the result of overwhelming stress due to saprophytic clostridial
organisms. In any case, it appears that such deaths represent a dis-
tinct syndrome, identifiable by the following 4 characteristics:
1. Sudden death.
2. Rapid post-mortem tympanites.
3. Crepitant subcutaneous swellings..
4. Isolation of bacteria of the genus Clostridium.
It remains to be determined if tocopherol deficiency is an 88883?
tial factor in the pathogenesis of this condition. .It appears that the
relationship of this syndrome in swine to tocopherol deficiency warrants
further investigation.
111
Feed Consumption..
Feed consumption was uniformly poor among pigs fed the basal 6%
protein ration. Supplementation with selenium, high level vitamin E,
or combinations of supplements as employed in Experiment II all resulted
in enhancement of appetites. Pigs fed a 20% protein ration ate greater
amounts than those fed 6% rations, with no appreciable change in appe-
tites when supplements were added to the ration.
Pigs A-5 and C-l3, both of which died with lesions of dietary
liver necrosis, were eating comparatively well up to the times of death.
The appetite of Pig B-9 was slightly reduced for about 24 hours before
the pig was found comatose with DLN.
Growth~
Weight gains were extremely poor among pigs of Experiment I. Pig
ArS, whose diet was supplemented with methionine, was growing at.a
better rate than the other pigs of Experiment I when it died of dietary
liver necrosis. This suggests that the increased growth rate with
additional dietary methionine might have acted as a stress factor in
precipitating fatal DLN.
In Experiment II, the supplements appeared to act synergistically
to promote growth in many cases (e.g., pigs B-l, B-2, B—4, B-8).
In Experiment III, it was obvious that increased protein in the~
ration was much more effective in promoting growth than any of the
supplements, including tocopherol. The data of Experiment III support
the findings of Pellegrini (1958) and Stokstad g£_§l,.(l958), who
reported that tocopherol-deficient pigs grew as well as controls. The
death of C-l3, like that of A-S, was an example of sudden death in an
112
animal which appeared to be among the thriftiest and healthiest in the
trial.
Liver Weights
It was not possible to demonstrate any consistent relationship
between liver weight or the ratio of body weight to liver weight and
dietary liVEr disease.
Hematology
The rather uniform reduction in packed cell volume and hemoglobin
values in Experiments I and II were probably as much a reflection of
protein deficiency as of toc0pherol deficiency. In Experiment III,
there was little change in the hemograms. Thus, these experiments do
not support the findings of Nafstad (1965), who.reported a definite
anemia in pigs fed vitamin E-deficient diets for 4 weeks. Stdkstad
__£._l. (1958) found no changes in packed cell volume or hemoglobin
values in vitamin E-deprived pigs.
The hematocrit value of pig G-l6 declined from 41% to 33%, probably
as a result of gastric hemorrhage.
Serum Ornithine Carbamyl Transferase Activity
Although the highest values for serum OCT activity were observed
in pigs with dietary liver necrosis, the figures were all well within
the normal ranges reported by Wretlind gtnal. (1959). It is possible
that there was a uniform decline in enzyme activity while the samples
were stored in the frozen state or that significant deterioration
occurred before freezing. In any case, the values obtained are regarded
as a useful index of dietary liver necrosis when viewed in relation to
each other.
113
The sharp rise followed by an abrupt decline in OCT activity in
the serums of pigs B-3 and B-8 is puzzling. One possible eXplanation
for this would be a temporary remission of the acute necrotic process
and a period during which the proliferative processes were dominant.
Pathology
Dietaryfliver‘necrosis. The results of these experiments indicate that
either selenium or alpha-tocopherol in adequate amounts in the diet
affords pig. complete protection against dietary liver necrosis. One of
4 pigs fed ethoxyquin had lesions Of DLN. The absence of necrosis in
pigs C-23 and C-24 is contrary to the findings of Eggert gtngl. (1957),
who reported that 4'of 6 pigs fed vitamin E-deficient diets containing
40% Torula yeast died of liver necrosis within 53 days. Pellegrini
(1958) observed fatal liver necrosis within 50 to 70 days in pigs fed
32 to 45% Torula yeast diets. It is possible that dietary liver necrosis‘
would have occurred in C-23 and C-24 if the pigs had been fed the
experimental ration beyond the 52nd day. Another possible explanation
for the fact that the livers of C-23 and C-24 were free of necrosis is
that Torula yeast may vary in selenium content; Some rations containing
40% Torula yeast might contain enough selenium to prevent DLN“
Gross and microscopic study of livers in various stages of DLN
enables the pathologist to draw some conclusions regarding the sequence-
by which the described lesions occur. Pro-necrotic changes are probably
not a prominent feature of this disease, although mineralization and
edema of the space of Disse may.occur. The necrosis occurs suddenly,
and, in acute, fatal cases, the liver is swollen and scattered with
numerous spots resembling petechiae. Microscopically, these are found
ox
114
to be lobules which have filled with blood after necrosis of the normal
cellular elements. The interlobular connective tissue is intact, and
characteristically limits the necrosis within individual lobules,
although adjacent lobules may be affected simultaneously. Within a
necrotic lobule cytOplasmic and nuclear fragments may be seen, but
there is apparently prompt lysis of much of this cellular debris, so
that the lobule consists of little more than a blood—filled reticulum.
Infiltration by polymorphonuclear leukocytes is seen in some lobules.
The portal structures are relatively free of lesions. At this point,
the necrotic lobule apparently collapses, and is partially replaced by
proliferating connective tissue and bile ducts. A few surviving hepato-
cytes are often seen arranged in small, gland-like groups surrounded by
connective tissue.- The roughened.surface observed grossly in chronic
DLN results from the contraction of the new scar tissue. Various combié
nations of these lesions in the same liver bear testimony to the fact
that dietary liver necrosis in the pig is often characterized by repeated
attacks of acute necrosis with subsequent fibrosis.
Atrophy of the thymus. Atrophy of the thymus is a common finding in
animals fed diets deficient in protein (Follis, 1958). The data of
Experiment III suggest that tocopherol as well as protein may have some
protective effect against atrophy of the thymus.
Hydropic degeneration of the liver.. The occurrence Of this lesion indi-
cated that it was associated with protein deficiency. This is in agreement
with the reports of Hove and Seibold (1955) and Kosterlitz (1944).
115
Hutritional.muscular.dystrophy. .The results of these experiments sug-
gest that adequate dietary vitamin E affords pigs complete protection
against NMD. Due to the fact that the muscles of only 2 of the 4 pigs ‘
fed ethoxyquin were examined, no inferences were drawn regarding the
effect of a synthetic antioxidant on the occurrence of this lesion.
The results of these trials support the findings of Grant and Thafvelin
(1958) and Swahn and Thafvelin (1962), who reported that selenium did
not completely protect vitamin E-deficient pigs from nutritional muscular
dystrophy. This is in contrast to the effectiveness of selenium in the
prevention of NMD in lambs and calves reported by Schubert 35.31, (1961)
and Hartley and Grant (1961).
ngions of the;epitheliumuof the esophagealerggion of the stomach. The
stomach lesions observed were classified as:* (1) epithelial degenera-
tion characterized by hyperkeratosis, parakeratosis, and ballooning
degeneration; (2) erosions; and (3) ulcers. These were regarded as*
representing different degrees of severity ofla single disease process,
with degenerative changes appearing as a precurser.to actual_desquama-
tion. Candida albicans was considered an opportunist in the diseased
tissues. Obel (1953), in her comprehensive treatise on dietary liver
necrosis (hepatosis diaetetica), considered ulceration of the squamous
epithelium of the stomach an important and fairly constant feature of
the disease. The results of these experiments do not support this view.
The incidence of these lesions approached 100% among the pigs of these
3 experiments, irrespective of diet or the occurrence of other lesions.
Although the number of animals involved does not-permit absolute con-
clusions, the data suggest that the lesions are less severe in pigs
116
whose diets contain.adequate.protein.‘ In this connection, Nafstad (1967a)
reported that high protein rations reduced the severity but not the in—
cidence of gastric ulcers in pigs. However, these experiments do not
support Nafstad's view that vitamin E exerts a partial protective effect
against ulcers (Nafstad, 1967a,b). The results of these.experiments are
in agreement with Nuwer gtyal, (1965), who found no protective effect
when toc0pherol was added to an ulcerogenic swine ration.
The degenerative lesions observed in blood vessels in tiSsues under-
lying ulcerated areas of gastric mucosa were also reported by Bicknell
(1965). These unusual changes are probably the result of ulceration
of the nearby mucosa. However, one cannot ignore the possibility that
the reverse is true: that vascular lesions play an important role in
the pathogenesis of gastric ulcers in swine.
There were several lesions which Obel (1953) reported as frequently
associated with hepatosis diaetetica{which were not observed in these
studies. Some of the more important of these were: acute nephrosis,
purulent cholangitis, yellow fat, and fibrinoid degeneration of small
arteries in the mesentery, heart, liver, and kidneys. No explanation
can be given for the failure of these changes to occur in the pigs used
in this research.
Bone Marrow Smears -
Study of the bone marrow smears of the pigs of Experiment III re-
vealed nuclear abnormalities in cells of the erythroid series of many
pigs similar to those described by Nafstad (1965). Such cells were
somewhat less numerous in the marrows of.pigs fed selenium or ethoxyquin
than in the marrows of those fed the basal or low level vitamin E diets.
117
However, in pigs fed high level vitamin E, abnormal erythroid elements
were not seen except in one animal (C-lO), and then only rarely.
These results are in general agreement with the report of Nafstad
(1965). However, she observed no reduction in the number of abnormal
cells in the bone marrow of pigs fed selenium supplements. She regarded
these lesions as evidence of a block in red cell maturation in tocopherol-
deficient pigs.
In the examination and interpretation of the marrow smears of pigs
in Experiment III, it was often difficult to determine whether an
apparently binucleated erythroid cell was indeed such a cell or merely
the result of an artifact of preparation.
Feed and Liver Analyses for Alpha-Tocopherol .
The slightly higher level of alpha-tocopherol in the basal ration
with added ethoxyquin is probably due to a protective effect of the
antioxidant upon toc0pherol. Until further data are available, the
significance of the tocopherol content of the livers of pigs of Experi-
ment I is not clear.
SUMMARY
Three experiments were conducted to study the pathology.and patho-
genesis of dietary liver necrosis (DLN) in the pig and to clarify the
roles of vitamin E, selenium, the sulfur amino acids, and total dietary
protein in the prevention of this disease. A total of 48 pigs was used
in the 3 trials. The basal 6% protein diet contained Torula yeast as a
source of protein and vitamin E-free lard as a source of fat. The
various supplements employed included vitamin E at 2 levels, selenium,
and methionine, each alone, and in several combinations. Other pigs
were fed a diet containing the antioxidant ethoxyquin, and 6 pigs were
fed rations containing sufficient Torula yeast to provide a 20% pro-
tein diet.
Dietary liver necrosis occurred consistently in pigs fed the basal
6% protein ration, or the basal 6% protein ration supplemented with
low levels of vitamin E or with methionine. Supplementation with
selenium, high levels of vitamin E, or additional protein completely
prevented dietary-liver necrosis during the periods of these trials.
Lesions of DLN were observed in l of 4 pigs fed ethoxyquin.
Acute dietary liver necrosis appeared grossly as scattered red
spots on the surfaces and in the-parenchyma of affected livers. Chronic.
changes were represented by irregularly shaped, roughened areas resulting
from the contraction of scar tissue.
118
119
Microscopically the most striking features were necrosis of hepato-
cytes, pooling of blood in the necrotic lobules, and a characteristic
limitation of the necrosis by the interlobular septa. Connective tissue
and bile duct proliferation occurred as a sequel to necrosis.
Microscopic lesions of nutritional muscular dystrophy (NMD) were
observed in some pigs of all groups except those fed the higher level of
vitamin E or ethoxyquin. Muscle tissue of only 2 of 4 pigs fed ethoxy-
quin was examined for this lesion.
Ulcers or pre-ulterous changes of the squamous epithelium of the
stomach were observed in virtually all the pigs in these experiments.
These lesions were somewhat less severe among pigs fed 20% protein
rations.
HydrOpic degeneration of hepatocytes and extreme atrophy of the
thymus occurred consistently in pigs fed 6% protein diets.
Serum ornithine carbamyl transferase activity of pigs with die-
tary liver necrosis was generally higher than that of pigs free of
DLN. However, none exceeded values reported as normal for pigs by
Wretlind £5 31. (1959).
Nuclear abnormalities in erythroid cells in bone marrow smears
were relatively common among pigs maintained on vitamin E-deficient
diets, whereas such lesions were extremely rare among the group fed a
high level of vitamin E.
Growth rates were very poor in pigs fed 6% protein diets, but
tocopherol deficiency appeared to have little effect on_growth.
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VITA.
Robert L. Michel was born in Philadelphia, Pennsylvania, on
July 20, 1924. He attended the public schools of that city and had
completed the preveterinary course and the first year of.veterinary
school at the University of Pennsylvania when his education was
interrupted by a period of service in the U.S. Army during World War
II. Returning to veterinary school after the war, the writer completed
his training, receiving the V.M.D. degree in 1949.
Following his graduation from veterinary school, the author
practiced for one year with Dr. H. B. Prothero in Johnstown, Pennsylvania.
For the next 13 years, the writer conducted a general veterinary prac-
tice in the rural Pennsylvania community of Troy, Bradford County.
In the fall of 1963, he entered the graduate school of Michigan State
University, majoring in veterinary pathology.
The author married Marjorie R. Kephart of Philadelphia and Ocean
City, New Jersey, in 1948. They have 4 children: Virginia, 15,
Janice 13, Andrea, 11, and Richard, 9.
128
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