PATHOLOGY OF TOCOPHEROL-DEFICIENT MINK AND SWINE Thesis In: the Degree of Ph. D. MICHIGAN STATE UNIVERSITY Howard Denison Stowe I962 This is to certify that the thesis entitled Pathology of Tocopherol-Deficient Mink and Swine presented by Howard Denison Stowe has been accepted towards fulfillment of the requirements for P_ho11-__ degree inmy Pathology Major professor L/ji I . pm July 10, 1962 0-169 LIBRARY Michigan State University ABSTRACT PATHOLOGY OF TOCOPHEROL-DEFICIENT MINK AND SWINE by Howard Denison Stowe Three experiments, two mink and one swine, were conducted to study the pathology of toc0pherol deficiency. Semipuri- fied type rations were employed and the deve10pment of the tOCOpherol deficiency was evaluated by means of growth rates, symptomatology, biweekly hematological and serological studies, gross pathology and histOpathology. Also investigated were the effects of tocOpherol deficiency upon Salmonella pullorum antibody production, the tocOpherol-depleting activity of cod liver oil (mink) and ethyl linoleate (swine), the tocOpherol- sparing activity of selenium and ethoxyouin and the species requirement for tocopherol. Sudden deaths among the tocOpherol-deficient mink and swine were considered significant manifestations of tocopherol deficiency. The sudden deaths of mink usually followed an exposure to a stress factor. No growth alterations or con- sistent physical symptoms were observed in surviving tocOpherol- deficient animals. The most significant hematological alteration that coin- cided with tOCOpherol depletion was an increased erythrocyte fragility that was indicated in both species by a #8-hour layered hemolysis in refrigerated saline. The increased fragility was also detected in swine by the dialuric acid Howard Denison Stowe hemolysis test. An absolute neutrOphilia was associated with tocOpherol-deficient mink that had steatitis. Packed cell volumes and hemoglobin concentrations increased with the age of mink and were unaffected by toconherol deficiency in either mink or swine. Prolonged tocOpherol deficiency of mink (Experiment II) resulted in decreased serum albumin and increased alpha and beta globulin fractions while the serum protein fractiOns were not altered by tocOpherol deficiency in swine. Elevated serum glutamic-oxalacetic and glutamic-pyruvic transaminase values and serum tocOpherol values between 50 and 70 micro- grams/100 ml. were associated with toc0pherol deficiency lesions. Grossly, internal intercostal and adductor myOpathy was common in toc0pherol-deficient mink while subcutaneous edema, hemorrhagic myositis and a perilobular to generalized fatty infiltration of the liver were noted in the tocopherol-deficient swine. Histologically, the skeletal myOpathy of tocOpherol- deficient mink consisted of swollen, differentially stained fibers, vacuolar degeneration, sarcolemmal and myoblastic prolflbretion and calcification of the non-phagocytized myo- fibrillae. Also associated with the deficiency in mink were calcified necrotic myocarditis, centrolobular hepatic hemorrhage, coagulation necrosis with calcification of the convoluted tubules and calcified necrotic foci in the adrenal cortex. Microscopic lesions characteristic of tocopherol defi- ciency in swine were centrolobular hepatic hemorrhage, Howard Denison Stowe endomysial edema, hemorrhagic myositis, a slight myoblastic proliferation, hyalinized adductor fibers containing rowed internal nuclei, and Purkinje fiber degeneration. Sarcolemmal proliferation and calcification of the myofibrillae were not observed in swine while the internal nuclear rowing of skeletal muscle was not characteristic of tocOpherol deficiency in mink. TocOpherol deficiency did not impair or enhance the antibody response of mink to Salmonella pullorum antigen; however, tocopherol-deficient swine demonstrated a greater ability to produce Salmonella pullorum antibody than did tocOpherol-supplemented swine. Isocaloric supplementation of 8% cod liver oil to toco- pherol-deficient ration fed to mink caused the deposition of a yellow, acid-fast pigment in the interstices of the adipose tissue. An isocaloric supplement of 5% ethyl linoleate to the tocopherol-deficient ration fed to swine, on the other hand, did not cause the acid-fast-pigmented steatitis. Neither supplement hastened tocOpherol depletion as measured by biweekly serum tocopherol values. Selenium supplementation at the rates of 0.1 and 1 p.p.m. prevented fatal tocOpherol deficiency lesions in mink. Neither selenium nor ethoxyquin was effective in preventing the 48- hour hemolysis phenomenon in mink or swine. Alpha-tocOpherol SUpplementation of the basal mink ration at the rate of 25 ~D-p.m. and the swine ration at 100 p.p.m. was adequate to ‘nPevent all lesions associated with a tocOpherol deficiency. PATHOLOGY OF TOCOPHEROL-DEFICIENT MINK AND SWINE By Howard Denison Stowe A THESIS ‘ Submitted to ~ Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology 1962 0‘ r- I ... .— .—r ACKNOWLEDGMENTS The author is grateful to the fellowing for helping to make this research and thesis possible: Dr. C. K. Whitehair, my major professor, whose encour- agement for research and whose counsel in the preparation of the manuscript were invaluable. Dr. C. C. Merrill, department chairman, for granting department facilities for this research and for his guidance in writing this thesis. . Dr. R. F. Langham for his assistance with the histo- pathology. Dr. M. L. Calhoun for her helpful suggestions in the preparation of the manuscript. Drs. R. W. Luecke and D. E. Ullrey for constructive criticisms of the manuscript, help with analytical procedures and the use of laboratories under their supervision. Mr. Robert Maronpot for his assistance with analytical determinations and photography. Mrs. Nancy Anderson, Mrs. Ann Goatley, Mrs. Delorise Palombo, Mrs. Jackie Bradley and Mr. William Truitt for their technical assistance. National Institutes of Health and the Mink Farmer's Research Foundation of Milwaukee fer their financial support. Distillation Products Industries, Rochester, New Ybrk, and Lake States Yeast and Chemical Division of St. Regis 11 Paper Company, Rhinelander, Wisconsin, respectively, for providing the molecularly distilled lard and torula yeast for these experiments. 111 TABLE OF CONTENTS INTRODUCTION 0 C O . . C . . C O C C . mmwormrmrvas . . . . . . . .‘ . MINE EXPERIMENTS Materials and Methods . . . . . . Experiment I . . . . . . . . . . Experiment II . . . . . . . . . Results . . . . . . . . . . . . . Experimentleeeeeeeeee Experiment II . . . . . . . . . Discussion . . . . . . . . . . . . SWINE EXPERIMENT Materials and Methods . . . . . . Results . . . . . . . . . . . . . Discussion . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . iv Page 98 102 127 .134 137 Table 10. 11. 12. 13. 14. 15. 16. LIST OF TABLES Mink Experiments Materials and Methods Experiment I Composition of the tocOpherol-deficient diet Amino acid and vitamin supplementation rate Experiment II Composition of the tocopherol-deficient diet Assignment to experimental lots . . . I . . Results Experiment I Growth rates . . . . . . . . . . . . . . . . Probable causes and number of deaths . . . . Leukocyte counts . . . .I. . . . . . . . . . Packed cell volumes . . . . . . . . . . . . Hemoglobin values . . . . . . . . . . . . . Differential leukocyte counts . . . . . . . Serum albumin and globulin fractions . . . . Serum glutamic-oxalacetic and glutamic-pyruvic transaminase values . . . . . . . . . . . Serum tocopherol values . . . . . . . . . . Urinary creatine and creatinine excretion values Specific gravity and surface tension values for urine 0 O O O O O O O O O O O O O O 0 Incidence of urinary incontinence . . . . . Experiment II Growth rates . . . . . . . . . . . . . . . . Probable causes and number of deaths . . . . V Page 29 33 33 36 37 -38 39 40 42 45 47 49 50 51 65 66 Table 19. 20. 21. 22. 23. 24. 25. 26. 27. 29. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Leukocyte counts . . . . . . . . . . . . . . . . Packed cell volumes . . . . . . . . . . . . . . Hemoglobin values . . . . . . . . . . . . . . . Differential leukocyte counts . . . . . . . . . Erythrocyte fragility indexes . . . . . . . . . Serum albumin and globulin fractions . ... . . . Serum glutamic-oxalacetic and glutamic-pyruvic transaminase values . . . . . . . . . . . . . Serum tocopherol values . . . . . . . . . . . . Salmonella pullgzum antibody titers . . . . . . Swine Experiment Composition of tocopherol-deficient ration . . . Amino acid and vitamin supplementation rates . . Assignment of pigs to experimental lots . . . . Observations pertaining to deaths of tocOpherol- deficient swine . . . . . . . . . . . . . . . Leukocyte counts, packed cell volumes and hemoglobin values . . . . . . . . . . . . . . Differential leukocyte counts . . . . . . . . . Dialuric acid hemolysis values . . . . . . . . . Serum albumin and globulin fractions . . . . . . Serum tocopherol values . . . . . . . . . . . . Serum glutamic-oxalacetic and glutamic-pyruvic transaminase values . . . . . . . . . . . . . Salmonella pullorum antibody titers . . . . . . Responses to transmissible gastroenteritis virus vi Page 67 68 69 7o 72 74 75 75 77 98 98 99 103 104 104 107 108 109 110 111 112 LIST OF FIGURES Figure 1. .2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18, 19. 20. 21. Mink Experiments Toccpherol-supplemented mink erythrocyte fragility t.3tt e e e e e e e e ta at O O O O O O O O O O O O 0 Gross internal intercostal myOpathy- Gross adductor myopathy . . . . Vacuolar muscular degeneration Sarcolemmal proliferation . . . Adductor myopathy . . . . . . . Focal adductor calcification . Internal intercostal myOpathy . Gross myocarditis . . . . . . . Focal calcified necrotic myocarditis . Extensive calcified necrotic myocarditis . Calcified necrotic focus in adrenal cortex Non-acid-fast-pigmented steatitis Pneumonitis . . . . . . . . . . Normal portal triad . . . . . . Edematous portal triad . . . . . Kidney of tocOpherol-deficient mink Adductor of a tocOpherol-deficient cod supplemented mink . . . . . . Normal adipose tissue . . . . . . I Adipose tissue from a tocopherol-deficient selenium-supplemented mink . . vii .TocOpherol-deficient mink erythrocyte fragility Page 43 43 55 56 57 57 58 59 6O 61 61 62 63 64 64 80 80 81 82 83 83 Figure go 23. 24. Gross "yellqu fat " O O O O O O O O O O O O O O O Acid-fast-pigmented steatitis . . . . . . . . . Kidney of toc0pherol-deficient cod liver oil- supplemented mink . . . . . . . . . . . . . . Kidney of tOCOpherol-deficient cod liver oil- supplemented mink . . . . . . . . . . . . . . Swine Experiment Gradations of the 48-hour refrigerated hemolysis Acute gastritis . . . . . . . . . . . . . . . . Perilobular and generalized fatty infiltration Of liver 0 O O O O O O O O I O O O O O O O O O Suffusion hemorrhage in the gracilis . . . . . . Subcutaneous and fascial hemorrhage . . . . . . Hemorrhage in the gracilis . . . . . . . . . . . Endomysial edema . . . . . . . . . . . . . . . . Adductor from a tocopherol-deficient pig . . . . Internal nuclear rowing in an adductor . . . . . Adductor from a tocopherol-deficient pig . . . . Purkinje fibers from a toc0pherol-supplemented‘ p18 0 O O O O O O O O O O O O O O O O C O O O Purkinje fiber from a toc0pherol-deficient pig . PurkinJe fiber from a tocopherol-deficient pig . Liver from a tocopherol-deficient pig . . . . . Adrenal from a tocOpherol-supplcmented pig . . . Vacuolar degeneration of a zone glomerulosa . . Adipose tissue from tocopherol-deficient pig . . Lung from a toc0pherol-deficient pig . . . . . . Pneumonitis of a tOCOpherol-deficient pig . . . viii 105 116 117 D.) O‘\ INTRODUCTION The nutritional importance of tocopherol has been exten- sively investigated in many species since Evans and Bishop (1922) identified the fat-soluble substance. Recently, semi- purified toc0pherol-deficient and unsaturated-fatty-acid-low rations were fed to mink to determine if a relationship existed between tocOpherol deficiency and urinary incontin- ence. Although no such relationship was found, several cases of tocOpherol-deficiency-myOpathy were produced without any evidence of "yellow fat" which Gorham gt al. (1951) considered characteristic of tocopherol deficiency in mink fed high levels of fish products. Thus, the mink was considered to be a suitable animal in which to study the pathology of toco- pherol deficiency uncomplicated by steatitis. Mink Experiment I was undertaken to re-examine the uncomplicated tocopherol deficiency, to study the effects of selenium supplementation and to obtain information relative to the tocOpherol requirement of mink under the conditions of the experiment. Mink Experiment II was designed to demonstrate the differences between the uncomplicated tocOpherol deficiency and one complicated by the effects of an elevated intake of unsaturated fatty acids. The study of a chronological re- lationship between the onset of increased fragility of tocopherol-deficient erythrocytes (observed in Experiment I) 1 2 and the onset of other clinical pathology of toc0pherol deficiency was also of interest. The importance of toc0pherol in swine nutrition was emphasized when Adamstone 2; al. (1949) noted a hemorrhagic condition in 10 mm. pig embryos identical histologically to that observed in tocOpherol-deficient chick embryos. Most investigators of tocopherol deficiency in swine have used unsaturated fatty acids as tocopherol-depleting agents. The resulting tocOpherol deficiencies were complicated with a steatitis related to unsaturated fatty-acid toxicity. The uncomplicated tocOpherol deficiency produced in mink suggested the feasibility of studying the same deficiency in the baby pie. REVIEW OF LITERATURE TOCOpherol in Nutrition General The fat-soluble dietary factor necessary for repro- duction in rats was discovered by Evans and BishOp (1922) and called vitamin E. For this biological alcoholic anti- oxidant, George Calhoun coined the term 'toc0pherol' from tokos - childbirth; phero - to carry; and 01 - alcohol. Of the seven known toc0pherol isomers, alpha, beta, gamma, delta, epsilon, zeta and eta, Dam (1957) reported that alpha is physiologically the most important, accumulates to the greatest degree in animal tissues and, lg ZlEEQ: elicits the least antioxidant effect. While the rat and chick have been the most frequently used laboratory animals for tocopherol experiments, the following species have also been utilized: monkey (Marvin 23 al., 1960), calves (Maplesden and Loosli, 1960), cattle (Andersson, 1960), sheep (Muth 23 al., 1959). foals (Dodd 33 al., 1960), dogs (Brinkhous and Warner, 1941), cats (Cordy and Stillinger, 1953), rabbits (Borgman, 1959), ham- sters (West and Mason, 1958) and guinea pigs (Bender g3 al., 1959). The major manifestations of toc0pherol deficiency in- clude exudative diathesis, encephalomalacia, adipose tissue , - -: ‘, ' -- ‘ p, I ~ ~~ 1"‘3 TT-f‘C-tiu. . .fr i ._. .J‘E , a liver necrosis, lung hemorrhage, erythrocyte fragility; and muscular degeneration. Some less frequently observed effects include adrenal necrosis, renal tubule degeneration, arter- ioscleroses, Purkinje fiber degeneration, reticular and elastic fiber discontinuity, duodenal and gastric ulcers, increased cellularity of bone marrow, sarcolemmal prolifer- ation and altered clotting time. Metabolic Role That the toc0pherols inhibit autoxidation of fats in contact with molecular oxygen is evidenced by two facts: certain signs of tocOpherol deficiency occur only when easily oxidized unsaturated fatty acids are present in the diet; and many antioxidants and redox substances structur- ally unrelated to tocOpherol afford partial to complete pro- tection against certain manifestations of tOCOpherol defi- ciency. Bouman and Slater (1957) indicated that the toco- pherols are also associated enzymatically with tissue respiration, specifically with the cytochrome 0 reductase activity in heart mitochondria where large quantities of cellular tocopherols are found. Contrary to the reports of Bouman and Slater, Pollard and Bieri (1959) found the DPNH cytochrome 0 reductase activity was not significantly affected by tocopherol deficiencies. Simple homogenization of the enzyme extracts was found to affect their inactivation, which could be overcome by the addition of lipid substances, by lyophilizing or by centrifuging. Also, Pollard and Bieri found that the iso-octane phase used by Bouman and Slater 5 had a narcotizing effect upon the enzymes. Removal of the bound isooctane from the respiratory chain preparations re- sulted in their reactivation. Zalkin and Tappel (1960) found tocopherol inhibited mitochondrial lipid peroxidation and concluded that tocOpherol functions solely to stabilize cellular unsaturated lipids against oxidative deterioration, thus maintaining their functional integrity at the subcellular level. Schwarz (1961) reported that the only detectable defect in tocOpherol deficient rat mitochondria is in their succinate utilization in the presence of diphosphopyridine nucleotide and was evi- denced by respiratory failure. An inhibitory factor, pos- sibly iron, was found to hasten the respiratory failure. Schwarz concluded that iron inactivates various sulfhydryl dehydrogenase substrate systems and that the physiologic function of tocOpherol is related to thiol or dithiol groups of enzyme systems because sulfur amino acids decrease the tocopherol requirement to approximately one-tenth that of normal. The metabolically active form of tocopherol may act at active sulfhydryl group sites, force the equilibrium toward the 3-8 form and eliminate the point of attack for inhibitory heavy metals. Corwin (1960) has also indicated alpha tocOpherol functions in maintaining sulfhydryl groups. Considerably more work is necessary to elucidate positively the metabolic roles for the various tocOpherols. 6 Tocopherol-Sparing Compounds Selenium Schwarz and Foltz (1957) demonstrated that the so- called Factor 3, which prevented hepatic necrosis in rats fed tocopherol deficient rations, was actually selenium. Since 1957, sodium selenite has been reported therapeutically effective against exudative diathesis of chicks by Dam and Sandersaard (1957), stiff lamb disease by Muth at 5;. (1959). 111 thrift in lambs by Drake 23,51. (1960), white muscle disease of calves by Sharman 23 gl. (1959) and Jolly (1960) and muscular dystrophy, necrotic hepatitis, hemorrhagic lymphadenitis and microangiopathy in swine by Grant (1961). Yang 2; al. (1959) and Lam g; 5;. (1961) suggested that some metabolic relationship exists between selenium and co-enzyme A. Dietary selenium at .05 p.p.m. was found by Yang and associates to stimulate co-enzyme A biosynthesis from S35 labeled cystine and to elevate co-enzyme A levels in the livers of rats on necrogenic diets. Rosenfield (1961) found that seleno-cystine and seleno-methionine are formed by bacteria in the ovine rumen from inorganic selenium. Rosenfield (1960) also showed that selenite interfered with protein synthesis by inhibiting the transmethylation of homocystine or nomocysteine by betaine or choline. Corwin and Schwarz (1959) did not find selenium effective in pre- venting the decline of succinate peroxidation which is ob- served in livers of tocOpherol-deficient rats. Schwarz (1960) reported that, in cystine, a trace contamination of 7 1 atom of selenium in 350,000 atoms of the sulfur ion is ade- quate to protect against hepatic necrosis in the rat. Selenium, as well as alphaetocOpherol, has been shown by Edwin 22.§l¢ (1961) and Green gg'gl. (1961) to increase the ubiquinone levels in rat heart, liver and ovaries. The ubiquinones are considered by Diplock 23,2l. (1960) and Redfearn and Punphrey (1960) to function in oxidative phos- phorylation or electron transport and the essential biologi- cal role of selenium may be associated with its ability to increase ubiquinone levels ln_vl!g: Bieri (1961) indicated that selenium increases the antioxygenic potential of cells by, in some wa$,altering the composition of the cell proteins. Bieri, however, observed no correlation between the dietary concentration of selenium and its antioxidant effect.- Selenium supplementation low- ered tissue thiobarbituric acid values,indicating decreased peroxidation. Redngubstanges Dam and coworkers (1951) reported the following to afford considerable protection against symptoms of exudative diathesis in tocopherol-deficient chicks: methylene blue @ .126% of diet, thionine @ .089%, thiOphenylamine @ .675%, disulfiram @ .025%, and ascorbic acid @ .5%. If any pro- tection were afforded, it was reflected by elevated toco- pherol values in adipose tissue. Against liver necrosis in rats, Dam and Granados (1951) found methylene blue to afford complete protection and magnesium supplementation afforded 8 slight protective action with the necrogenic diets. Antioxidants The tocOpherol sparing effects of NN'-diphenyl-p- phenylenediamine (DPPD) have been extensively studied by many including Sherman and Moore (1958), Draper (1959)and Crider 23 al. (1961). In tocOpherol deficient rats DPPD was found to prevent the brown uterus, testicular degener- ation and the abnormal tendency of the erythrocytes to hemolyze. Oser and Oser (1956), found DPPD, however, caused prolonged gestations which led to dystocias in rats. Machlin and Gordon (1960) found that .1% ethoxyquin added to a toco- pherol-deficient-torula-yeast ration and fed to chicks pre- vented both exudative diathesis and encephalomalacia. Others Moore and Sharman (1958) found isoniazid, an agent effective against tubercle bacilli, prevented the rapid post mortem autolysis in kidneys and the mottling of the incisor teeth of tocOpherol deficient rats. Mason and Rao (1960) showed that alpha-tocapherolhydroquinone, thought only to contain antidystrophic activity, does in fact possess antisterility activity approximating 1/20th that of alpha- tocOpherol. Corwin (1960) found S-H glutathione and 2,3- dimercaptOprOpanol (EAL) prevented the decline in oxidation rates of alpha-ketoglutarate or succinate in liver homogenates from tocOpherol deficient rats. Green g;_§l. (1960) found that intraportal injections of the cobalt ion have high 9 activity in preventing respiratory decline in liver slices from tocOpherol-deficient rats. Tocopherol Antagonists Unsaturated Fatty Acids Although Agduhr (1926) first reported the harmful effects of cod liver oil fed to children, Goettsch and Pappen- heimer (1931) were among the first to find that the unsatur- ated fatty acids contained in cod liver oil aggravated a tocOpherol deficiency. Madsen (1936) experimented with fats and oils in synthetic rations and concluded that fats alone were not responsible for the muscular dystrophy produced. Morgulis and Spencer (1936) reported two factors were prob- ably involved in nutritional muscular dystrophy, one in wheat germ oil and the other in green lettuce and dry alfalfa. Leaf fate of pasture herbage have been found by Hilditch (1956) to be highly unsaturated yet Keith and Schneider (1957) found no correlation between the dystrophogenicity of roughages and their tocOpherol content. Although Brown (1953) has shown cod liver oil to con- tain appreciable tocOpherol, the polyenoic fatty acids, especially in cod liver oil, have been used extensively as' tocOpherol depleting agents in nutritional eXperiments since about 1945. Filer and associates (1947) found C18 acids with 3 double bonds most effective in producing histological changes in the tocOpherol-deficient rat. Kokatnur 22.2;- (1960) found the most effective polyenoic acid for the 10 acceleration of encephalomalacia in chicks is 12 oxo sis-9- octadecenoic acid, especially when used with corn oil. Nishida 23 al. (1960) produced encephalomalacia in chicks in from one to five hours by intravenously injecting 10 mg of linoleic acid previously emulsified in 1 ml. of serum. Parenterally administered alpha-tocopherol prevented this encephalomalacia which seemed to be initiated by the ln ylyg accumulation of lipohydroperoxides. Horwitt and coworkers (1961) indicated that the re- quirement for toc0pherol is a function of the amounts of polyunsaturated fatty acids in the diet and body tissues and that past dietary habits affect tocopherol needs. Diet changes were found to alter the mitochondrial lipids of all tissues including the brain. In studies undertaken to inves- tigate the relationship between tocOpherol and various types of dietary fat for the rat, Alfinslater 22 al. (1961) found that toc0pherol was required by the rat in the absence as well as in the presence of dietary fat. Other TocOpherol Antagonlggg Sulfathiazole and sulfaguanidine therapy, according to Daft 23 al. (1943), resulted in lesions similar to tocopherol deficiency. Pindborg (1949) confirmed this and speculated that bacteria of the alimentary tract synthesized some toc0pherol and when the gut is sterilized by sulfa therapy, tocOpherol deficiency may result. Tedeschi and De Cicco (1953 and 1954) found o-cresol succinate and.gunacol acetate, injected into rats, produced fetal resorption, germinal 11 epithelial degeneration and muscular dystrophy. All these were preventable with simultaneous administration of alpha- tocopherol. Hove (1953) found pyridine administered to tocopherol- deficient rats caused liver damage and death which could be prevented by alpha-tocOpherol, methylene blue and yeast nucleic acids. Pyridine was found to depress the antioxi- dant activity of alpha—tocopherol. Hove (1955) included carbon tetrachloride and sodium sulfate as tocopherol stress factors and found tri-o-cresyl phosphates to interfere with tocopherol absorption from the gut. Sherman and Richards (1960) noted chlorine dioxide-bleached commercial flours, when fed to rats, were able to cause positive dialuric acid hemolysis values indicative of a tocOpherol deficiency. Clinical Indications of TocOpherol Depletion Insane Tocopherol Direct tissue tocopherol analyses provide the most direct indicators of tocopherol depletion. However, the number of different procedures available is indicative of their variable reliability. Determinations for total toco- pherol in use presently are basically of two types. One measures the reducing capacity of ethanolic extracts cf tocopherol in the presence of ferric chloride and alpha- alpha dipyridyl (Quaife 23 gl., 1949). The other method by Duggan (1959) is based upon the ability of tocOpherol ex- tracts to fluoresce. -_...., ,.,_..~ .h. .. ._._..,...¢-_...- -——~ 3. ~- 7 ‘- ' ~ 12 For specific tocopherols, two basic methods are also available. The method of Bro-Rasmussen and Hjarde (1957) depends upon the capacity of chromatographic columns of activated secondary magnesium phosphate to adsorb the tocopherols. These are then selectively eluted depending upon the percentage of ethyl ether in the petroleum ether elutriant. The second and reportedly more reliable method (Edwin 23 al., 1960) incorporates the purification of the tocOpherol extracts on a fluoricil column followed by two dimensional paper curtain chromatography to separate the individual tocopherols. Creatine-Creatinine Excretion The presence of creatine and its anhydride, creatinine, in the urine has been used as an indicator of tocopherol deficiency. Bicknell and Prescot (1948) considered creatin- uria, at least in the rat, to be the first clinical sign of tocopherol deficiency. Although Melville and Hummel (1951) indicated that a creatinuria preceded histological changes in a tocopherol deficiency, they were unable to correlate degrees of creatinuria positively with the severity of the deficiency. Butturini (1949) judged hypercreatinuria to be a nonspecific clinical symptom found in all vitamin defic- iencies. Hove and Harris (1947) with rabbits and Bauer and Berg (1943) with mice demonstrated altered urinary creatinine levels during a tocopherol deficiency: however, Bacigalupo (1952) found tocopherol deficiency in lambs had no effect upon the creatinine excretion. 13 Blood Enzymes Blood enzyme determinations have become diagnostic aids in nutritional myopathies since alterations of some blood enzyme levels were observed in the human following myocardial infarction and viral hepatitis. Possibly instigated by the work of White and Hess (1957) on blood enzymes and muscular dystrophy in the human, Kuttler and Marble (1958) and Blincoe and Dye (1958)found elevated serum glutamic oxalacetic transaminase (SGOT) values in natural and artificially in- duced cases of white muscle disease in calves. Swingle 33 El. (1959) have found similar results in lambs with nutri- tional muscular dystrOphy. Blincoe and Marble (1960) found a significant positive linear correlation between SGOT and serum lactic dehydrogenase levels in lambs having nutritional myopathy induced by feeding cod liver oil. In severe dys- traphy, serum alkaline phosphatase decreased to one-half its normal value. Normal values for SGOT and serum glutamic pyruvic transaminase (SGPT) in domestic animals have been compiled by Cornelius 23 a1. (1959). Only in the dog liver did these workers find an appreciable SGPT activity and likewise only in the dog was there an elevation of SGPT following a hepatic necrosis induced by carbon tetrachloride. Erythrocyte Fragility An increased susceptibility for erythrocytes to hemolyze is indicative of tocopherol depletion. Dialuric acid was found by Christensen and Dam (1951) in Denmark to produce 1 vitro hemolysis of erythrocytes from tocOpherol-deficient 14 rate. This hemolysis was inhibited by alpha-tocOpherol and partially by methylene blue. These workers postulated that alpha-tocopherol inhibited the hemolysis by preventing a free radical, involved in the autoxidation of dialuric acid, from reacting with a substance within the erythrocyte. Rose and GyBrgy (1952) showed intravenously administered dialuric acid to cause intravascular hemolysis. These workers also found that 1.5 micrograms of alpha-tocOpherol added to a substrate of erythrocytes gave complete protection against the hemolytic action of dialuric acid; that in the order given, beta, gamma and delta tocopherols showed a decreasing protective action against dialuric acid and that hydrogen peroxide caused hemolysis to both tocOpherol-deficient and normal erythrocytes. Friedman gt al. (1958) established a bioassay for vitamin E by the dialuric acid hemolysis test and indicated that the activity of dialuric acid is readily destroyed upon contact even with such inert substances as polyethylene. Gitler g£,al. (1958) found selenium, methionine, cystine, methylene blue and butylated hydroxy toluene (BHT) non- effective, under the conditions employed, in altering the susceptibility of tocopherol-deficient erythrocytes to hemo- lyze with dialuric acid. Bunyan 23 gl. (1960) indicated that the presence of methyl groups, ortho to the hydroxy group in tocOpherol, enhanced the lg ylggg potency of the tocopherol in preventing dialuric acid induced hemolysis. Ions found active in this regard were Co, Mn, Sn, Cr04, and _,_..,_. a; 15 Cr207. The former two appeared to act synergisticly with alpha-tocopherol. Alfinslater 23 al. (1961) correlated plasma tocOpherol, erythrocyte tocopherol and erythrocyte fatty—acid compos- ition with the susceptibility of tocopherol-deficient ery- throcytes to dialuric acid hemolysis. Last (1960) indicated that dietary fat plays a more important part in the hemolytic susceptibility than does dietary tocopherol in swine rations. Erythrocyte Survival Marvin ggflgl. (1960) and Horwitt g§,§l. (1960),using Cr51 tagged autologous erythrocytes, demonstrated an accel- erated turnover of erythrocytes in the tocopherol-deficient monkey and man respectively. In monkeys, survival times of 35-49 days were associated with tocopherol deficiency while normal erythrocytes had a survival period of 100 days. Glucose Tolerance Another indirect tocopherol depletion indicator was reported by Mertz and Schwarz (1955) to be an impaired intravenous glucose tolerance. Although not significantly affected by 50 mg. of alpha-tocopherol acetate, 2.15 mg. of Schwarz's Factor 3 fraction increased glucose clearance rate to that of the control animals. In this regard, Horn 23 al. (1955) concluded that tocOpherol protects liver reserves of glycogen. 16 TocOpherol Deficiency Myopathy Biochemical Lesions One of the first biochemical lesions associated with tissue from tocOpherol deficient animals, an increased requirement for oxygen, was described by Victor (1934). Houchin and Mattill (1942) confirmed Victor's work and re- ported that striated muscle from tocopherol-deficient animals required from 125-250% more oxygen than normal muscle and that following oral administration of alpha-toco- pherol, normal oxygen consumption was restored in 22 hours.) Roderuck (1949) also confirmed this increased oxygen require- ment in cavia. Further proof of an increased oxygen require- ment was shown somewhat differently by Teleford 22,5l. (1954) who subjected tocOpherol-deficient, normal and tocopherol- supplemented rabbits to a series of three decompression exposures. During the course of these decompressions, all the tocopherol-deficient animals, one supplemented animal and no normal animals died. ' Bonetti 23,5l. (1952) noted that the content of con- tractile elements (myosin and actomyosin) in tocopherol- deficient rabbit skeletal muscle decreases gradually during the deficiency. Keeler and Young (1961) electrOphoretically' characterized the protein extracts from normal and dystrOphic ovine muscles. Actomyosin, myosin and a rapid moving myoal- bumin were found elevated in extracts from affected longissi- mus dorsi and quadriceps femoris muscles while the myogen fraction was decreased. In blood serum from the affected animals, the alpha globulin fraction increased while beta and 17 gamma globulins decreased. The myoglobin concentrations of both pale and red skeletal muscle of toc0pherol-deficient cavia have been found by Schottelius 33 al. (1959) to decrease fol- lowing 15 days on tocopherol low rations. Dinning and Day (1957), at Arkansas, have shown the tocopherol-deficient monkey to have an increased skeletal muscle DNA and a reduced skeletal muscle creatine. Lawrie (1960) found a rapid post mortem decline in the pH of dys- trOphic longissimus dorsi muscle of swine to an ultimate of 5.11 compared to 5.5 for the normal longissimus dorsi. Pathogenesis The best documented and most convincing study relative to the pathogenesis of toc0pherol deficiency myOpathy has been made by West and Mason (1358) using hamsters. Animals were sacrificed at ten day intervals during an 80-day defi- ciency period and a subsequent 20-day recovery period. Sections were taken from the adductor magnus, sacrospinalis, subscap- ularis, masseter and tongue muscles. In order to observe much longer segments of unsectioned muscle fibers, thin stained spreads of the skeletal muscle from the cheek pouch were pre- pared. The microsCOpic changes were classified as reversible and irreversible. The former were characterized by alignment of muscle nuclei in chain-like rows within the fibers. The phenomenon, called internal rowing, could remair static for indefinite periods. The irreversible injury included changes in this order: focal degeneration of myofibrils C1) 1 which were converted into highly cellular masses composed of surviving muscle nuclei, investing sarcoplasm, macrophages and leukocytes; formation of contraction clots, similar to those produced instantaneously by trauma and coagulation necrosis of segments of fibers. Macrophages then enter to remove the necrotic material. Muscle elements or myoblasts form a syncytium from which reconstitution of the fiber segment occurs. Regeneration occurs, to a lesser extent, through terminal budding or plasmodial outgrowth from viable portions of affected fibers. Later phases of regeneration are more or less indistinguish- able from the sublethal reaction described as internal rowing. Following tocopherol therapy, degenerative changes are promptly prevented, all evidence of previous necrosis is abolished in 5-10 days, and there remain only normal, regenerating and rowed fibers. The latter gradually trans- form into normal fibers and the tissue restoration, at least structurally, appears complete. West and Mason (1958) preferred to consider the prom- inent feature coagulative necrosis rather than Zenker's or ,hyaline degeneration because of the chances for misinterpre- tation in overstained iron and phosphotungstic acid-hematox- ylin sections. Adams 22.9lo (1953), it is interesting to note, did not include tocOpherol deficiency in their list of factors producing waxy, hyaline or Zenker's degeneration in the human, nor did they consider tocOpherol deficiency 19 myopathy a true muscular dystrOphy because, in the latter, no regenerative activity is present. Recognition must be given the fact that all striated muscle does not react alike during tocOpherol deficiency. In this regard, for instance, West and Mason mentioned nothing about the calcium infiltration of muscle fibers in myopathy due to tocopherol deficiency, reported especially in calves by MacDonald glygl. (1952). According to Semenova (1958) the neuropathology assoc- iated with a nutritional myopathy, at least in the rat, is confined to the motor end plates and neuromuscular spindles. Following the early degenerative changes in the muscle, changes occurred which consisted of separation of terminal ramifications together with some thickening and an increase in nuclei at the base of motor end plates. As the myOpathy progressed, the number of nerve endings was reduced and degenerative changes occurred in the neuromuscular spindles. Upon toc0pherol supplementation, the muscle fibers regener- ated and then the nerve endings were restored. Tocopherol Deficiency in Mink Relation to Steaglglg The existing information relative to the tocopherols in mink nutrition, however meager, has evolved almost entirely from studies of steatitis in mink. Natural cases of yellow fat in mink in the United States were first reported by McDermid and Ott (1947). Hartsough and Gorham (1949) 20 considered this disease to be histologically similar to that experimentally produced by Dan (1944) in the rat. According to Hartsough and Gorham, the disease in mink is clinically manifested by a leukocytosis of 25-35,000, an erythrocytOpenia of approximately 3 million and a hemoglo- binuria. The affected mink, usually kits, appear unusually wide through the abdomen, have an unnatural gait, have been ravenous eaters and may die suddenly following a short anorexic period. Morbidity ranges up to 50% and mortality to 75%. The gross pathology consists of lumpy palpable abdominal fat, marked subcutaneous edema, and brownish yellow coloration of the subcutaneous and visceral fat which may have a rancid odor. Less common findings include Spleno- megaly, mesenteric lymphadenitis, mesenteric and omental hyperemia and petechial hemorrhages in the affected adipose tissue. Quortrup at al. (1949) described the micrOSCOpic path- ology of early field cases as a disseminated non-suppurative inflammation of the subcutaneous and visceral fat which is infiltrated primarily with polymorphs. Fat necrosis is mini- mal and the interlobular septae of the adipose tissue are edematous. More advanced steatitis, according to Quortrup, shows fibroblastic proliferation and many macrophages con- taining fat in the inflammatory area. An atrOphic epidermis, often only one cell thick, and dermal edema may also accompany the steatitis. Gorham gt alo (1951) found that 20 mg. tOCOpherol/mink/ day added to a basal ration consisting of 855 fresh frozen 21 fish scrap, 13% commercial mink cereal and 2% brewers yeast were sufficient to prevent any acid-fast pigmentation of adipose depots. Lalor g3 gl. (1951) reported a daily intake of 5 mg. of alphartocOpherol was required to prevent steatitis on rations containing relatively high amounts of trienoic acids as are present in horse fat to the extent of 16%. Dalgaard-Mikkelsen 2;,gl. (1958) demonstrated that 50 mg. tocopherol/kg. of feed, 3% hardened pig fat and methylene blue at the rate of 50-100 mg/kg.of feed. protect against yellow fat even when the diet contains 10% fish oil. They also showed that if the fish oil were limited to 3-4% of the diet, no antioxidants are required to prevent steatitis. I Leekly and Cabell (1959) found that 112 gm. DPPD/ton of ration composed largely of frozen stored fish canner waste prevented steatitis. However, reproductive performance was affected by DPPD. Butylated hydroxytoluene was tested with similar diets and found to prevent steatitis without any reproductive impairment. Relation to Myogathy Zenker's degeneration or "white heart" disease was reported by Benson (1959) to be most common in older mink and tocOpherol supplementation of mink rations provided effective prevention of the cardiac degeneration. TocOpherol Deficiency in Swine Reproductive Failure Early investigations relative to the tocopherols in swine 22 nutrition were designed to demonstrate the effectiveness of tocopherol in preventing reproductive failures in farm swine. In this regard, Bay and Vogt-Miller (1934) found wheat germ oil effective in combating sterility in sows. Considerably later, Carpenter (1949) found no benefit from supplementing the sow's ration with wheat germ oil shortly before farrowing; however, supplementation prior to concep- tion and during gestation did improve reproductive performance and the livability of the pigs. Garton and Naftalin (1953) produced an exudative diathesis of swine with tocopherol- low rations containing lard and cod liver oil as the fat BOUPC O 8 e Unstable Pork With regard to the role of tocopherols in stabilizing animal tissues, Burr (1945) and Watts 23 al. (1948) found supplemental tocOpherol decreased the susceptibility of pork fat to rancidity. Fish-product-rich swine rations became associated with unstable pork fat which Breirem (1952) attributed to oxidative destruction of the available toco- pherol. Dammers 23 gl. (1958) found 40 mg. tocOpherol/day adequate for maximum keeping quality of pork fat. Zaehringer 23 El, (1959) found thiobarbituric acid values of frozen pork varied inversely with the number of weeks on a tocOpherol Supplemented ration. The maximum rate of alpha-tocOpherol supplementation was 530 IU/day for six weeks prior to s1 aughter . 23 Steatitis As the antioxidant role of the toc0pherols became realized, cod liver oil became the most common tOCOpherol depleting agent in experimental tocopherol deficiencies and porcine steatitis became associated with these deficiencies. Robinson and Coey (1951) fed a tocOpherol-low ration supple- mented weekly with five ounces of cod liver oil and produced a brown fat which was prevented by 50 mg. tocopherol/day. These workers also found the iodine values of carcass fat were elevated in proportion to the cod liver oil intake. Gorham EL gl. (1951) and Davis and Gorham (1954) pro- duced yellow fat in swine with a diet containing 85% fish scrap and found that 500 mg. tocOpherol/day prevented the deposition of the acid fast pigment. Burr (1945) had postu- lated that the tocopherols act through an oxidase system to effect oxygen uptake of tissue and in turn determine the stability of meat products. Garton and Duncan (1954) raised pigs from weaning on tocOpherol-low rations containing up to 50% cod liver oil and lard. The resulting fats were dark brown and contained the absorbed lard and oil practically unchanged. Gedigk and Fischer (1959) postulated the origin of the lipid pigments, of the depot fats during tocOpherol deficiency. They stated that, in the absence of tocOpherol, the unsaturated fats accumulate gradually in the area of protein containing metabolically active cytOplasmic foci and are then oxidized and polymerized. The lipid pigments were found in macrophages 24 following their absorption of fat containing tissue parti- cles. Necrotic Hepatitis A naturally occurring disease, necrotic hepatitis of swine, which Hjarre (1951) reported to affect ten percent of all the pigs seen annually at the Stockholm Veterinary Medical Institute, is clinically similar to that reported by Dam and Granados (1951) in vitamin E deficient rats and to that re- ported by Naftalin and Howie (1949) to result from the stresses of a cold and damp environment upon swine. Obel (1953) has extensively studied and experimentally produced the disease to which she has given the name "hepatosis diaetetica" (h.d.). It occurs in pigs under six weeks of age and is associated with anemia, discolored adipose tissue, subcutaneous edema, moderate ascites, gastric ulcers, renal and splenic congestion, mottled liver and waxy degeneration of skeletal and cardiac musculature. Histologically, the liver changes include centrolobular necrosis, hemorrhage, dystrophic calcification of the hepatic cells, anoxic vacuoles at the periphery of the lobules, purulent cholangiolitis and thrombi in the bile canaliculi. Reparative processes appear early and consist of neutrophilic and lymphocytic infiltration, bile duct proliferation and apparent hepatic cell regeneration. Histologically associated with hepatosis diaetetica is a steatitis, fibrinoid degeneration of medium sized arteries, nephrosis of Henle's loop and edematous lymph- adenopathy. 25 Obel produced h.d. with a basal diet of 73% carbohydrates, 18% dried brewer's yeast, 6% cod liver oil and 3% minerals. Supplementation of the basal ration with tocopherol failed to elicit a protective effect on the liver but if lard re- placed the cod liver oil and alpha-tocopherol were added, h.d. was prevented. With neither torula yeast or baker's yeast could h.d. be produced. Supplementation of the basal diet with .5% methionine or cystine prevented the h.d. Unfortunately, the significance of selenium was not recog- nized at the time of Obel's experiments. She considered hepatosis diaetetica to be caused by a combination of toxic products, reflex anoxia and diminished detoxifying property of the liver due to limited sulfur-containing amino acids and tocOpherol. Hove and Seibold (1955) described a fatal liver necrosis which developed in growing swine fed a diet deficient in vitamin E and containing 6% protein (soybean meal) and 2% cod liver oil. Deaths were attributed to acute hemorrhagic liver necrosis and the liver fat had less concentrations of linoleic and pentaenoic acids than did the liver fat from tocOpherol supplemented animals. In New Zealand, Dodd and Newling (1960) reported a natural outbreak of hepatosis diaetetica in swine receiving a diet consisting of cheese' whey, barley, meat meal and a fish liver oil supplement of 8 ounces/500 gallons of whey. Myopathy Forbes and Draper (1957) experimentally produced skeletal 26 and cardiac muscle degeneration in baby pigs receiving semi- purified type rations containing 20% methionine-supplemented casein, 45% cerelose, 29% vitamin E free lard, minerals and vitamins. Lannek 93 gl. (1961) in Sweden demonstrated that a stress factor such as cod liver oil is required to exper- imentally provoke muscular dystrOphy in swine and in field cases of the dystrOphy in swine; they' considered the stress factor to be a toxic substance in the natural grains. General Characteristics Pelligrini (1958) characterized the manifestations of vitamin E deficiency in growing pigs receiving a semipurified type, 32-45% torula yeast ration from 10 days of age and studied the effectiveness of alpha-tocopherol acetate, sodium selenite and L cystine in preventing the deficiency symptoms. A fatal liver necrosis accompanied by degeneration of the semitendinosus, semimembranosus and latissimus dorsi muscles, developed in growing pigs after 50-70 days on the basal diet. L-cystine supplementation prevented the liver necrosis, but not the muscle degeneration. Fifty mg. alpha-tocOpherol acetate and .045 p.p.m. sodium selenite prevented deaths and the pathology of liver and muscles. Rates of gain and feed efficiency were satisfactory in all groups prior to death. Occurring with lesser frequency in the fatal cases were splenic infarcts, portal triad endarteritis, bile duct proliferation, ascites, hydrotherax, lymphadenitis and hyper- emia of the intestines. In a similar study in swine, Eggert 33 gl. (1957) reported sudden deaths, liver necrosis, steatitis, 27 hemorrhagic lymphadenitis, and hemorrhagic gastroenteritis, all of which were prevented by 40 p.p.m. alpha-tocopherol acetate or 1 p.p.m. sodium selenite. Plasma Enzymes With reference to plasma enzymes and tocOpherol defic- ient swine, Orstadius 2;,al. (1959) demonstrated elevated SGOT and SGPT and ornithine-carbamyl transferase values in cases of spontaneous liver dystrOphy of swine while spontan- eous muscular dystrOphy provoked only an elevation of the transaminases. Augustinsson 33 gl. (1960) showed aryles- terase, a phenotypically characteristic enzyme of swine plasma, to be unaffected by an induced hepatic dystrophy preventable by sodium selenite. Erythrocyte Fraglllly Fbrbes and Draper (1957) and Leat (1961) reported that tocOpherol deficiency in swine was not accompanied by an increased susceptibility of the erythrocytes to hemolyze with dialuric acid. Leat (1961), however, reported that a spontaneous hemolysis in .9% saline occurred earlier in the erythrocytes from the animals given 2% olive oil than from animals on a low-fat diet. Only once though was this hemolysis affected by tocOpherol supplementation. The material presented in this review in no way repre- sents a complete coverage of the available literature rela- tive to the tocopherols in nutrition. Rather, it is intended 28 to highlight some current concepts relating especially to the metabolic roles of tocopherol and selenium and to review the literature most pertinent to the experiments reported herein. MINE EXPERIMENTS Materials and Methods Experiment I Forty six-weeks-old dark male mink kits were obtained from the Michigan State University Fur Project on June 11, 1959. and placed on the semipurified tocOpherol-deficient ration described in Table 1. Table 1.--Composition of the tocOpherol-deficient diet. W Ingredient Per Cent Vitamin free casein 16.0 Isolated soybassay proteina 8.0 Torula yeast 16.0 Sucrose ' 25.5 Melecularly distilled lardc d 24.0 Solka-Floc (alpha-cellulose) 6.0 Phillips and Hart Salt Mix (IV)° 4.0 Amino acid and vitamin supplement 0.5 aFrom Archer-Daniels-Midland Company, 2795 Sharon Rd., Cincignati, Ohio. Furnished by Lake States Yeast and Chemical Division of St. Regis Paper Company, Rhinelander, Wisconsin. °Furnished by Distillation Products Industries, Rochester, New Yark. From Brown-Company, 150 Causeway St., Boston 14, Mass. 9Phillips and Hart (1935). The amino acid and vitamin supplement for this ration was prepared to furnish the constituents at rates shown in Table 2, page 30. The ration was prepared dry in a Hobart mixer and stored in polyethylene bags under refrigeration at all times. Quantities sufficient for each day's feeding were removed from the refrigerator immediately prior to feeding, mixed with water to the consistency of mashed potatoes and fed on 29 30 Table 2.--Amino acid and vitamin supplementation rate. Component Rate* Component Rate* Arginine HCl 250.0 Inositol 50.0 DL methionine 100.0 p-amino benzoic acid 100.0 Riboflavin 1.0 Folic acid 0.2 Pyridoxine 0.5 Cyanocobalamin 0.16 Calcium pantothenate 3.6 Biotin 0.05 Nicotinic acid 5.0 Vitamin A acetate 0.52 Choline chloride 400.0 Vitamin D2 0.01 Thiamine HCl 0.5 Menadione 0.5 *"g.7100 gm. dry diet metal feed doors once per day with additional feedings as indicated. The feed doors were scraped regularly to prevent the mink from having access to any rancid feed. All animals remained on the above ration for four weeks at which time eight animals were assigned to lot A and their basal ration was supplemented with alpha-tocOpherol at the rate of 150 p.p.m. on a dry basis. Four weeks later, when sudden deaths were frequent among the toc0pherol-depleted animals, lots B, C and D were established with eight animals per lot. The basal rations for lots B and C were supplemented with alpha-tocopherol at the levels of 50 and 25 p.p.m. respectively. The basal ration for lot D was supplemented with selenium, as sodium selenite, at the level of 1 p.p.m. Eight other kits of the same age but which had been adapted to a semipurified type ration on another experiment were acquired late in July. These kits were placed on the tocopherol depletion diet along with the remaining unallotted tocopherol-deficient kits from the original forty animals. 31 These unallotted animals were used as replacement animals. All animals were observed daily and at the time of weighing each week, all were examined for indications of urinary incontinence. Biweekly’ blood samples of 10-12 ml. were taken via cardiac puncture from one-half of the animals in each lot, thereby permitting biweekly' analyses but sub- jecting each animal to the bleeding stress only once monthly. The differential counts, total white counts, packed cell volumes and hemoglobin values were obtained from the whole blood samples according to the procedures described by Coffin (1953). Near the termination of the experiment, a few preliminary red blood cell fragility determinations were made in saline according to the procedure of Dacie 23 gl. (1938). The following determinations were made on the blood serum fraction: total serum tocOpherol according to a method of Quaife 22 gl. (1949) modified to a macro-technique, serum glutamic-oxalacetic transaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT) according to the methods of Reitman and Frankel (1957) as outlined in Sigma Chemical Company's 1959 Technical Bulletin No. 505, and paper electro- phoresis analyses of serum proteins on a Spinco Model R paper electrOphoresis system at room temperature. Biweekly 24-hour urine samples were obtained and ana- lyzed for creatine, creatinine and specific gravity according to the procedures of Coffin (1953). Surface tension deter- minations were also made on the urine samples using a du Nouy tensiometer. 32 Animals that died or were sacrificed were examined via standard necrOpsy procedures. Sections from the following organs were preserved in acetate-buffered ten per cent formalin for histological examination: internal intercostal, adductor magnus and cardiac muscles, trachea, lung, parotid salivary gland, small intestine, pancreas, liver, kidney, adrenal gland, ureter, urinary bladder, urethra and any other tissue considered important at the time of necrOpsy. Testicular tissue was preserved in Bouin's fluid. Appropriate sections from each of the tissues were stained with hematoxylin and eosin for general characteristics and with Von Kossa's stain for demonstrating calcium deposition within the tissue. The histological procedures followed were according to the Armed Forces Institute of Pathology Manual of Histologic and Special Staining Technics. II (0 ..:'Yr‘ r_ *5. w D“ d—v'l ‘ave .Aue-vet Thirty nhxrweeks-old brown male mink kits were pur- chased from the J. S. Dyer Mink Ranch, Delta River Rd., Lansing, Michigan, on June 27, 1961, and were placed on the semipurified toCOpherol-deficient ration shown in Table 3, page 33. The ration was prepared as described for Experiment I. Three weeks later, on July 18, 1961, when the mink had become fully accustomed to the ration, the mink were assigned to the experimental lots as indicated in Table 4, also on page 33. The ‘biweekly hematological and serological analyses completed in this experiment were the same as those completed 33 Table 3.--Composition of the tocOpherol-deficient diet. m Ingredient Per Cent Vitamin free casein 8.0 Isolated soy assay protein 16.0 Torula yeast* 20.0 Sucrose 26.5 Molecularly distilled lard* 20.0 Solka-Floc* 5.0 Phillips and Hart Salt Mix (IV)* 4.0 Amino acid and vitamin supplement* 0.5 *As previously described in Experiment I. Table 4.--Assignment to experimental lots. W Lot Number of Mink Ration Per Lot A 10 Basal tocOpherol deficient B 8 Basal plus eight per cent cod liver oil to replace an equal amount of lard. C ' 8 Basal plus .1 p.p.m. selenium as sodium selenite. D 4 Basal plus 25 p.p.m. alpha- tocopherol. _ in Experiment I. Red blood cell fragility determinations were made during the entire course of this experiment according to the method described by Dacie (1953) but modified to in- clude a 48-hour refrigeration period. This was found neces- sary in Experiment I to demonstrate the increased fragility as a layering hemolysis (LH) of the erythrocytes from the tocopherol-deficient mink. The onset of LH was compared chnxkflogically with the onset of alterations in other hematological and serological determination values. Dialuric acid hemolysis determinations 34 by the method of Friedman 23,3l. (1958) were also conducted routinely on the erythrocytes from the tocopherol-deficient and supplemented animals during the experiment. Near the termination of this experiment, the ability of the various lots of mink to produce antibody in response to injected antigen was studied. Four mink from each group were given intraperitoneal injections of Salmonella pullorum antigen daily for six days. The amount of antigen injected was determined by the formula Lbs bod wt x e cent blood volume _ 10 x dilution of the antigen 7 Amt. of antigen. The blood volume was estimated at seven per cent and the antigen was diluted 1/80 with sterile saline. After the last injection of antigen, all animals in the study were bled to obtain post-antigen-injection serum samples. The agglu- tination titers of these samples, together with those of the pre-antigen-injection serum obtained on the last routine bleeding before this study was started were determined via the method of Stafseth at s_l_. (1959). Also, at the termination of the experiment, the ability of an orally administered commercial antioxidant, ethoxyquin (Santoguin)*; to eliminate the LH of erythrocytes from tocOpherol-deficient mink was studied. The results of this study were compared to those obtained from a study of the rapidity with which oral alpha-tocOpherol was able to elim- inate LH in previously tocopherol-deficient mink. The animals for this experiment, unlike the first experiment which ¥ *Monsanto Chemical Company, St. Louis, Missouri. 35 was conducted at the Michigan State Fur Project area, had to be maintained at the Dyer Mink Ranch. Facilities were not available there for collecting urine samples: therefore, the urinalyses conducted during Experiment I were not repeated. Both formalin and Zenker's fixatives were used as general fixatives for tissues from this experiment and sections of liver were fixed in Carnoy's fixative to be later stained with Best's carmine for glycogen. The same tissues taken at necropsy in Experiment I were also taken in Exper- iment II. Both the formalin and Zenker's fixed tissues were stained with hematoxylin and eosin for general char- acteristics. Appropriate sections were stained with Best's carmine, Von Kossa, periodic acid-Schiff, Sudan IV and Ziehl-Neelsen stains. Results Experiment I Growth The data on weight gains for the tocopherol-deficient, tocOpherol-supplemented and tocopherol-deficient-selenium- supplemented mink for the course of the experiment are given in Table 5. The surviving tocopherol-deficient mink at 102 days of age had grown as rapidly as the tocopherol-supplemented animals fed 150 p.p.m. alpha-tocOpherol. Even follOwing tocopherol supplementation in lots B and C and selenium sup- plementation of lot D, no significant growth differences were observed by 147 days of age. Table 5.--Average weights in grams of tocOpherol-deficient mink of different ages and fellowing their tocopherol and selenium supplementation. Lo—t 'j'" 'Feat—men't" ' ' "" """' " _ -Da—ys r' A‘s" " ' 41 192r' "_"'11IZ W A 150 p-P.m. tocopgerol 275 (0) 984 (34) 1193—Ci9) B 50 p.p.m. tocopherol 285 (O) 1015 (0) 1136 (45) C 25 p.p.m. tocopherol 294 (O) 968 (O) 1258 (45) D 1 p.p.m. selenium 285 (0) 1010 (0) 1170 (o) 45* Values in ( ) are number of days of tocOpherol supple- mentation. *value indicates number of days of selenium supplementa- tion at 1 p.p.m. Mortality Twenty animals died during the experiment and the cir- cumstances pertaining to their deaths are presented in Table 6. 36 37 0f the animals that died following a stress factor, three had grossly visible myopathy. Of the fifteen tocOpherol- deficient animals to succumb during the eXperiment, five died within a three-day period after approximately 70 days on the tocOpherol-deficient regimen. No animals died due to a tocopherol deficiency after their tocopherol-deficient rations were supplemented with as little as 25 p.p.m. of alpha-tocOpherol or with 1 p.p.m. of selenium as sodium selenite. Table 6.--Probable causes and number of deaths among toco- pherol-deficient and supplemented mink receiving semipurified type rations. m Probable cause of death Number of deaths for each statug Deficient_ Supplemented Stress of bleeding procedure 3 Stress of metabolism cage 4 Extensive myopathy 2 Urinary calculi 1 Cardiac tamponade 2 Undetermined 3 UMIIII Hematology Total white blood cell counts, packed cell volumes and hemoglobin determinations were not significantly different among the experimental groups during the experiment. Values for each determination, however, increased gradually during the course of the experiment as had been observed in a previous mink experiment utilizing similar rations. Maximum values were reached when the mink were between 116 and 137 days of age. The data are recorded in Tables 7, 8, and 9, pages 38, 39, and 40. 38 'I .poa some new codempnoseHQQSm .aoaao pamedmamn obaaeeamma no panama omen came omen omen om_mw omea omen 0mm“ seam mm.m mmmm meme mmmm a wasp emmm meme ewme n oe.« omo.« ones same case omen omen omen .m.m mmom comm some poem * mmom comm ooem mean 0 1 o_eP« ommw Odo—w omne came omma oven oeom omee anew e_mm meme a amps m__e mace mmen m coop“ on" oemw 00m.“ ommpw onhn Ohm“ o—efl mPNm meme ewme ooeo «mom cone omen a cmme comm < uwm es. mm. an. m_. 0.. mo. 11 mm mm es om 111 om< no name eon OAQV OEOQOQ P on azdcoaem one .on .s.d.o mm .Amv .a.a.a om .Adv .a.d.a om— um pceaeauec Hoaoncooop you .a.a.o\mac:oo epheomoca :m6211.~ Hoaonaooopumndam no“: coflpmpooseadaom macs» wcfizoaaom can mean manna 39 .poa mode you oodumpocaoaddom ohOthQ Ufidflgfimuh seapomamoh we assume o..« m.o« m.o« m..« m.o« m.o« m._« m.o« 0.»: m.mm m.nm o.mm e._m s n.me o.¢e m.oe e.mm a m.—« v.0..." m.o« n.mu 0.3" n.m« m...“ ¢.—fl m.>q p..m _.mm m.om m.mm * >.>q m.oe m._e >.mm o 1 e._« m..« o._« m._« e.n« m._« a..« e.me m.mm ..em w.em m.mm s m.ee e.me e.oe m.mm m c..« m.o« m.o« m.o« Show >._« m.o« o..« _.me m.mm m.mm m..m e.mm o.om e.oe . _.om1 _.sn e .>< an. mm. um. wp— 0?. mo. mm mm c» ow , 11 ow< we when #04 .AQV .s.a.d F on snacmamm one gov .s.d.a mm .Amv .e.a.a om..A¢V .e.a.a om. no actondooouuenaac so“: scammecmscaddzm mums» mcdzofiaou can mean vcefioaumpuaommsnoooa pom fiasco away mmadao> dame pagoda om0211.w canoe 40 .Aoaao camccmpmw .pOH some you codvmadosoaaodm e>aaeoanoa no uampm* _._« w.o« N.OH m.—« m.ou m.on m._« w.o« m.m— m.>. w.w— ..S_ «.m— * m.>. e.¢_ m.w— m.—— a m.on m.ow w.ow m._« p.pw m.mn _.—H ¢.0H m.m_ n.>— m.m. n.». 0.0— * p.p— m.m_ o... o.—— o 1 w.o« v.0“ n.0« m.ow m.o« n.o« ¢.o« m.o...h.o. m.m_ .—.m_ m.mp * m.b. b.<. n.m— m.o_ m n.0w n.0n c.o« n.0fl ¢.o« v.0“ w.o« ¢.o« o.w.11e.m— <.w' v.5. m.m, ".mp m.m— * m.m_ 0.0. 4 .>< e>_ mm. an. m.. 0.. mo. mm om an om. mm< no when veg eAQv eBeQeQ — 96 addendum one .on .s.a.a mm . my .s.a.a om .Adv .a.a.o om. pm Homage noOOQImnaHd and: ceapmasmsea one hams» mausoaaom one made anoaoaueo Hoaonaoooa you Aeooan .Hs ooF\awv menace canoamosen ucozuu.m canes 41 Data relative to the differential counts during the experiment are summarized in Table 10, page 42. The pre- and post-tocOpherol and selenium supplementation values show no characteristics indicative of a differential response to these supplements. The data for all lets do indicate a shift in the lymphocyte/neutrophil ratios from 49:47 at 60 days of age to 34:63 at 153 days of age. In the preliminary study of erythrocyte fragility, after the recommended 24-hour refrigeration period, no differences were observed between the sets of tubes with erythrocytes I from the tocopherol-supplemented animals and the sets of tubes with erythrocytes from the tocopherol-deficient animals. On occasion, however, sets of these fragility tubes were observed following a 48-hour refrigeration period. Little change had taken place in the set of control tubes (see Figure 1, page 43) containing the tocopherol-supplemented erythrocytes. Intact erythrocytes were still visible at the bottoms of the tubes of nearly isotonic saline while hemoly- sis occurred gradually in the more hypotonic saline. In tubes of equally hypotonic saline containing erythro— cytes from the tocopherol-deficient mink, lysis had taken place and the lysed fraction remained concentrated (layered) at the bottoms of the tubes as illustrated in Figure 2 (see page 43). It was noted that the lowest level of alpha- tocOpherol supplementation (25 p.p.m.) was adequate to pro- tect the erythrocytes from this type of hemolysis and that 1 p.p.m. of selenium, as sodium selenite, did not protect the erythrocytes from this type of hemolysis. 42 .mvoa Obdfiomnmoen 3.3 boy Godpdvcmamfinadm-mo fudvm... m. 0 mm. mm». mm. - 0 0 10M. 0 mfl.nmonsm 0.. 0.. m. 0.. 0.. 0.. 0.. m. m. maanoooamom 0.. mm. mm. mm. mm. mm. 0.. 0.m 0.e eeoaooeoz 0.0m 0.0m 0.0m o.mm o..¢ o.>m 0.0n o.m¢ o.me manhoonaamq a 0.0 0.0 0.0 0.» 0.0 0.0 0.0 0.e 0.0 assessmeesoz 0.0m 0.em 0.a0 0.0m 0.me * 0.00 0.00 0..0 0.0e emeoeamom maanaoaaoez m. 0 0 0.m mm. 0 0 m. 00. oaanaomem 0.0 0.. 0.0 0.m 0.. 0.0 0.. 0.0 mm. nHHeooeeoom 0.. 0 mm. o o.— m. w. 0.0 o.m mophoocoz 0.00 0.:0 0.0m 0..e 0.an 0.00 0..m 0.ee 0..m monsooeosaq 0 o.m o.m 0.0 o.m o.m o.» 0.. o.m o.m popnoamemqoz 0.00 0..0 0.00 0.00 0.mm . 0.mm 0.00 0.04 0.0a ooeoosmem mafindoaasoz a. 0 0 m. m. 0 0 mm. 0 maaooomem 0.m 0.m me. 0 0.. 0.0 0.0 0.m 0.. mflanoos.nom 0.. 0 m. 0 0 m. 0.. 0.0 0.: monsoocoa 0.0a 0.00 0.00 0.00 0.00 0.00 0.00 0.me 0.04 eoeaoomosan m 0.e 0.m 0.0 0.0 0.5 0.0 oflm 0.0 0.0 eopcoswomooz 0.00 0.0a 0.00 0.00 0.00 e 0.00 0.00 0.0a 0.0a eoesesmem madmaoaasoz mm. mm. mm. 0.m 0 0 0. mm. ea.soomsm 0.m 0.. 0.m m». 0.0 0.e 0.0 mm. eaanooa.eom mm. mm. 0 me. 0. 0.. 0.0 0.0 neeaooeoz e 0.mm 0.0m 0.ee 0.ee 0.00 0.00 0.04 0.00 meeaooeosan a 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 eepoesmonooz e 0.0: 0.00 0.0a 0.0: 0..m 0.00 e 0.00 0.sm oeeoeamem oaasoonesez on. mm. an. 0.. 0.. mo. 00 00 en 00 om< no mama camooxsmq we; AQV .e.a.d . pm enacoaom one .on .a.d.d mm .Amv .e.o.a om .Amv .e.d.a om. am Hoaondoeoaumnoam and: sodaeuuoscaoasm pause wcdzoaaom one news asoaodmoouaoaonaooop Beau mpcsoo opheoxsoa Heapooaomuflo no mamesom11.o. oHnee 43 5' 1 14" ‘9 RE. 0.70 0.65 0.60 0.55 0.50 0.45 Per Cent Saline Figure 1.--Tocopherol-supplemented mink erythrocyte fragility test following 48-hour refrigeration period. 0.70 0.65 0.60 0.55 0.50 0.45 Per Cent Saline Figure 2.--Tocopherol-deficient mink erythrocyte fragility test following 48-hour refrigeration period. Note lysed fraction concentrated (layered) at bottom of tubes. 44 Serology The pre- and post-tocopherol and selenium supplementation data from the electrOphoretic studies are presented in Table 11, page 45, and reveal no consistent pattern alter- ations associated with either supplement. However, as in the hematology data, trends associated with the age of mink were noted. Combining the data from all the lots during the course of the experiment, the per cent albumin increased from 48.6 to 60.1, the per cent alpha globulin decreased from 20.3 to 11.4, the per cent beta globulin decreased from 22.8 to 14.4 and the per cent gamma globulin increased from 8.2 to 14.3. The alterations in the average serum values for glutamic- oxalacetic and glutamic-pyruvic transaminase before and fol- lowing tocOpherol or selenium supplementation are given in Table 12, page 47. Considered of significance in the SGOT data are the elevated values for lots B, C and D on day 116. Sera for these determinations were obtained only six days after the start of supplementation for the respective lots. Insufficient time had elapsed to correct the probable myOpathy and thereby lower the SGOT values as was accomplished by the succeeding bleeding. The total serum toc0pherol values for the experimental animals are presented in Table 13, page 48. An immediate response followed the tocOpherol supplementation of lot A while a more moderate response followed the supplementation of lots B and C. Values of less than 50 micrograms/100 m1. of serum were observed during the time the sudden deaths among the tocopherol-deficient animals were most frequent. 174 _ 153 137 116 *— (D). g their supplementation with 110 Days of Age 50 p.p.m. (B), 25 p.p.m. (C) and 88 102 epome (A), 86 selenium at 1 p.p.m. deficient mink and followin 74 Lot 6O —— Table 11.-Average per cents for albumin and globulin fractions of serum from tocopherol- alpha-tocopherol at 150 p Serum Fraction % C0 0 0 Ln 0 e e e \o 4- r- -e 1.0 L0 in LG C‘OOON 1.000" e e e. e e e e O i .10 .03 .16 .12 .01 Creatinine(‘) 1.40 1.54 * 1.47 1.35 1.41 1.43 10.16 0.15 0.14 0.16 0.28 1.61 1.63 1.55 1.35 * 1.51 1.53 10.06 0.26 0.17 0.12 0.26 1.59 2.29 1.75 1.61 * 2.65 1.98 10.12 0.29 0.13 0.25 1.1 1.86 1.44 2.12 1.53 * 1.76 1.74 10.12 0.12 0.14 0.18 0.10 *Start of supplementation of respective lots. (1)Mg./100 gm. body weight/24 hours. tStandard error. 50 Table 15.--Average specific gravity and surface tension values for urine from tocopherol-deficient mink and following their supplementation with alpha-t000pherol at 150 p.p.m. (A) 50 p.p.m. (B), 25 p.p.m. (C) and selenium at 1 p.p.m. (D . m Deter- Lot Days of Age Av. mination 67 81 86' 95 109 110 143 Specific A 1.061 1.040 * 1.029 1.035 1.044 1.042 Gravity I .003 .006 .004 .005 .006 (gm./ml.) B 1.052 1.036 1.028 1.035 * 1.047 1.040 t .005 .002 .004 .006 .009 C 1.056 1.042 1.032 1.044 * 1.038 1.042 I .002 .008 .009 .007 .011 D 1.058 1.042 1.049 1.046 1.051 1.049 I .003 .004 .007 .003 .005 Surface A 51.6 45.2 * 39.8 45.2 46.8 45.7 Tension I 0.6 2.8 1.1 0.7 1.2 (dynes/cm.) B 51.2 42.9 39.2 46.5 43.9 44.7 I 1.0 1.4 1.0 1.3 1.8 C 48.2 43.4 47.3 45.3 49.9 46.8 I 0.6 0.6 0.6 1.6 2.7 D 49.9 47.7 47.0 46.8 49.8 48.2 I 1.0 1.4 3.1 0.7 0.9 __ tStandard error. _W*Start of supplementation of respective lots. 51 Table 16.--Incidence of transient and chronic urinary incon- tinence in mink fed purified type rations supplemented with alpha-tocOpherol at 150 p.p.m. (A), 50 p.p.m. (B), 25 p.p.m. (C) and selenium at 1 p.p.m. (D). Lot Duration of Number of Incontinent Mink Respective Supplement Transient Chronic A 79 days 5 1 B 45 " 4 1 c 45 " 3 1 D 45 " 5 2 incontinence in mink. Although the experimental average for the urine surface tension in lot D was 2.5 dynes greater than the average for lot A, no consistent alterations in surface tension followed the tocOpherol supplementation of lots B or C or the selenium supplementation of lot D. Symptomatology Three t000pherol-deficient mink that died with gross evidence of myopathy showed ante-mortem symptoms. One had lost weight during the last weigh period, one had been . anorexic for 24 hours prior to death and the third had a partial posterior paralysis. Tocopherol-deficient mink without gross evidence of myopathy at necropsy manifested no unusual physical symptoms. Gross Pathglggy Skeletal myopathy and what appeared to be a peri- lobular fatty infiltration of the liver were observed with equal regularity in the tocOpherol-deficient mink at necr0psy. The my0pathy involved the internal intercostal muscles (Figure 3, page 55). the insertion and of the adductor 52 muscles (Figure 4, page 56) and the diaphragm. Although the myopathy was usually bilateral, some unilateral lesions were observed in the intercostal muscles. Gross cardiac myopathy was observed in two tocopherol- deficient mink. One case was observed on the endocardial surface of the right ventricle (Figure 10, page 61) and the second case was visible on the epicardial surface of the right ventricle near the apex. Histogathology Skeletal muscle.--Three phases of skeletal my0pathy were observed in the tocOpherol-deficient mink. The first phase was characterized by swollen, differentially stained muscle fibers, vacuolar degeneration (Figure 5, page 57) and/or an increased number of irregularly spaced sarcolemmal nuclei (Figure 6, page 57). In the second phase, illustrated in Figure 7, page 58, the previous features were present along with an extensive nuclear element composed primarily of sarcolemmal nuclei and myoblasts. Lymphocytes and myophages were often present. The third or reparative phase was char- acterized by focal calcium deposits amid normal muscle fibers (Figure 8, page 59). Occasionally the myophagic reaction and calcification of individual myofibrils shown in Figure 9 (page 60) were observed. While gross evidence of myopathy was more frequent in Idle internal intercostal muscles, microscOpic lesions were Observed with greater frequency in the adductor muscles. The increase in sarcolemmal nuclei was more characteristic 53 of myopathy of the adductors than of the internal inter- costals or diaphragm. Internal nuclear rowing was not a prominent feature of skeletal myopathy in mink. Cardiac_muscle.--Early cardiac myopathy was distinguished by a relative increase in the number of internal nuclei which showed a tendency to row. Advanced cardiac my0pathy (Figures 11 and 12, pages 61 and 62) was characterized by calcified necrotic foci immediately adjacent to normal cardiac muscle. No intermediate stages of cardiac myOpathy were observed. llyg§.--The perilobular infiltration of fat was con- firmed Upon histopathological examination. In addition, there was centrolobular congestion and the veins of the portal triads were unusually large. Tococherol-deficient- selenium-supplemented mink sacrificed at the end of the experiment had a lymphocytic infiltration around the portal triads. Kidney.--Coagulation necrosis of the proximal and distal convoluted tubules was observed in mink with extensive myop- athy. T000pherol-deficient mink with early myOpathy had hemorrhagic peritubular foci in the renal cortex and medulla. Hemosiderin was evident in the cytoplasm of the proximal and distal convoluted tubule cells. A rather homog- enous sero-sanguinous fluid was present in the intertubular Spaces of the kidneys of sacrificed tocopherol-deficient Wink and around Bowman's capsules of the sacrificed tocopherol- deficient-selenium-supplemented mink. Bowman's capsules were cfiften distended with a proteinaceous material which appeared to 54 have produced some glomerular atrOphy. There was evidence of hyperplasia of the Juxta-glomerular apparatus cells of one mink that had an early myOpathy. Adrenal gland.--A basophilic reaction of the acinar' cells of the medulla, a sero-sanguinous exudate at the cor-- tico-medullary Junction and focal necrosis with calcification of the cortex (Figure 13, page 63) were observed and believed to have resulted from tocOpherol deficiency. Adipose tissue.--Mink that survived a prolonged toco- pherol deficiency had a form of steatitis illustrated in Figure 14 (page 64). Tiny non-acid-fast drOplets were present in the interstitial spaces of the adipose tissue. LEEE§-"A pneumonitis (Figure 15, page 64) was present in all the experimental animals but was more extensive in the tocOpherol-deficient mink. This pneumonitis was accompanied by atelectasis, emphysema and congestion of the alveolar capillaries. 55 Figure 3.--Bilatera1 internal intercostal myopathy in a tocopherol-deficient mink. 56 Figure 4.--Necrosis near the insertion end of an adductor from a tocopherol-deficient mink. Figure 5.--Vacuolar degeneration of internal intercostal muscle fibers from a tocopherol- deficient mink. (x660) Figure 6.--Sarcolemma1 proliferation in an adductor from a tocopherol-deficient mink. x300 58 Figure 7.--Adductor from a tocopherol-deficient mink; A. Swollen, differentially stained fibers; B. Vacuolar degeneration; and C. Prolifer- ating sarcolemmal cells and myoblasts. (x350) 59 Figure 8.--Calcified foci (black) in an adductor from a tocOpherol-deficient mink. (Von Kossa 1350) Figure 9.--Interna1 intercostal muscle from a tocopherol-deficient mink; A. Early myo- fibrillar calcification; and B. M ophagia of calcifying muscle fiber. (x5#0{ 61 Figure 10.--Ca1cified necrotic right—ventricular myocarditis of a toc0pherol-deficient mink. Endo- cardial surface is exposed. Figure 11.--Focal calcified necrotic myocarditis of a tocopherol-deficient mink. (x320) 62 Figure 12.--Extensive calcified necrotic myo- carditis of a tocopherol-deficient mink. (x320) 63 Figure 13.--Calcified necrotic focus in the adrenal cortex of a tocOpherol-deficient mink. x750 Figure 14.--Steatitis of a mink made chronically tocopherol-deficient without unsaturated fatty acid supplementation. Spheres in the interstices are not acid-fast. (Ziehl-Neelsen x860) Figure 1S.--Pneumonitis characteristic of both tocopherol-deficient and tocopherol-supplemented mink. (x300) 65 Experiment II Growth The data on weight gains of the mink on their respective rations are presented in Table 17. Table 17.--Mean weights in grams for mink fed purified type tocopherol-deficient ration (A) supplemented with 8% cod , liver oil (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha- toc0pherol (D). M Days of Age Lots .32 A _‘ §_ ::__ ’6‘ D :_ 60 484 (10) 467 (8) 423 (8) 503 (4) 67 669 " 692 " 686 " 699 " 74 733 " 744 " 733 " 771 " 8‘ 832 N 869 I. 847 u 853 N 89 _ 937 " 981 " 956 " 978 " 96 1003 " 1043 " 932 " 988 " 103 1005 " 1094 (7) 1014 “ 1094 " 110 1086 (9) 1174 " 1049 " 1119 " 129 1107 (8) 1106 (6) 1123 " 1063 " 144 1279 (7) 1255 " 1321 " 1228 " 165 1275 (5) 1216 " 1281 " 1256 " Values in ( ) indicate number of animals per 15ti‘ Among the surviving animals at 165 days of age, no growth differences were observed. A growth lag attributed to the' severely hot summer weather occurred in all lots when the mink were between 90 and 110 days of age. (The owner of the ranch indicated feed consumption and growth for all his mink were decreased during this same period.) Slight weight losses in lots A, B and C occurred during the last weigh period. The heaviest surviving mink in lots A, B, C and D- weighed 1550, 1440, 1755 and 1435 grams respectively. Mortality During the course of the experiment, six animals died 66 and the circumstances pertaining to their deaths are given in Table 18. Table 18. --Probable causes and number of deaths among mink fed tocOpherol-deficient ration (A) supplemented with 8% cod liver oil (B), 0.1 p. p. m. selenium (C) and 25 p. p.m. alpha- tocOpherol (D). Probable Causes Number of Deaths in Each Lot A B C D Stress of bleeding procedure 2 (57)(109) -- -- Stress of antigen- antibody reaction 2 (96)(98) -- -- Steatitis 1 (38) _- -- Cardiac tamponade 1 (77) -- -- Values in ( ) indicate number of days on the respective rations. No selenium or alpha-tocOpherol supplemented mink died during the experiment. Hematology The mean leukocyte counts for all lots are presented in Table 19, page 67. Based upon the total leukocyte counts considered normal from EXperiment I, the counts for all lots- at 74 days of age were apparently elevated. Lot A, the uncomplicated tocOpherol-deficient lot, except at 74 days of age, had higher total leukocyte counts than lots 0 and D for the duration of the experiment. Let B, that was fed 8% cod liver oil, had a significant leukocytosis between 102 and 132 days. The packed cell volume and hemoglobin data are summarized in Tables 20 and 21 respectively (see pages 68 and 69). The packed cell volumes for lot B were substantially lower from 102 to 164 days_of age than lots 67 Table 19.--Mean leukocyte counts/cmm for mink fed tocopherol- deficient ration (A) supplemented with 8% cod liver oil (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha-tocOpherol (D). c... - ~ ...- - ~- ....—— .— _‘ Av. 102 11 132 164‘ ;f7? A 8533 7933 9175 6237 8388 5217 8770 7750 t 520 435 871 685 964 1855 1232 B 8950 6875 14790 15470 10450 5021 7383 9848 t 1552 1349 640 3091 360 1122 591 c 9950 4375 6281 4913 7250 3117 5808 5956 t 317 510 600 640 425 142 1170 D 10200 4850 5500 5700 4400 4650 5950 5893 t - 20 1249 948 1649 1007 1012 tStandard_;rror. —— —_ A, C and D. The hemoglobin values for lot B remained approx- imately 1 gram lower than the control lot (D) for the dura- tion of the experiment. There was a tendency for packed cell volume and hemoglobin values to increase with the age of mink. These values reached maximum at 164 days of age. The differential count data presented in Table 22, page 70, indicate that the relative leukocytosis in let B resulted from an absolute neutrOphilia and was accompanied by a decrease in the percentage of lymphocytes. The differential counts for lots A, C and D were not considered altered by the respective treatments. As in Experiment I, the lymphocyte/neutrophil ratio reversed during the experiment with a ratio of 60:37 at 74 days of age changing to 39:55 at 164 days of age. 68 Table 20.--Mean packed cell volumes (per cent) for mink fed toc0pherol-deficient ration (A) supplemented with 8% cod liver oil (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha- tocopherol (D). Lot Da s of e Av. 74 88 1 2 11 132 164 T71- A 40.4 46.3 50.1 50.7 53.1 56.0 54.0 50.1 t 0.7 ‘04 006 ‘01 007 300 003 B 40.7 44.0 47.3 40.8 46.6 45.3 51.0 45.7 1 1.4 2.0 1.0 5.5 4.3 2.4 1.2 C 40.1 42.7 51.5 49.7 52.0 53.2 50.8 48.2 t 0.3 1.1 1.5 1.5 1.2 1.0 2.4 0 40.6 45.0 52.4 50.9 54.2 53.1 51.7 48.0 t - 1.4 2.0 2.5 0.7 0.6 1.0 tStandard error. 69 Table 21.--Mean hemoglobin values (gm./100 ml. blood) for mink fed toc0pherol-deficient ration (A) sup lemented with 8% cod liver 011 (B), 0.1 p.p.m. selenium (C? and 25 p.p.m. alpha-t000pherol (D). Lot Da s o e Av. 74 88 102 11 132 164 77? A 13.9 14.9 16.9 16.5 17.9 19.2 17.8 16.7 t 0.2 0.2 0.3 002 0.1 008 "' B 13.8 13.9 15.9 '16.2 16.9 17.4 16.8 15.8 1 0.4 0.7 0.1 0.5 0.6 2.6 0.6 c 13.6 13.6 17.5 16.9 18.0 18.3 17.6 16.5 t 0.2 0.2 0.6 0.6 0.3 0.4 0.7 D 15.5 14.7 17.4 16.7 18.6 18.5 18.1 16.8 t - 003 0.4 0.6 0.1 008 0.3 tStandard error. 70 Table 22.--Summary of the differential leukocyte counts for mink fed tocopherol-deficient ration (A) supplemented with 8% cod liver oil (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha-toc0pherol (D). W Lot Leukocytes Day§_g£_ggg_ ___ Av. 74 88 102 118 132 1T4 171 NeutrOphils Segmented 31 51 33 4C 44 54 34 51 Noneegmented 1 0 0 1 2 3 1 1 A Lymphocytes 64 47 64 55 49 32 57 53 Monocytes 2 2 1 0 0 2 1 1 EosinOphils 2 1 2 4 5 5 6 3 Basophils 0 0 0 0 0 1 1 0 Neutrophils Segmented 35 46 51 63 50 52 54 52 Noneegmented 0 0 0 4 5 2 2 2 B Lymphocytes 60 56 45 28 41 39 41 44 Monocytes 1 0 1 1 O 1 1 1 Eosinophils 3 O 2 3 3 5 1 2 Basophils 0 0 0 0 0 1 0 0 NeutrOphils Segmented 42 48 53 41 50 53 54 47 Nonsegmented 0 1 0 3 4 2 1 2 C Lymphocytes 53 47 60 49 38 42 43 47 Monocytes 1 1 2 1 0 1 1 1 Eosinophils _ 5 3 5 6 5 1 1 3 Ba80phils 0 0 O O 1 1 0 O Neutrophils Segmented 39 46 33 4O 4O 50 51 43 Nonsegmented 0 0 O 5 3 3 2 2 D Lymphocytes 64 53 61 52 55 43 38 52 Monocytes 2 0 2 1 0 2 1 1 Eosinophils 0 0 5 2 2 2 2 2 Basophils 0 0 0 1 1 1 1 1 71 The 48-hour erythrocyte fragility test for LH was con- ducted on each blood sample taken. All samples were negative on the first bleeding. By the time the mink in lots A, B and C were 88 days of age, the 48-hour LH occurred as illustrated in Figure 2. All the remaining fragility tests for LH of erythrocytes from these animals were positive for the duration of the experiment. The longer animals in lots A, B and C remained on the experiment, there was an increasing tendency for LE to occur in less than 48 hours. Layering hemolysis never occurred in the fragility tubes containing erythrocytes from lot D, the control animals; however, erythrocytes from these animals did become more fragile during the experiment. At 74 days of age, hemolysis began in 0.47 per cent saline and was complete in 0.35 per cent saline. At 171 days of age, hemolysis began in 0.67 per cent saline and was complete in 0.37 per cent saline. Near the end of the experiment, several animals from lots A and C were changed to the tocopherol-supplemented ration and bled every 24 hours for several days thereafter. Within 48 hours after these animals consumed their first tocopherol-supplemented ration, their erythrocytes were protected from the layering hemolysis. In a similar trial, Santoguin,* a commercial antioxidant, added to the basal toc0pherol-deficient ration at a level of *Monsanto Chemical Company, St. Louis, Missouri. 72 100 p.p.m., was found ineffective in preventing LH even after the mink had had access to the antioxidant-supplemented rations for seven days. The dialuric acid hemolysis pro- cedure, with all the precautions listed by Friedman gt g1. (1958) proved an unreliable indicator of t0c0pherol depletion in mink. From the optical density readings from tube 3 of each determination, however, erythrocyte fragility indexes for each group were obtained and are shown in Table 23. Table 25.--Average indexes of fragility for erythrocytes from mink fed toc0pherol-deficient ration (A) sup lemented with 8% cod liver oil (B), 0.1 p.p.m. selenium (C and 25 p.p.m. alpha-tocOpherol (D). c 86 81* 125 185 296 i 1 7 15 98 72 D 65 49 63 56 67 i 4 1 10 4 2 *Layering hemolysis was presgntfigt this bleeding. tStandard error. Although the fragility indexes for lots A, B and C on day 88 were little different from those for day 74, LB had already occurred by day 88. From 102 days the fragility index for lot B fed 8% cod liver oil was greater than the index for all other lots. Supplementation of the tocopherol- deficient ration with 0.1 p.p.m. selenium (lot C) was inef- fective in preventing the increased fragility index. 73 Serolo Information obtained from the electrophoretic study of the serum proteins is presented in Table 24 on page 74. Toc0pherol deficiency appeared to decrease the serum albumin fraction and increase the alpha and beta globulin fractions. The addition of cod liver oil to the tocopherol-deficient ration (B) accentuated the above changes while selenium supplementation (C) minimized the changes. Erythrocytes from lot B, the cod liver oil supplemented group, hemolysed so readily that difficulty was encountered in obtaining non-hemolysed serum samples from this group.‘ The presence of free hemoglobin in the sera interfered with evaluating the paper strips for beta and gamma globulin, especially the latter. As a result, the gamma globulin data for lots A and B are inconsistent. 0n the basis of the average gamma globulin values for lots C and D, however, gamma globulin was not affected by tocopherol deficiency. The average serum glutamic-oxalacetic (SGOT) and glu- tamic-pyruvic transaminase (SGPT) data are presented in Table 25, page 75. Significant increases in both SGOT and SGPT values in lots A and B occurred by 118 days of age and persisted for the remainder of the experiment. Both selen- ium and alpha-tocOpherol supplements in lots C and D respect- ively prevented elevation in the transaminase values for these groups. The mean serum tocopherol values for each bleeding of the respective groups are shown in Table 26, page 75. At 11' III II Ill 11" r 11'] I ‘II III, I I ll (illlji II [III III; 74 Table 24.--Mean per cents for albumin and globulin fractions of serum from mink fed toc0pherol-deficient ration (A) sup- plemented with 8% cod liver oil (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha-tocOpherol (D). Serum Lot __: Days of Age Av. Fraction 74' 887' 102 118 3T32 164 Albumin A 34.0 35.7 42.1 40.1 50.1 46.3 41.6 t 307 20? 106 2.0 008 007 8 B 37.4 37.6 59.6 45.4 - - 39.0 i 4.8 2.2 107 ‘- C 42.0 58.4 42.1 52.9 49.1 52.4 45.3 i 0.5 2.4 1.0 1.0 0.8 2.4 D 40.3 43.1 40.5 55.6 53.7 53.3 48.8 t - 109 306 ‘03 203 008 Alpha A 22.8 22.9 18.5 19.8 15.5 15.3 19.2 Globulin t 2.4 2.7 1.1 3.5 1.7 2.8 B 2109 2007 1709 2509 - - 2004 I 1.3 0.9 1.2 - C 22.9 21.3 19.4 14.6 16.1 12.4 18.2 t 1.0 1.2 0.8 0.7 1.7 0.9 D 21.5 24.1 17.4 15.7 14.0 15.9 17.7 t - 4.7 0.6 2.5 0.3 1.8 Beta A 24.1 23.5 23.6 24.7 19.2 21.7 22.8 Globulin i 1.9 0.3 0.5 1.7 1.1 1.0 B 26.2 26.1 29.6 18.1 - - 26.7 i 004 107 009 ‘ C 22.1 21.9 22.7 20.8 19.7 17.1 21.0 i 1.1 0.8 0.9 0.1 1.8 0.1 D 18.3 18.5 24.2 17.2 17.0 14.0 17.8 i - 101 201 004 103 1.0 Gamma A 1809 1708 1508 1503 1502 1607 160a Globulin 1 3.1 1.2 1.4 2.0 1.7 0.8 B 14.3 15.6 12.6 10.6 - - 13.8 t 401 100 107 - c 12.9 18.3 15.7 11.6 15.1 18.1 15.3 1 2.1 1. 1.0 0.4 1.4 1.4 D 19.9 14.2 17.8 11.4 15.3 16.6 15.6 i - ‘07 202 106 102 009 iStandard error. 75 Table 25.--Mean serum glutamic-oxalacetic and glutamic- pyruvic transaminase values in Sigma Frankel units for mink fed tocOpherol-deficient ration (A) supplemented with 8% cod liver 011 (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha-t000pherol (D). ‘- _.-_--—_._A._._--» Serum Lot Days of Age Av. Transaminase 74‘ 88 102 118 1 2 157 Glutamic- A 116 173* 117 349 356 309 237 Oxalacetic t 16 18 4 43 20 9 B 124 168* 146 375 401 400 280 1 - 21 26 73 44 - C 198 163* 124 171 160 107 154 i 26 23 8 20 6 28 D 150 120 124 176 142 143 143 z - 36 4 12 18 30 Glutamic- A 63 73* 49 168 226 89 111 Pyruvic t 6 8 4 80 9 ' - B 33 66* 53 361 270 239 170 t - 8 _ 4 116 - 70 C 85 79* 59 62 73 44 67 t 23 15 4 6 - 6 D 52 67 48 77 73 42 60 t - 24 10 24 “ - 4 *Layering hemETysis was preéSnt:_fi_ tStandard error. Table 26.--Mean serum t0c0pherol values in micro rams/100 ml. serum from mink fed toc0pherol-deficient ration %A) supple- mented with 8% cod liver 011 (B), 0.1 p.p.m. selenium (C) and 25 p.p.m. alpha-tocOpherol (D). W Lot Days of Age ‘741'; 88** 102 118 “‘ 732‘ 157 A 85 41 35 75 14 -- 1 7 12 13 14 6 B 117 68 35 25 29 ~- : 40 13, 20 22 5 ‘ c 130 96 27 26 17 9 t 10 1 12 2 4 4 3 D 164 213 464 536 1420 1221 i - 19 14 19 230 307 ;All animals had been 6n'depletion7ration 14 days. **Anima1s had been on respective rations for 7 days. tStandard error. 76 the first bleeding, when all the animals had been on the same basal tocopherol depletion ration for two weeks, the average was 120 micrograms/100 ml. serum and no layering hemolysis was present. By the succeeding period, when layering hemolysis was present in lots A, B and C, the average.toc0pherol value for all animals in these lots was 68 micrograms/100 ml. serum. There is little evidence from the serum tocopherol data that the 8% cod liver oil hastened tocopherol depletion under the conditions of the experiment. The tocOpherol values in lots A, B and 0 continued to decline during the experiment while those for lot D, the toc0pherol-supplemented group, continued to increase to the unexpectedly high level of 1.4 mg./100 ml. serum. The pre-and post-Salmonella pgllorum antigen injection titers are presented in Table 27, page 77. Little indication was present relative to an association between tocopherol deficiency and the ability of mink to produce antibody in response to Salmonella pullorum antigen. Pathology Tocogherol-deficient lot A.--These mink showed no deficiency symptoms prior to death or being sacrificed. Two mink at necr0psy had gross my0pathy of the internal intercostal muscles as illustrated previously in Figure 3. Histological examination revealed skeletal myopathy in the adductor muscles as well as the intercostal muscles. The myopathy observed was characteristic of phase two des- cribed in Experiment I and depicted in Figure 7. (I‘ll IIIIIIII'II. (II 1.} il‘ll'll. 1].! IIJIJIIII‘II' I II] (I: II III 1 J1 1' II III (III II.1 I 'll. 1 77 Table 27.--Pre-and post-Salmonella pullorum antigen injection titers for mink fed tocopherol-deficient ration (A) sup le- mented with 8% cod liver 011 (B), 0.1 p.p.m. selenium (C3 and 25 p.p.m. toc0pherol-(D). _Lot Mink Pre-inJection Post-injection Titers No. Titers . W 1f10‘ 1/20 1720 “1740 1780 1F60 73—25 A 1 .. - - + r - - .- 2 - - - + + + - - 3 + + - + + i - 4 Fourth animal died during the study. B 5 - - - -+* 4- + 1 t 6 + - - 1 + + - - 7 - - - +*- + + 1 1 8 + - - 1 + 1 - - C 9 - - - + + + 1 - 10 - - - + + - - - 11 + 1 - + + t - - 12 + - - + + t 1 - D 13 1 - - + + 1 - - 14 1 - - + + - - - 15 - - - + 1 - - - 16 - - - + + 1 - - +positive agglutination 1questionable agglutination -no agglutination *serum from hemolyzed blood sample ! I‘lllilll'. III. I Il.l|.lil.|11‘ll III. I III I! 1| 1"! I 78 Perilobular fatty infiltration plus centrolobular congestion and hemorrhages were the prominent pathological features in the liver. When compared to the normal portal triad (see Figure 16, page 80), the triads for the t000pherol- deficient mink were larger than normal and edematous (Figure 17, page 80). Glomerular congestion, focal capillary congestion and pyknosis of the proximal and distal convoluted tubules were observed. Hemosiderin was prominent in the cytoplasm of the cells of some convoluted tubules (Figure 18, page 84). The micro-droplets of non-acid-fast material were again observed in the interstitial spaces of the adipose tissue (see Figure 14, page 64). Varying degrees of the pneumonitis shown in Figure 15 on page 64 were present. Hemorrhagic foci were observed in the adrenal cortex and medulla. Tbcophergl;deficient cod2livgr oil-supplemented lot B.-- Two of these mink had gross skeletal myopathy at necropsy. Histologically, the my0pathy was more extensive than that observed in lot A. Many fibers, especially of the adductor muscles, were undergoing calcification in the presence of many sarcolemmal nuclei as in Figure 19 on page 82. On the basis of the classification presented in Experiment I, this myopathy was considered to be early phase three. The adipose tissue of all mink in lot B was very yellow (Figure 22, page 84). Acid-fast pigment droplets, lympho- cytes, and some macr0phages were observed in the inter- stitial spaces as in Figure 23 on page 84. 79 Calcified foci were present in the cytoplasm of necrotic cells of proximal and distal convoluted tubules (see Figures 24 and 25 on page 85). The hepatic, adrenal and pulmonary lesions were present as described in lot A. Toctherol-deficient selenium-supplemented lot C.-- While selenium supplementation of the tocopherol-deficient mink prevented the sudden deaths and gross myopathy observed in lots A and B, microsc0pic toc0pherol-deficiency lesions were not completely prevented. Sacrificed mink from lot C had an early adductor myopathy characterized by an increase in the number of sarcolemmal nuclei as in Figure 6'on page 57. The adipose tissue, when compared with the normal (Figure 20, page 83), contained interstitial evidence of a very early steatitis (Figure 21, page 83). Hepatic and renal congestion were noted. Lymphocytic foci were observed in the zona fasciculata and some degree of pneumonitis was present. Tocopherol-supplemented lot D.--Twenty-five p.p.m. of alpha-toc0pherol prevented all the symptomatic, clinical, gross and microscopic pathology associated with tocopherol deficiency in mink. One of the control mink, however, had a slight pneumonitis as observed in the previous lots. 80 Figure 16.--Normal portal triad of mink. (x300) Figure 17.--Edematous portal triad from tocopherol- deficient mink. (x230) 81 Figure 18.--Kidney of a tocopherol-deficient mink; A. Hemosiderosis; and B. Coagulation necrosis in convoluted tubules. (x620) 82 Figure 19.--Adductor of a tocopherol-deficient cod liver oil-supplemented mink; A. Sarco- lemmal proliferation; B. Myofibrillar necrosis; and C. Early calcium deposition. (x350) 83 \ k y» . r ‘1; i A ’ “"I’ ‘1 _fi 1 1 Figure 20.--Normal adipose tissue from toc0pherol- supplemented mink. (x900) 1‘} 4 Figure 21.--Adipose tissue from tocopherol-deficient selenium-supplemented mink. Interstitial spaces contain amorphous, non-acid-fast material. (x860) Figure 22.--Groes steatitis "yellow fat" of tocopherol-deficient cod liver oil-supple- mented mink. Figure 23.--Adipose tissue from tocopherol- deficient cod liver oil-supplemented mink; A. Acid-fast-pigment droplets; and B. Lymphocytes in the interstitial spaces. (Ziehl-Neelsen x860) Figure 24.--Kidney of tocopherol-deficient cod liver oil-supplemented mink; A. Coagu- lation necrosis; and B. Calcification of the convoluted tubules in longitudinal section. (x250) Figure 25.--Kidney of tocopherol-deficient cod liver oil-supplemented mink; A. Coagulation necrosis; and B. Calcification of the convo- luted tubules in cross section. (x250) Discussion Mink Experiments I and II Growth Data obtained from these experiments provide little evidence that mink require tocopherol for growth. Although these experiments were conducted long enough for normally fed mink to attain over 80 per cent of their adult weight, two factors suggest that the experiments may not have been of sufficient length for growth differences to appear. First, Mackensie and Mackensie (1959) reported a toc0pherol requirement for growth of both male and female rats main- tained longer than four months on toc0pherol-deficient rations. Second, slight weight losses were observed among the tocopherol-deficient mink, lots A, B and C, during the last weigh period of Experiment II. Mortality 0f significance in the mortality information is the number of spontaneous deaths of tocopherol-deficient mink following such stress factors as being moved from the normal rearing cage to the less familiar but roomy metabolism cage or responding to an injection of Salmonella pullorum antigen. EXplanation for these deaths cannot be made entirely on the basis of cardiac insufficiency because only two of the nine animals that died following such stresses had evidence of cardiac myopathy. 86 87 The observed adrenal pathology, however infrequent and inconsistent, supports the contention of other workers that the adrenal cortex may be the site of primary insufficiency during tocOpherol deficiency. Raymondi (1958) demonstrated that the action of toc0pherol is comparable to that of ACTH in that toc0pherol deficiency influenced the zonae glomerulosa and fasciculata with cortical changes related to hyperemia. Hiisi-Brummer (1955) presented evidence that the stress of prolonged tocopherol deficiency results in a cortical dys- plasia from a hyperfunction of the adrenal cortex. Histo- chemical studies are required to further examine the possible insufficiency of the adrenal gland during a tocopherol deficiency. , Deaths among the tocopherol-depleted mink in Experiment I were prevented when alpha-tocOpherol and selenium supple- mentation was started. Therefore, in a semipurified ration containing 24 per cent molecularly distilled lard, adequate antioxidant activity to arrest and/or prevent fatal toc0pherol deficiency lesions is provided by 25 p.p.m. of alpha-tocopherol or 1 p.p.m. of selenium as sodium selenite. The same toco- pherol-sparing effects of selenium have been demonstrated by Schwarz g; 2;. (1957) in the rat, by Eggert g§|§;. (1957) in swine, by Dam (1957) 1n the chick and by Muth (1959) in sheep. In Experiment II, 0.1 p.p.m. of selenium was also found adequate to prevent fatal tocopherol deficiency lesions. Hematology The relationship between the total leukocyte counts and mink age observed in Experiment I, and an earlier unpublished 88 study, was not demonstrated in Experiment II. However, the alterations in packed cell volumes and hemoglobin values during the course of both experiments indicate a relation- ship normally exists between these factors and mink age. This relationship was not indicated by Kubin and Mason (1948). These workers, however, did not indicate the ages of the mink from which their information was collected. Although 25 p.p.m. of alpha-t000pherol in the dry diet maintained erythrocyte integrity in Experiments I and II, neither 1 p.p.m. or 0.1 p.p.m. of selenium used in Experi- ments I and II respectively was able to maintain the eryth- rocyte integrity in the tocopherol-deficient mink. This seleniim-erythrocyte relationship has not been previously studied in the mink; however, these findings agree with Gitler g; 2;. (1253) who used the rat and chick. In fact, from the results of an unpublished survey for compounds which might cause the layering type hemolysis in normal erythrocytes after a 48-hour refrigeration period in saline, sodium selenite was found effective. On this basis then, additional selenium in an already t000pherol-deficient ration would increase erythrocyte fragility rather than decrease the tendency for hemolysis. The layering type hemolysis of mink erythrocytes appears to be a more practical indirect indicator of toc0pherol deficiency than does the dialuric acid hemolysis test of Friedman gt gl. (1958). No doubt, with adequate modification for species, the dialuric acid test might be adaptable to the mink erythrocytes. 89 Serology The serum protein data obtained from the alpha-t000pherol supplemented dark and brown mink from Experiments I and II respectively are considered normal. The alterations in the albumin and alpha globulin fractions, observed in the toco- pherol-deficient mink sera during Experiment II and not observed during Experiment I, suggest that in the latter experiment, the tocooherol depletion period was insufficient to affect the serum protein fractions. Other investigators are not in agreement regarding the effects of tocopherol deficiency upon the serum protein fractions. Bottiglioni and Vannini (1957) found no serum protein alterations assoc- iated with tocopherol deficiency in the rat, while Keeler (1961) reported dystrOphic sheep to have an increased alpha globulin fraction and decreased beta and gamma globulin fractions. In Experiment I, when toc0pherol depletion was started in mink at least three weeks younger than in Experiment II, it is important to note that spontaneous deaths occurred prior to significant changes in SGOT values. It is for this reason that the transaminase determinations are not con- sidered as valuable aids for determining the presence of tocopherol deficiency my0pathy in mink as Swingle g3 gl. (1959) have reported them to be in sheep. The SGOT and SGPT values for the control animals in each trial are in close agreement and again, in the absence of other data relative to the serum transaminases for mink, 90 the SGOT range of 103-176 and SGPT range of 42-90 with the respective averages of 136 and 60 Sigma Frankel units/ml. of serum are considered normal for mink receiving purified type rations. The serum t000pherol data from Experiment I indicate that 25 p.p.m. of alpha-toc0pherol in the semipurified ration containing 24 per cent molecularly distilled lard are ade- quate to maintain serum t000pherol levels above those assoc- iated with tocOpherol deficiency myopathy. The requirement for toc0pherol by the mink has not been previously estimated using purified type rations but the value of 25 p.p.m. in a diet containing a relatively saturated fat compares favorably with the range 10-20 mgs. of alpha-tocopherol/animal/day which Wilton (1958) indicated would prevent steatitis in mink fed a ration, the meat portion of which was frozen fish scraps. The serum t000pherol data for Experiment II compare closely with those from Experiment I except for the uneXpect- edly high values in lot D by 156 and 171 days of age. These values indicate the mink were accumulating total body toco- pherol from the dietary sources. Serum tocopherol values of less than 50 micrograms/100 ml. of serum were associated with sudden deaths of non- selenium-supplemented mink in Experiment I. Based upon this experience, a high incidence of mortality was anticipated in Experiment II when the mink in lots A and B were 102 days old and had serum t000pherol values below 50 micrograms/100 91 ml. of serum; however, deaths were not frequent at this time. The fact that mink in Experiment II were three weeks older than those in Experiment I before tocopherol depletion began may account for the lower-than-anticipated incidence of mortality during EXperiment II. Information obtained from the antigen-antibody study, however limited, tends to indicate that a toc0pherol deficiency does not impair the ability of mink to produce antibodies, at least to Salmonella pullorum antigen. This information lends support to the report of Axelrod and Pruzansky (1955) who reported that tocopherol-deficient chicks produced normal anounts of antibodies to porcine gamma globulin. Urinalyses Normal creatine excretion values for mink were not available for comparative purposes and the available crea- tinine data are not in agreement. Leoschke (1959) reported the normal 24-hour excretion of creatinine per kilogram of mink as 31 mgs. while Oldfield (1950) reported the figure of 15.5 mgs. The latter is within the range found in Exper- iment I. Neither the creatine or creatinine excretion data appear to be as satisfactory an indirect tocopherol depletion indicator as does the layering hemolysis test. .The specific gravity of mink urine was reported by Kubin and Mason (1948) to range between 1.018 and 1.036. The hydrometric values obtained in this study were usually above the upper limit of the above range. The unnatural type of 92 ration and the inherent fecal contamination problem may have affected the specific gravity values. The surface tension values obtained in this study were approximately 4 dynes/cm. greater than the average of 42.7 which Leoschke (1959) reported for normal mink. Because only five animals, during Experiment I, were considered to have the chronic type of incontinence considered damaging to pelts and because of the fecal contamination problem, Dr. Leoschke's contention that urine from incontinent mink has a lower surface tension and thereby an increased affin- ity for the fur around the urethral orfice can neither be disputed nor supported. Pathology Skeletal muscle and adipose tissue were the most frequently affected tissues in experimental t000pherol deficiency in mink. My0pathy was observed, however, in the presence and absence of any form of steatitis described by Hartsough and Gorham (1949) and steatitis was observed in the absence of my0pathy. The internal intercostal and adductor muscles appeared to be predilection sites for toc0pherol deficiency my0pathy in mink. Extensive myopathy was often present without gross evidence of "white muscle" commonly associated with toco- pherol deficiency. In such cases, the myopathy was histolOg- ically identified more readily in the adductor muscles than in the intercostal muscles. The skeletal my0pathy, in general, followed the pattern of nutritional my0pathy 93 described by Adams and Denny-Brown (1953). However, in mink, vacuolar degeneration appeared more characteristic of the early myopathy phase than was emphasized by Adams and Denny- Brown. Also, the internal nuclear rowing, described by West and Mason (1958) as a prominent feature of tocOpherol deficiency myopathy in hamsters was not a common feature of the same myopathy in mink. Gross cardiac myopathy, a condition which Benson (1959) reported in adult mink and called white heart disease, was observed only twice during these experiments. This incidence of tocopherol-deficiency cardiac myopathy is in contrast to that reported in lambs by Bacigalupo (1952). He indicated. that right ventricular lesions were frequent in the experi- ' mentally-produced stiff-lamb syndrome. The overall incidence of toc0pherol deficiency my0pathies in Experiment II was less than that observed in Experiment I. This is attributed to the fact that in Experiment I, the kits were weaned onto the tocopherol-deficient rations, while in Experiment II, kits were completely weaned and eating standard ranch rations before they were obtained for the experiment. Consequently, considerably more muscle develOpment had occurred in the latter mink prior to starting them on the tocopherol depletion ration than had occurred in Experiment I. With the variety of muscle lesions observed, it should be easy to formulate the steps in the pathogenesis of tocopherol deficiency myopathy, but the gamut of lesions is seldom observed in a single animal or a single muscle. 94 Swollen fibers and vacuolar degeneration are often present without signs of my0phagic infiltration or focal calcifi- cation. Fibers may be differentially stained or even infil- trated with my0phages without evidence of vacuolar degeneration present. And, individual calcified fibers can be present without any myopathy of the surrounding fibers or fasciculi. When the internal rowing of skeletal muscle, described by West and Mason (1958), is present, it is believed to follow vacuolar degeneration, the nuclei accumulating in the vacuoles. Vacuolar degeneration, however, does not preclude internal rowing for internal nuclear rowing is seldom observed in toc0pherol-deficient mink where vacuolar degeneration is a prominent characteristic. A sequence for the pathogenesis of t000pherol deficiency myopathy in mink may be similar to the following: 1. Serum toc0pherol values below 50 micrograms/100 ml. serum. 2. Swelling of individual fibers of the active , voluntary muscles, especially the internal inter- costals and the adductors. 3. Alteration of the pH of these swollen fibers indicated by differential staining characteristics. 4. Vacuolar degeneration and/or proliferation of sarcolemmal nuclei. 5. Fragmentation and lysis of the degenerate sar- 00plasmic masses. 6. Attempted regeneration of the muscle by prolif- eration of myoblasts in intact endomysium. 7. Phagocytosis of the necrotic myofibrils and sarcolemmal cells. 8. Calcium deposition in the phagocytized or non- phagocytized sarc0plasmic debris. 95 9. Restoration of function to regenerated muscle fibers. Unfortunately, the reasons why the decreased serum toc0pherol starts this chain of events in the musculature remain more speculative than the above sequence would sug- gest. Skeletal and cardiac myopathy in the absence of any form of steatitis was produced within 60 days in mink kits that were weaned onto the toc0pherol-deficient and unsaturated fatty-acid-low basal rations used in these experiments. The situation wherein my0pathy was present in the absence of steatitis was considered the uncomplicated tocopherol defi- ciency. Mink depleted of toc0pherol in the above manner and that survived a prolonged toc0pherol deficiency (three months) developed the steatitis observed in Figure 14 on page 64. Micro-draplets of non-acid-fast material infiltrated the interstices of the adipose cells without changing the gross color or the physical characteristics of the fat. Mink kits that survived a prolonged period of tocopherol depletion while being protected with sodium selenite showed evidence that the selenium afforded partial protection against the previous form of steatitis. However, another form of steatitis resulted which was characterized by the accumulation of amorphous material in the interstices of the fat (see Figure 21 on page 83). This steatitis appeared to ' be an earlier stage of the form previously described. The 96 evidence that selenium afforded partial protection against adipose tissue changes during toc0pherol deficiency isvnot in full agreement with the report of Edwin g; 3;. (1961) who found that selenium supplementation of necrogenic torula yeast diets, fed to rats, gave no protection against peroxi- dation of the body fats. When mink kits were fed the basal tocopherol-deficient ration with an isocaloric addition of 8% cod liver oil, the steatitis described by Hartsough and Gorham (1949) and Gorham g§,g;. (1951) was produced. As in Figure 23 on page 84, the interstices of the affected adipose tissue were in- filtrated with acid-fast-pigment dr0plets, a few lymphocytes and some macr0phages. The acid-fast-pigment rendered the fat yellow. Although there was a neutrophilia in these mink so affected, possibly resulting from the concurrent myopathy, the neutrOphilic infiltration of the adipose tissue, char- acteristic of steatitis in cats (Gordy and Stillinger, 1953), was not observed. The hepatic, renal and adrenal hemorrhages observed indicate a relationship exists between capillary permeability and toc0pherol deficiency. Such evidence was provided by Grant (1961) who reported upon a microangiopathy associated with tocopherol deficiencies and yellow fat disease in swine. The angiopathy was identified by the presence of periodic- acid-Schiff positive material accumulating in the sub-endo- thelial area of small arterioles and capillaries, especially in the heart. The findings of Grant in this regard were not 97 confirmed in the mink experiments either histochemically or_ histopathologically. Evidence has been presented by other researchers to indicate that the renal lesions previously attributed to toc0pherol deficiency actually were the result of post-mortem autolysis which is more rapid in toc0pherol-deficient animals than tocopherol-supplemented animals. Figures 24 and 25 on page 88, however, are considered evidence that ante-mortem renal lesions may be present following prolonged tocopherol deficiency, especially if the tocopherol depletion ration is relatively high in unsaturated fatty acids. Although a pneumonitis, similar to the viral feline pneumonitis, was observed during these experiments in both the tocOpherol-deficient and tocopherol-supplemented mink, the former were more severely affected. If this pneumonitis were proven to be of viral origin, the results of these experiments would indicate that toc0pherol-deficient mink have a lower resistance to pneumonitis virus than do toco- pherol-supplemented mink. The pneumonitis may have served as the primary stress factor for the uncomplicated toc0pherol-deficient animals and ultimately caused the sudden deaths that occurred among these mink. In fact, the venous distention and edema of the portal triads and the centro-lobular congestion in the liver, in conjunction with the pneumonitis, lend support to the theory that the sudden deaths among the tocopherol-defi- cient mink were caused primarily by hypoxia which in turn resulted in cardiac insufficiency. SWINE EXPERIMENT Materials and Methods Fifteen Chester White-Yerkshire crossbred baby pigs were taken from their dams at four days of age and placed on the semipurified type ration (Table 28) prepared in the form of a synthetic milk. Table 28.--Composition of tocopherol-deficient swine ration. Per Cent Ingredient Vitamin free casein 10.0 Isolated soy assay protein* 12.0 Torula yeast* 20.0 Glucose (cereloee) 37.5 Molecularly distilled lard* 10.0 SOlka-FIOC'I‘ 500 Phillips and Hart Salt Mix (IV)* 4.0 Amino acid and vitamin supplements 0.5 l I *As described in Mink Experiments I and II. The amino acid and vitamin supplements were prepared to furnish the constituents at the rates given in Table 29. Table 29.-~Amino acid and vitamin supplementation rates. Component Rate* Component Rate* Arginine 801 100.0 p-amino benzoic acid 3.0 Thiamine HCl 0.5 Folic acid 0.2 Riboflavin 1.0 Cyanocobalamin 0.16 Pyridoxine ’O.5 Biotin 0.05 Calcium pantothenate 3.6 Vitamin A acetate 0.52 Nicotinic acid 5.0 Vitamin D2 0.01 Choline chloride 200.0 Menadione 0.5 Inositol 25.0 16.0 Ascorbic acid *mg./1OO gm.solids. 98 99 The milk was prepared by suspending the dry ingredients, melted lard and fat-soluble vitamins in sufficient, con- stantly stirred 160° F. water to constitute 15 per cent total solids. The suspension was immediately homogenized in a two stage Manton Gaulon homogenizer at 500 and 2508 pounds pressure. After the mix was cooled in a standard spray-type milk cooler, the water-soluble vitamins were added. The pigs rapidly learned to drink from a pan and were fed five times per day for the first two weeks and four times per day for the third week. Thereafter, they were gradually converted to the dry basal diet, fed three times per day and assigned to the experimental lots shown in Table 30. Table 30.--Assignment of baby pigs to experimental lots. ‘— TL Lot Number of Pigs Ration Per Lot “ A 5 Basal tocOpherol deficient. B 4 Basal plus ethyl linoleate to replace an amount of lard equal to five per cent of the caloric content per unit weight. C 4 Basal plus 100 p.p.m. alpha- tocopherol. _' _~_ iw—t —_ Allotting had been postponed because of a diarrhea problem which was fatal to two pigs prior to allotting and to one pig in lot B immediately following allotting. Biweekly blood samples of 10-12 ml. were obtained from the anterior vena cava and all the hematological and 100 serological determinations described for the Mink Experiment II were made on the swine blood. Likewise, the necr0psy and histological procedures used in the swine experiment were those described for the Mink Experiment II. Near the termination of the swine experiment, a prelim- inary study was made to compare the responses of tocopherol- deficient and supplemented swine to the stress of an infec- tion of transmissible gastroenteritis virus (T. G. E.). The T. G. E. virus was obtained from Purdue University in the form of homogenized infected intestines (lot 1206-9) harvested January 12, 1961. Three milliliters of the infected material was diluted to 20 ml. with sterile saline. Aliquots of this diluted material were administered orally, by means of a grain bolus, to two tocopherol-deficient and two tocopherol-supplemented pigs on the basis of body weight. The temperature of each animal was taken daily, and in addition, each animal was checked for evidence of anorexia, vomition and diarrhea. These observations were continued until all surviving animals appeared normal again. Also near the termination of the swine experiment, the rapidity with which intramuscularly administered alpha- tocopherol was able to prevent the layering hemolysis (LH) in previously tocopherol-deficient swine was studied. Animals whose erythrocytes were positively demonstrating LH were given single intramuscular injections of alpha-tocopherol at the rate of one mg. per pound of body weight. Thereafter, the animals were bled daily to observe how soon LH ceased 101 following the t000pherol supplementation and how rapidly it returned. This repletion-depletion technique was used as a means of estimating the daily toc0pherol requirement for swine under the conditions of this experiment. Results Growth Uniformly poor growth was made by animals in each experimental group. An insidious diarrhea of unknown origin affected each group from about 7 to 21 days of age. Growth during this period was negligible and normal gains were never attained. No differences, however, were detected in the growth rates of the different lots which might be con.- strued to be due to the effects of toc0pherol deficiency. Mortality During the course of the experiment, six of the eight tocopherol-deficient pigs died suddenly while no toc0pherol- supplemented pigs died. Table 31 presents the information pertaining to these deaths. With the exception of one, these deaths occurred sometime during the night and the dead ani- mals were first observed at the next morning feeding. The second pig to succumb in lot B had a tympanites sufficient to rupture the abdominal wall and skin, permitting exter- iorization of the small intestine. Gastric tympanites, in the second pig to succumb in lot A, caused gastric rupture and forced the gastric contents into the subcutaneous tissue as far posterior as the stifle area. It is possible that this death was a result of the stress of the T. G. E. 102 103 Table 31.--Observations pertaining to deaths of tocopherol- deficient swine. m Lot Ration Days on Associated Expt. Observations A Basal tocopherol-deficient 80 Sudden death. " " " 130 Sudden death 15 hours after exposure to T. G.E. virus. Rapid post- mortem tympanites. A " " " 131 Sudden death. A " " " 140 Sudden death and rapid post-mortem tympanites. B Toc0pherol-deficient, ethyl linoleate-supplemented 50 Sudden death and rapid post-mortem tympanites. B Toc0pherol-deficient, ethyl linoleate-supplemented 56 Sudden death and rapid post-mortem tympanites. infection, however, a second toc0pherol-deficient animal so exposed survived and elicited only a minor anorexic response to the virus. Hematology Alterations attributable to toc0pherol deficiency were not observed in the packed cell volumes or hemoglobin values among the experimental groups. However, a variable response to the early diarrhea problem among the groups was reflected by a slight range in the average white cell counts not correlated with toc0pherol deficiency. The data from the white cell counts, packed cell volumes and hemoglobin deter- minations are summarized in Table 32, page 104. 104 Table 32.--Mean white cell counts, packed cell volumes and hemoglobin values for swine fed tocopherol-deficient ration (A), supplemented with 5% ethyl linoleate (B) and 100 p.p.m. ' alpha-toc0pherol (C). W Lot White cells/cmm Per cent packed Gms. hemoglobin/ fi_fi_ cell volumes _iOO ml. blood A 14.976 37.7 11.4 i 2.070 1.1 0.2 8' 11,438 38.2 11.7 1 1,890 1.6 0.3 0 13.130 38.3 11.5 1 1,400 1.1 0.2 1Standard error. The average differential counts for each lot for the duration of the experiment are presented in Table 33. The counts were not affected by the presence or absence of toco— pherol in the ration. The lymphocyte: neutrOphil ratio was 64:34. ' Table 33.--Mean differential counts for swine fed a toco- pherol-deficient ration (A) supplemented with 5% ethyl linoleate (B) and 100 p.p.m. alpha-toc0pherol (C). Leukocytes Lots . A B C_* NeutrOphils Segmented 33 32 . 35 Noneegmented 1 >1 1 Lymphocytes 64 65 62 Monocytes 1 1 2 Eosinophils 1 1 1 Basophils >1 1 )1 The 48-hour layering type hemolysis of erythrocytes from the tocopherol-deficient swine of lot A occurred when the animals were 77 days old and continued for the duration of the exper- iment (See Figure 26 on page 105). Erythrocytes from the 105 .monsa no msopvon neon eonohmHv ecpmspcoomoo one spasm cacao 1amod1aososaooop soak mop ooanahpo no msofiuosam comma .m op .m .mnda Ho aoavon ca panama> one man coanosoHQQSmnaohonaooop a scam mophoohnphso eomhaosonuaoz .< .csfiasm uses and v.0 ca mfimhaosos copssowahuos ssonums on» no msoapsemaw11.mm shaman h . m a u m d 106 two animals that died in lot B never elicited the layering phenomenon and the surviving pig in lot B was 105 days old before its erythrocytes showed LH. Erythrocytes from the tocopherol-supplemented swine never elicited the response. Near the termination of the experiment a lot A tocopherol- deficient pig whose erythrocytes were demonstrating the lay- ering hemolysis, was given intramuscularly 1 mg. of alpha- tocopherol acetate/pound of body weight and bled daily for the next 11 days. The erythrocytes taken from this pig 24 hours after the injection of tocopherol failed to elicit the layering hemolysis. Thereafter, erythrocytes taken daily from this animal did not elicit the hemolysis phenomenon until the eighth day post-injection after which the hemolysis persisted. From this repletion-depletion technique, a tocopherol requirement value of 125 micrograms/pound of body weight/day was obtained. As in Mink Experiment II, ethoxyquin (Santoguin*), a commercial antioxidant, was added at the rate of 100 p.p.m. to the toc0pherol-deficient ration of a lot A pig whose erythrocytes showed the layering hemolysis. In the seven- day trial, during which time the animal was bled daily, the antioxidant was ineffective in preventing the hemolysis phenomenon. The data obtained from the dialuric acid hemoly- sis determinations are summarized in Table 34 on page 107. The first significant elevation in the dialuric acid values occurred in lot A when the pigs were 73 days old and 14 days “Monsanto Chemical Company, St. Louis, Missouri. 107 Table 34.--Average dialuric acid hemolysis values (per cent) for erythrocytes from swine fed toc0pherol deficient ration (A) supplemented with 5% ethyl linoleate (B) and 100 p.p.m. alpha-toc0pherol (C). W Lot __ Days of ggg :g1 28 35 42 5 3 70 7 *84—’98 105 _ A - - - 6.9 1.8 - —64.3 - 27.9*‘: 14.5 25.2 B 1.5 0.6 5.1 4.4 16.0 — _ s C "’ 008 108 O01 007 108 - *Layering hemolysis present. ahead of the onset of the layering hemolysis for this lot. When the remaining animal in lot B was 70 days old, there was an indication that it had an increased dialuric acid value. Unfortunately, no later valid dialuric acid deter- minations were obtained from this lot to compare with the onset of layering hemolysis which occurred at 105 days of age. The data from the paper electrophoresis analyses of the serum protein fractions are presented in Table 35. page 108. The gamma globulin was consistently lower in lot_A than in lots B or C. However, because the same change relative to gamma globulin did not occur in lot B, the alterations in this serum fraction were not considered associated with the presence or absence of tocopherol in the ration. The other fractions were not significantly altered by treatment. Elevations in the serum glutamic-oxalacetic and glutamic- pyruvic transaminase values occurred in lot A simultaneously with the onset of the layering hemolysis when the animals Expt. p.p.M. 126 Av. 91 105 84 77 5% ethyl linoleate (B) and 100 70 alpha-tocopherol (C). 53 _——._———~—-— Days of Age —-=- 56 49 14 21 28 35 42 flat— Table 35.-Mean per cents for albumin and globulin fractions of serum from swine fed t000pherol deficient ration (A) supplemented with Serum Fraction [CD 0 M <1 '0 M r1 KN u\ F0- MN 10 10(1)!“ 0# 000| 00 (NF-tn MN m M n 00 0 0 mm m 0| 0 0 LB 01'- M M PO 0 0 LOO) n d‘ r-ox 0| 0 0 7 co awn «x -¢ 1,2,. 0 0 '00) n 04'0“!) 0 0 0 0 I CDPOO h 41 [mm 0 0 \001 M O\§'¢O\Wn 0 0 0 0 0 0 0-0—0-0-O'xo n n (\J ou~ N 10 [‘0 0| 0 0 F \O\O (\1 01 +1 4*! m 0 108 O\ (\1 F 0 0 0 O 00 1‘ M (\J 0.1 N [‘0- (\1 NCDO FO‘ 000| 00 0010 COO M M <10 00 (1)01 01 41' \OQ) 0| 00 CD ON 01 \OK\ 00 GNP CU -¢ «no 0| 00 (\1 \00 h (\1 O\(“ 00 <11“ m 04'me 0 0 0 0 —~mw M 01 (1)0- 00 (1)0- (\1 \omcomoxo .0 O... 1.0-#63004“) I“ (\J M \OCD O O 00 n annex-=1 0 0 0 0 QFNO N (\I FLOOQDOCD N—hou— r1 cu (N p.100 (I) [‘01 v- (H +0 +0 +1 <1 m U C! H H 11333 $2.0 Q0 1-11-1 43¢ t‘ (D in O\ (I) O‘ Q) 0 0 Ck O [‘ GO 0 0 0| 0 0 v-v-m m0- CU 0- 0- 00 OO O\ M'- 0| 00 ON ON '- CU I‘d) ONO <1 01“ 0| 00 \o sw— \041’ 0 0 GO \or~¢ 0 0 0 0 (\OCDO 0- 0- MON 0 0 mo t~ (Dd 0| 0 0 0 F0) (\1 (\1 4‘! +1 +1 d a) L) 5:: H ...; 3 (15.0 «PO 0H mt!) 20.3 Gamma Globulin 19.5 41m 00 b-O exp own MM iStandard error. Y~ 109 were 77 days of age. In the ethyl linoleate-supplemented tocopherol-deficient group (lot B), the SGOT values were first elevated after 84 days of age and the SGPT values were not significantly elevated until 105 days of age. At this age, the layering hemolysis was also observed in lot B. The average serum transaminase values for each bleeding period of the respective groups are given in Table 37 on page 110. The data from the serum tocopherol determinations made' during this experiment are summarized in Table 36. Table 36.--Mean serum tocOpherol values (micro rams/ml.) for swine receiving toc0pherol-deficient ration (A supplemented with 5% ethyl linoleate (B) and 100 p.p.m. alphaetocopherol(C). Lot‘ '1 ' _ DJso'w _,__ 14 21 28 35 712 49'5663'1370'77m84‘91—T’T10 12 ‘ “— A 134 138 547—172 60* 66;; 56 42 46 1 5 10 - 26 4 22 15 10 16 B 109 180 180 74 50 85* ' 39 704* 1 11 21 34 17 6 -' - - C 220 152 240 216 221 226 245 260 1 - 6O 27 20 34 16 12 17 *Onset of elevated dialuric acid hemglysis values. **Onset of layering hemolysis. Illogically high total tocOpherol values for lot A at 49 days of age were noted. Had this value been close to the 42 day value, the toc0pherol depletion rates for the two lots would have been very similar. According to these data, ethyl linoleate supplementation of lot B did not hasten tocopherol depletion. In lot A, excepting day 49 data, the layering hemolysis occurred after 35 days of serum tocopherol values 110 .aoaho esoesmamfl e m m n m a 1 a an we on as an .e on 1 o 1 1 1 a a 1 m 1 n 4.. mm mm an me on mm mm m o. m .m o. __ m . aoaesnasa . n 61>:aam am .m mmr.11, oe 1rmm 1.1. on an canno1. 1, 1m« 1.< 1edeeeeao : _ a a n m «.1». mm an an cm as .e we mm o 1 1 1 1 m 1 a 1 a cum on em we es mm me an m n_ m m. n_ m m 1 assessaea _ u canoesaswo 0.. m1, so, we. he en es oases .n 4 1odssseflo WW; mop .or1wm 5 11ON n 1. m WW 11ww1111 WWHH_N (Wm 1 ommnassmqmaa o -o m-ma ,uoq . sssom .on Hoaosaooo¢1mnmam .s.d.n 00— and Amv oamoaocaa Hague Rm and: eoasosoamdsm A ommcfisomssso oa>samnuodss¢sam one capoomamx01oassasa news: Housman semam ea Epsom mmoz1l.bm canes 111 less than 66 micrograms/100 ml. In lot B, an average of 63 micrograms of tocopherol/100 ml. serum had been present for more than 60 days before the layering hemolysis phe- nomenon appeared. In the antigen-antibody response study conducted near the termination of this eXperiment with three tocOpherol- deficient and two toc0pherol-supplemented swine, there was an indication that the deficient pigs elicited a greater antibody response than did the controls. The evidence is presented in Table 38. 1 Table 38.--Pre- and post-Salmonella pullorum antigen injection titers for t000pherol-deficient and supplemented swine. m TocOpherol Anim. Pre-in ection Titers Post-injection Titers Status No. 1/5 1/10 y/@_y/—40 11766 11320 11640 141280 Deficient 1 + + + + + ' 2 + 1 1 — + + 1 - 3 + i - + + + i Supplemented 1 + + - - + i - 2 + 1 - + 1 — +Positive agglutination. 1Questionable agglutination. -No agglutination. The titer for the tocopherol-deficient swine appeared to be between 1/640 and 1/1280, while the titer for the supple- mented swine appeared to be between 1/320 and 1/640. The results of the preliminary study to compare the effects of the stress of a T. G. E. infection on tocopherol- deficient and supplemented animals are presented in Table 39. page 112. One t000pherol-deficient animal (N86) succumbed 112 Table 39.--Summary of responses of four-month-old toc0pherol- deficient and supplemented swine to an, exposure to trans- missible gastroenteritis virus (T. G. E.). ’ Obser- Date Tocopheggl-Degicient Tocopherol-Supplemented vations NBS N WB2 9/28 Temp. 103.2 102.7 102.8 102.7 Stool Normal Normal Normal Normal LH + + f + 2:00 p.m.--.5 cc. diluted 1206-9 T. G. E. virus / administered/pound body weight. 9 29 Temp. 103.5 Dead 102.5 103.6 Anorexia ++ - +++ Diarrhea - - - meition t I i - 9/30 Temp. 103.5 103.4 102.0 Anorexia + ++ +++ Diarrhea ++ _ +++ 10/1 Temp. 10107 ‘03.} ‘0390 Anorexia - - + Diarrhea - - +++ LB + - - 10/2 Temp. 103.0 103.8 102.3 Anorexia - - - Diarrhea - + +++ 10/3' Temp. - 101.4 102.8 102.5 Diarrhea - +++ +++ 10/4 Diarrhea - ++ +++ 10/4 Temp. 103.8 100. 102.0 Diarrhea - + ++ 10/6 Temp. - 103.6 102.4 103.0 Diarrhea - - + W? Diarrhea - - - -Absent +Mild 1Questionable ++M0derate +++Severe 113 sometime within 15 hours after the T. G. E. virus was admin- istered. An acute gastritis (Figure 27 on page 116) was noted in this pig. The other tocopherol-deficient pig, NBS, on the other hand, reacted mildly to the infection and recovered 3 days after the virus was administered. The tocopherol-supplemented animals elicited a greater enteric response following exposure to the virus than did the sur- viving toc0pherol-deficient animal. Unfortunately, suffi- cient tocopherol-deficient animals were not available to repeat this study for more conclusive data. Pathology Symptom§.--With the exception of two animals which had a pronounced cyanosis on occasion, especially of the ears, at about 90 days of age, no other symptoms associated with a tocopherol deficiency were observed in either lot A or B. Gross lesions.--The most consistent gross pathology observed in the toc0pherol-deficient swine was the hepatic involvement as shown in Figure 28 on page 117. The livers appeared to have a peri lobular to generalized fatty infil- tration with questionable necrotic areas on the non-diaphragm- atic surfaces. One liver had raised white 1 mm. nodules scattered over its diaphragmatic surface. Bilateral edema and crepitant swellings were observed in the posterior quarters and axillary regions of animals that died in both lots A and B. Two animals that died in lot A had ecchymotic to suffusion type hemorrhages in the gracilis muscle (Figure 29, page 118) and flank area (Figure 30, page 118). 114 Microscopic lesions.--Extensive myopathy was not a characteristic of the tocopherol-deficient swine that died or were sacrificed in this experiment; however, when myOpathy was observed, it involved the adductor and gracilis muscles similarly. The myOpathy in lots A and B was characterized by swollen fibers, interfascicular hemorrhage (Figure 31, page 119), edema of the endomysial spaces (Figure 32 on page 119), central nuclei in hyalinized fibers (Figure 33 on page 120), internal nuclear rowing, fragmentation and lysis of the muscle fibers (Figure 34, page 120), and myoblast pro- liferation (Figure 35. page 121). The latter was especially noticed in the sacrificed ethyl linoleate-supplemented swine. The myocardium of the tocOpherol-deficient animals appeared to have longer and more prominent chains of internal nuclei than was considered normal. Also, numerous bizarre nuclei were evident. Unlike the plump, foamy.Purkinje fibers of normal swine (Figure 36, page 122), thequrkinJe fibers of tocopherol-deficient swine were shrunken and atrOphic as in Figures 37 and 38 on page 122. The hepatic lesions of lots A and B were not unlike one another and consisted principally of centrolobulax~ hemorrhage as shown in Figure 39, page 123. Hemosiderosis was frequently observed as a result of the hemorrhage and the interlobular septae were often edematous. Renal lesions in both tocopherol-deficient lots were limited to medullary peritubular edema, cortical peri- 115 tubular hemorrhage and hemosiderosis of the convoluted tubule epithelium. Congestion and petechial hemorrhages were frequently observed in the adrenal cortex. On one occasion, what appeared to be vacuolar degeneration was noted in the zona glomerulosa of a tocopherol-deficient pig. The normal and degenerative glomerulosa zonae are illustrated in Figures 40 and 41 on page 124. Less frequently observed lesions which were considered associated with tocopherol deficiency included congestion and hemorrhage of the testes and a perivascular edema of the cerebrum. In the ethyl linoleate-supplemented group, a nonpigmented steatitis was observed that was character- ized by a segmented appearance of the fat cells, Figure 42, page 125. Pulmonary lesions were seen in swine from ‘ each eXperimental lot. Pulmonary congestion and interalveolar serous exudation (Figure 43, page 126) were observed in the toc0pherol-deficient swine that died suddenly. The pneu- monitis shown in Figure 44, page 126, was found in the toc0pherol-supplemented and deficient swine; however, it was more extensive in the latter group. 116 Figure 27.--Acute gastritis of a pig exposed to transmissible gastroenteritis virus. T17 \ Figure 28.--Perilobular and generalized fatty infiltration of the liver from a tocopherol- deficient pig. Figure 29.--Suffusion hemorrhages in a graci- lis from a tocopherol-deficient pig. Figure 30.--Subcutaneous and fascial hemorrhages of a tocopherol-deficient pig. 119 Figure 31.--Hemorrhage in a gracilis from a tocopherol-deficient pig. (x1000) Figure 32.--Endomysia1 edema in an adductor from a tocopherol-deficient pig. (x1000 120 Figure 33.--Adductor from a tocopherol-deficient ?1%. )A. Endomysial edema; B. Internal nuclei. x 50 Figure 34.--Interna1 nuclear rowing in a fiber of an adductor from a tocopherol-deficient pig. x390 121 -‘ Figure 35.--Adductor from a tocopherol-deficient pig. A. Myoblastic proliferation; B. Endo- mysial edema. (x1050) Figure 36. .--Purkinge fibers from a toc0pherol-supple- mented pig. (x26 Figures 37 and 38.--Purkinje fibers from tocopherol- deficient swine. (x700) . ‘ .7 , ‘- 1. .3; ‘ fr. ). ‘Vx‘ ’ " .,_ 1‘ ,>.& .A 5 1.5%. 'N 1. v "‘X' 1 ’7 ‘ ‘$ 14* ., ¥ a ‘ :72... x if -~\‘\-7 , A i g1 , - {2" , .... fl, ‘4 1". ' 3 '. ‘3 _ ;. s, , . '- uf-L- _ ' 33.3 agivv" ,5» . . re"; -3 - /--- 513-. . .'-“ \ ‘. ‘5. «2", G Q; . Figure 39.--Liver from a tocopherol-deficient pig. A. Centrolobular hemorrhage; and B. Interlobular septal edema. (x110) Figure 40.--Adrenal gland from a tocopherol- supplemented pig. (x260) Figure 41.--Vacuolar degeneration of the zona glomerulosa from a tocopherol-deficient pig. (x260) 125 Figure 42.--Adipose tissue from a tocopherol- deficient pig. Note segmented appearance of the fat cells. (x710) Figure 43.--Lung from a tocopherol-deficient pig. A. Serous, alveolar exudate; and B. Interalveolar pulmonary congestion. (x260) Figure 44.--Pneumonitis of a tocopherol- deficient pig.(x320) Discussion Growth Although poor gains were made by the swine in this experiment, there were no differences among the lots which could be attributed to toc0pherol deficiency. This finding concurs with the results of Pelligrini (1958) who used a similar semi-purified type ration containing torula yeast as the principal protein. Also, Dammers gt 3;. (1958), who studied tocopherol deficiency in swine fed a more conventional ration,found no differences in growth attributable to a toc0pherol deficiency. Mortality The sudden deaths and the rapidly developing post- mortem tympanites are considered significant among the mortality data. Evidence is available to the effect that at least two of these deaths, which were followed by the rapid post-mortem tympanites, were due to an overwhelming stress factor of saprophytic clostridial organisms. Large colonies of clostridium-like rods were found in the liver of one pig that succumbed in lot A and 9;. perfgingens was? was cultured from the intestine of another severely bloated _ lot A animal with hemorrhages and crepitating swellings in the subcutaneous tissues of the flank area. or the animals sacrificed in each group, only Salmonella newport, Salmonella oranienburg and Proteus vulgaris were 127 128 isolated from the intestine. Bacteria were not isolated from other organs. The highly concentrated rations being consumed might have predisposed these swine to deaths from clostridial infections. However, the resistance to such infections demonstrated by the tocopherol-supplemented lot is another indication of the inability of the tocopherol- deficient swine to withstand stress--in this case, the stress of saprophytic organisms. Hematology With the exception of the low number of band neutro- phils, the total white and differential counts are within the normal range for swine given by Calhoun and Smith (1958).. Likewise, the packed cell volume and hemoglobin values for each lot are within the ranges considered normal for swine by Miller gt al. (1961a). The absence of a tocopherol effect upon these hematological factors supports the findings of Pelligrini (1958) and Borgman (1959) who also found these factors unaltered during a tocopherol deficiency in swine and rabbits respectively. The layering hemolysis phenomenon required 77 days to elicit in swine. However, its occurrence coincided with the first elevated transaminase values and with serum toco- pherol values below 65 micrograms/100 ml. serum. Although the first elevated dialuric acid values for swine occurred two weeks prior to the layering hemolysis phenomenon, the latter is considered a more reliable indirect indicator of toc0pherol deficiency than the dialuric acid 129 test. This is because the hemolytic action of dialuric acid is inhibited by contact with such inert laboratory materials as polyethylene. Forbes and Draper (1958) were unable to demonstrate an increased susceptibility of toc0pherol-defi- cient swine erythrocytes to dialuric acid hemolysis. Two possible reasons for this are considered here. Either their experiment was not continued for a sufficient length of time or all the precautions for the dialuric acid test, as outlined by Friedman (1958), were not strictly observed. Serology Comparing the serum protein electrophoretic patterns obtained in this study with those for normal swine obtained by Miller gt 5;. (1961), the serum albumin is lower by approximately 10 per cent, alpha and beta globulin fractions are within the normal range and gamma globulin is 10 per 1 cent higher than normal. The high gamma globulin values may be_a reflection of a response to the severe diarrhea problem encountered in the initial phase of this experiment. The absence of an association between the electrOphoretic patterns of serum proteins and tocopherol deficiency is in agreement with the work of Bottiglioni (1957) in the rat. but it is in contrast to the report of Kesler (1961) who found dystrophic sheep to have an increased alpha-globulin fraction and decreased beta and gamma globulin fractions. Cornelius and associates (1959) reported normal serum glutamic-oxalacetic and glutamic-pyruvic transaminase values for swine as 31.1114.1 and 27.317.8 respectively. Wretlind 130 lg; filo (1959) gave normal SGOT and SGPT values for swine as) 28.03t15.39 and 20.37:5.54 respectively. In comparison to these values considered normal, the pre-layering hemolysis SGOT and SGPT values obtained during this experiment are elevated with means of 40.931.6 and 37.412.1 for SGOT and SGPT respectively. Finding elevated post-layering hemolysis transaminase values for the tocopherol-deficient swine in this experiment supports the work of Orstadius 11;. 3;. (1959) and Lannek _e_1:._ 5;. (1961) who demonstrated elevated SGOT and SGPT values in field cases of both toxic liver dystrOphy and muscular dystrophy of swine. Based on the extent of the pathology found in the muscles and liver of these tocopherol-deficient swine, the transaminase determinations appear to be as sensitive myopathy indicators for swine as Kutler and Marble (1958) have found them to be in lambs and calves. It is apparent that 100 p.p.m. of alpha-tocOpherol in semi-purified type rations are sufficient to maintain the toc0pherol level in swine serum near 225 micrograms/100 ml. of serum. This amount is adequate to maintain the integrity of erythrocytes and prevent lesions of muscle, adipose tissue and liver associated with toc0pherol deficiency in swine. These toc0pherol deficiency lesions in swine can be anticipated when total serum toc0pherol values fall below 65 micrograms/100 ml. The available data, however limited, pertaining to the serum toc0pherol values for swine appear to be in agreement 131 with the data obtained in this experiment. Lost (1961) reported plasma tocopherol values of 26-60 micrograms/100 ml. for swine fed less than .84 mgs. of mixed toc0pherols/100 grams of meal and values of 350 to 450 micrograms for swine fed 10 mg. of alpha-tocopherol succinate/100 grams of meal. Bratzler 23,51. (1950) reported tocopherol values of 248, 668 and 594 micrograms/100 ml. of plasma from swine fed concentrates supplemented with 3. 55 and 110 mg. of mixed toc0pherols/kg. of live weight respectively. These workers, therefore, drew the conclusions that plasma toc0pherol levels did not indicate the level of tocopherol supplemen- tation. Unfortunately, Obel (1953) Save no serum toc0pherol data in her extensive study of hepatosis diaetetica. The results of the preliminary studies relative to the responses of tocOpherol-deficient and supplemented swine to T. G. E. virus and Sglmonglla pullorum antigen are some- what parallel. Toc0pherol-supplemented swine appeared to have lower Salmgnella pullorum antibody titers than did the tocopherol-deficient swine fellowing the series of g. pullgrum antigen injections. Possibly because of associated reasons, tocopherol-supplemented swine showed a more severe enteric response (diarrhea) than did the surviving tocopherol- deficient pig. (Although one tocopherol-deficient pig died following exposure to the T. G. E. virus, this death occurred too soon--about 15 hours--after the T. G. E. was administered to have been directly caused by the virus.)' These results are not in agreement with the report of Axelrod and Pruzansky 132 (1955) who indicated that antibody production is not altered during tocopherol deficiency, at least in the chick. Pathology The cyanosis observed in this experiment is considered a manifestation of a stage of tocopherol depletion and was reported by Obel (1953) as a part of the clinical picture in spontaneous hepatosis diaetetica. The adequacy of the ration for the sulfur-containing amino acids is considered responsible for the relative absence of hepatic necrosis in the animals that died on the toc0pherol-deficient rations. These rations were designed to meet the requirement of swine for DL methionine as reported by the National Research Council (.6% of the diet on a dry weight basis) yet have the limiting amino acid in the sulfur-containing amino acid group. With specific reference to internal nuclear rowing of skeletal muscle, the myOpathy observed in swine was more characteristic of that described by West and Mason (1958) in the cheek pouch of the tocopherol-deficient hamster than has been described for other species. The hemorrhagic myositis, hepatitis, nephritis and orchitis observed are considered related to capillary and arteriolar permeability. As reported in the mink experiments, Grant ( 1961) has demonstrated a microangiOpathy (MAP), char- acterized by the sub-endothelial accumulation of periodic-. acid-Schiff positive material in tocopherol-deficient swine. However, sub-endothelial, PAS positive material was not 133 demonstrated in tOCOpherol-deficient swine on this exper- iment. The Purkinje fiber degeneration observed in swine has been reported by Xaplesden and Loosli (1960) to occur in the tOCOpherol-deficient calf; however, they did not illustrate their observations nor elaborate further on the pathology. The tocOpherol deficiency produced in swine from lot A was not associated with steatitis and was therefore considered uncomplicated. In swine from lot B, the isocaloric addition of 5 per cent ethyl linoleate provided insufficient unsatu- rated fatty acid to complicate the tocopherol deficiency with an acid-fast-pigmented steatitis but did result in an intermediate form of steatitis not previously illustrated. SUP-INARY Experimental tocopherol deficiency was produced in mink and swine fed semipurified type rations. DevelOpment of the toc0pherol deficiency was evaluated in terms of growth rates, symptomatology, biweekly hematological and serological studies, gross pathology and histopathology. The effects of toc0pherol deficiency upon antibody production were determined by com- paring the Sglmqgellg pullorum antibody titers of toc0pherol- deficient and supplemented animals following a series of Salmonella pullorum antigen injections. The t000pherol-deplet- ing effects of cod liver oil (mink) and ethyl linoleate (swine), the tocopherol-sparing effects of selenium and ethoxyquin, and the species requirement for tocopherol were measured by erythrocyte fragility tests and histologic tech- niques. Alterations in growth rates or consistent physical symptoms were not characteristic of tocopherol deficiency in mink or swine; however, sudden deaths were frequent among the toco- pherol-deficient animals. The deaths among the deficient mink usually followed exposure to a stress factor. The most significant hematological alteration that coin- cided with toc0pherol depletion was an increased erythrocyte fragility that was indicated in both species by a 48-hour layered hemolysis in refrigerated saline. The increased fragility was also detected in swine~by the dialuric acid hemolysis test. An absolute neutrOphilia was associated with 134 135 tocopherol-deficient mink that had steatitis. Packed cell volumes and hemoglobin concentrations increased with the age of mink and were unaffected by toc0pherol deficiency in either mink or swine. Prolonged tOCOpherol deficiency of mink (Experiment II) resulted in decreased serum albumin and increased alpha and beta globulin fractions while the serum protein fractions were not altered by toc0pherol deficiency in swine. Elevated serum glutamic-oxalacetic and glutamic-pyruvic transaminase values and serum toc0pherol values between 50 and 70 micro- grams/100 ml. were associated with tocopherol deficiency lesions. Grossly, internal intercostal and adductor myOpathy was common in toc0pherol-deficient mink while subcutaneous edema, hemorrhagic myositis and a perilobular to generalized fatty infiltration of the liver were noted in the tocopherol-defi- cient swine. Histologically, the skeletal myOpathy of tOCOpherol- deficient mink consisted of swollen, differentially stained fibers, vacuolar degeneration, sarcolemmal and myoblastic pro- liferation and calcification of the non-phagocytized myofi- brillae. Also associated with the deficiency in mink were calcified necrotic myocarditis, centrolobular hepatic hemorrhage, coagulation necrosis with calcification of the convoluted tubules and calcified necrotic foci in the adrenal cortex. Microscopic lesions characteristic of toc0pherol defi- ciency in swine were centrolobular hepatic hemorrhage, 136 endomysial edema, hemorrhagic myositis, a slight myoblastic proliferation, hyalinized adductor fibers containing rowed internal nuclei and Purkinje fiber degeneration. Sarcolemmai proliferation and calcification of the myofibrillae were not observed in swine while the internal nuclear rowing of skeletal muscle was not characteristic of tocopherol deficiency in mink. Toc0pherol deficiency did not impair or enhance the antibody response of mink to Salmonella pullorum antigen; however, tocooherol-deficient swine demonstrated a greater ability to produce Sglmggellg pgllggum antibody than did toc0pherol-supplemented swine. Isocaloric supplementation of 8% cod liver oil to the tOCOpherol-deficient ration fed to mink caused the deposition of a yellow, acid-fast pigment in the interstices of the adipose tissue. An isocaloric supplement of 5% ethyl lino- leate to the tocopherol-deficient ration fed to swine, on the other hand, did not cause the acid-fast-pigmented steatitis. Neither supplement hastened toc0pherol depletion as measured by biweekly serum tocopherol values. Selenium supplementation at the rates of 0.1 and 1 p.p.m. prevented fatal toc0pherol deficiency lesions in mink. Neither selenium nor ethoxyquin was effective in preventing the 48-hour hemolysis phenomenon in mink or swine. 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