r'r-p-r- lva r' '_r‘ wt-” 4- . . .-a7:.:-.1 t”. {We 2:41 If .3 4339":- {If 3/11.in ' " cm " "Qw‘ 51%;: 5353):?” '7 ”1%: I" 1:: ,1 2”" J, 75“ //-,, €39 0 3". 1,. (53101;: ‘t.'f5?p.‘luj ,‘951' “I 0;)? 94.)?“ ”J (-4,; :‘Af‘ (flu. . (C L ,1} 1:1 :2 - f( ,- "Saw.- . '5‘}? Iii-.I. «my. . :5” If») "1‘ .. u». ‘* I .vq‘k‘éu'? ‘ J ‘fL‘p'i? I "t ‘ (f 1" 1’: \I 5. )W _ €1.24 .: “' 551:1": an»? 3-21“; 1’)?" .w ' «1/1"! :1;V{ -.1.-,|:,'.. I”; .“P :tll‘hib 11;?! ‘1‘: 7‘ "4‘51,“ 515‘) ‘. 5 (1‘1" "‘3 :11 If 1 ‘5 3?; -25” 1'.le wiggwwws ‘ . 2. rL‘ t 1,5. a. 5.“ Ni. '15) v, ,‘ .i‘” ‘1; £11.: .,1 21-.- cr‘ sfiéyhgfim. ‘ 1, H. :33 €53} ‘ 1 k' , r1 .‘5' 5} ., .2 1% MM » ; #:1555949?“ - 1 2.3!}: . V9.45. ,1 - h‘ . “5131- . “1‘5 '1': ”‘41)“: .n 444 . (1'14" "" ’g'WE/ .I‘. J‘:\ ‘ SVA' 3‘!“ ‘ 'fil-y/T-N . ’13.? I .‘ ' . ‘ “ ”fits; V§V>{V“E‘gh’v\xv s’q‘f>\ 4:“) 3""1“, 133:1; 1&4; . ,, . _-< , 1'" . IA“ ‘7'“ I ’fl-"x‘s tan”; 1"" a f p. ‘5”. )\. '37, 1;; . ”as; '4'”? 553*:.'v3";"'2‘ ff (NW 3%. . s s "1. ’1 1" 1”“ . . 152' hm“- ' 121:: ,w "it"ifi 5" “{3}ng rui ”I Y ‘I ”'7‘; ' ("1”{4’71‘7‘ ‘ 1‘ . ‘ {73% ‘w "M. ’{v‘<"l M1". 11’,” ‘1 ‘ 7n: ‘ ”*‘(nyg‘w’V‘zfiggfifl 3‘0 7., 1' ‘ 193. “(:4 . l ‘ . 2 . ' [I ‘- N 1.‘ ., ,~'1,s, (HIV .' . . .1'1 ' 3hr. .(u. Pg}. n." . . ~‘2‘,: 2" .. . #541111“ If . 15:2} #641. 2313,? wig-'1‘:c .111;er9 . r" (({j'zx‘ fiw“ k] ; .pw‘q; '1,- 1 44-45.. - .4 «sffigfiéw‘i‘éséjx 3 ‘ "$112.";fiaz'1télfiflgifim fit "I“ ,, 31$? V7170 . ‘ . Jun ".11.._ thrgpg; "‘44? "WW “7'“ {3“ 33‘ N‘ 1"? 1" , “ I} “a ‘36 ”3"”; - 1g ' , .'=. :2; “9,5 133;?" 31391 fix 9‘5 "'3‘ figxqfigfi $4431; "451-4. firm I” v, f §»‘_‘xg‘£v‘i 2‘ 111.45: «21;. ' I I (“9.554. 1 .31..” . “C l "m I‘I‘N' 'r» / ,1". ' L r 51¢. 59.11.57: ’ ,1 A r -' J‘rv/j' {'1 111’? node: é’g» A “1‘41, ”I" / "71.93pm: ‘UVIJ u,, 1::‘9'51U.’ I". 5 72. {a . . l ‘1' ‘7 (1.," JJ, 3;: r. , .".n.-.‘.,{" H”! 5, ‘5." 1'1, 1... 1 .1" ' 21f; ' » , ..—, {[1 m ,. “r. n '- ‘21 v V ,II. 5/" .’5 1 q._ . (Mr-w». I; . .. ‘gémm" 74' ' ‘1’ . . m - «firs "wt; ”2* grain?!" . . , b15711 atm'vw‘ffx ‘ _ “(Ev-Q, A: -‘ ' , -' egg . % %u§441yfl¥_p&l 93V} "3 i5?” .4 13.3ch a. 4' And 335:6 (In. ~_ _" . 1.531.115 : IlllllHllIllllllllllllllllllllllllllllllllllllllll!IlllllHll a LIBRARY ‘ 3 1293 00788 1075 iMich’s’gan 31:33::- i 3 University E a This is to certify that the dissertation entitled EFFECTS OF VITAMIN E AND SELENIUM ON THE NUMBER AND IMMUNORESPONSIVENESS OF CELLULAR COMPONENTS OF SOW PERIPHERAL BLOOD, COLOSTRUM AND MILK presented by HASTARI WURYASTUTI has been accepted towards fulfillment of the requirements for Ph'D degreein %m Om Major professor Dr. H. D. Stowe DMe November 10, 1989 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 Large Animal Clin. Sci. 0" 9E“. L” ' z PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. ' DATE DUE DATE DUE DATE DUE .533. 2 6 YEW l || _ —_T‘ MSU Is An Affirmative Action/Equal Opportunity Institution eWMS-o: EFFECTS OF VITAMIN E AND SELENIUM ON THE NUMBER AND IMMUNORESPONSIVENESS OF CELLULAR COMPONENTS OF SOW PERIPHERAL BLOOD, COLOSTRUM AND MILK By Hastari Wuryastuti A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Large Animal Clinical Sciences 1989 053 (50 ABSTRACT EFFECTS OF VITAMIN E AND SELENIUM ON THE NUMBER AND IMMUNORESPONSIVENESS OF CELLULAR COMPONENTS OF SOW PERIPHERAL BLOOD, COLOSTRUM AND MILK By Hastari Wuryastuti Twenty four multiparous sows were bred as they came into postweaning estrus, and assigned to one of the following dried high moisture-corn / soybean meal—based gestation diets: unsupplemented (-E-Se); plus 0.3 mg selenium/kg (-E+Se); plus 60 IU vitamin E/kg (+E-Se) and plus 0.3 mg selenium/kg and 60 IU vitamin E/kg (+E+Se/ control). Blood samples for cell isolation and for vitamin E, selenium, glutathione peroxidase and cholesterol analy- ses were collected at 0, 30, 60 and 90 days of gestation and at parturition. Colostrum and milk samples were ob- tained at parturition and on postpartum day 4 , respective- ly, for cell isolation. Immunoresponsiveness of cellular components of peripheral blood, colostrum and milk were de- termined by measuring the lymphocyte proliferation response to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), and the phagocytic and microbicidal activities of polymorphonuclear (PMN) cells. At 90 days of gestation and at parturition, vitamin E-deficiency reduced (p<0.05) the response of peripheral blood lymphocytes (PBL) to PHA and PWM stimulation. Reduction in the response of PBL to PHA and PWM was also observed in sows fed the -E-Se diet, as early as 60 days of pregnancy. The -E+Se and -E-Se diets were associated with reduced (p<0.05) responses of colostral lymphocytes to PHA whereas only the -E-Se diet reduced (p<0.05) the response of colostral lymphocytes to PWM. Phagocytic activity of sow blood PMN cells was reduced (p<0.05) by 90 days of gestation and at parturition for sows fed -E+Se and +E-Se diets and by 60 days of gestation in sows fed -E-Se diet. Only sows fed the -E-Se diet had lower phagocytic activity of colostral PMN cells. Vitamin E-deficiency, selenium-deficiency and a combined vitamin E and selenium-deficiency reduced (p<0.0S) the microbicidal activity of blood and colostral PMN cells. A significant decrease in microbicidal activity of milk PMN cells was only observed in sows fed -E-Se diet. This study indicates that vitamin E deficiency signifi- cantly depressed functions of both B and T lymphocytes and PMN cells in gestating sows. Selenium deficiency, however, only depressed the PMN cell function. Dedicated with love to my mother, my father and my husband, R. Wasito ii ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. H. D. Stowe, my major professor, for his encouragement, patience and valuable advice during my course of study. I am also grateful to my committee members, Dr. R. W. Bull, Dr. E. R. Miller, Dr. S. D. Sleight and Dr. B. J. Thacker for their help in reviewing this manuscript and willingness to share their professional expertise. Special thanks go to the Government of the Republic of Indonesia, the MUCIA-Indonesia educational project and the College of Veterinary Medicine, Michigan State University for their financial support. I am indebted to Dr. R. W. Bull and Peggy Coffman for allowing me to use the immunology laboratory facilities for portions of the assays, and their professional assistance given in the laboratory. My appreciation also to Paul Matzat, Ray Kramer, Steve Mussell and DuWain Simon for their assistance with the research animals; to Anne House, Tonie Thiel, Beta Borer, Faith Manning and Kathy O'hare for their assistance with the laboratory work, their warmth and their friendship; and to Mary Ellen Shea for her help in preparing the figures. iii Finally, my deepest appreciation to my husband, Dr. R. Wasito who has tolerated and endured my mental and/or physical absence and most of all for his continous love and support making the completion of this degree a reality. iv TABLE OF CONTENTS LIST OF TABLES ..................................... LIST OF FIGURES .................................... INTRODUCTION ....................................... LITERATURE REVIEW .................................. Historical ..................................... Function ....................................... Deficiency ..................................... Toxicity ....................................... MATERIALS AND METHODS .............................. Animals and diets .............................. Sample collections ............................. Isolation of cells ............................. Peripheral blood lymphocytes .............. RBC decontamination ....................... Polymorphonuclear cells ................... Colostrum and milk ........................ Lymphocyte blastogenesis assay ................. Yeast phagocytic assay ......................... Preparation of yeast solution ............. Test procedure ............................ 10 19 26 29 32 32 35 36 36 37 37 38 39 40 41 41 Vitamin E analyses ............................ 42 Serum ..................................... 42 Feed ...................................... 43 Cholesterol analysis .......................... 44 Selenium analyses ............................. 44 Serum ..................................... 44 Feed ...................................... 46 Glutathione peroxidase activity ............... 47 Statistical analysis .......................... 48 RESULTS ........................................... 50 DISCUSSION ........................................ 83 SUMMARY AND CONCLUSIONS ........................... 93 LIST OF REFERENCES ................................ 96 APPENDIX .......................................... 115 vi Table 10 11 LIST OF TABLES Vitamin E and/or selenium responsive diseases in animals .......................... Experimental periods ......................... Composition of diets .......................... Differential white cell counts of colostrum from sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... Differential white cell counts of milk from sows fed vitamin E and/or selenium depletion diets between conception and parturition ...... 3H-thymidine uptake of mitogen-stimulated colostral lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition .................... Phagocytic activity of colostral and milk polymorphonuclear cells of sows fed vitamin E and/or selenium depletion diets between conception and parturition .................... Microbicidal activity of colostral and milk polymorphonuclear cells of sows fed vitamin E and/or selenium depletion diets between conception and parturition .................... 3H-thymidine uptake of mitogen-stimulated milk lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition ............................... Serum vitamin E concentrations of sows fed vitamin E and/or selenium depletion diets between conception and parturition ............ Serum selenium concentrations of sows fed vitamin E and/or selenium depletion diets between conception and parturition ............ vii Page 33 34 75 76 77 79 8O 82 Table Page 12 Serum glutathione peroxidase concentrations of sows fed vitamin E and/or selenium depletion diets between conception and parturition ...... 117 13 Serum cholesterol concentrations of sows fed vitamin E and/or selenium depletion diets between conception and parturition ............ 118 14 3H-thymidine uptake of unstimulated peripheral blood lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... 119 15 3H-thymidine uptake of FHA-stimulated peripheral blood lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... 120 16 3H-thymidine uptake of PWM-stimulated peripheral blood lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... 121 173 H-thymidine uptake of Con A-stimulated peripheral blood lymphocytes of sows fed vitamin E and/or selenium depletion diets between conception and parturition ............................... 122 18 Phagocytic activity of blood polymorphonuclear cells of sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... 123 19 Microbicidal activity of blood polymorhonuclear cells of sows fed vitamin E and/or selenium depletion diets between conception and parturition ................................... 124 20 Reproductive performance of sows fed vitamin E and/or selenium depletion diets between conception and parturition .................... 125 21 Normal cellular components of peripheral blood, colostrum and milk of sows, cows and ewes ..... 126 viii Figure LIST OF FIGURES Function of selenium and its relationship to the antioxidant function of vitamin E ...... The effects of vitamin E and selenium- dependent GSH-Px on the metabolic pathway of arachidonic acid .............................. Serum vitamin E concentrations in sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ................................ Serum selenium concentrations in sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ................................ Serum glutathione peroxidase activity in sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se from conception to parturition ................................ Serum cholesterol concentrations in sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ................................ Unstimulated peripheral blood lymphocytes from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ..................... PHA stimulatory responses of peripheral blood lymphocytes from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ...... PWM stimulatory responses of peripheral blood lymphocytes from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ..... ix Page 25 52 54 57 59 62 . 64 Figure 10 11 12 Page Con A stimulatory responses of peripheral blood lymphocytes from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception to parturition ..... 69 Phagocytic activity of blood polymorphonuclear cells from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E—Se and +E+Se diets from conception to parturition ........... 71 Microbicidal activity of blood polymorphonuclear cells from sows fed: (a) -E+Se and +E+Se diets; (b) +E-Se and +E+Se diets; (c) -E-Se and +E+Se diets from conception and parturition .......... 73 INTRODUCTION It is generally accepted that severe multinutrient de- ficiencies lead to immune system dysfunction. However, de- ficiencies, excesses or imbalances of a single nutrient can also adversely influence host resistance. For example, vi- tamin A deficiency in rats caused a significant reduction in their response to T- and B-cell mitogens, a situation reversible upon supplementation with vitamin A (Beisel, 1982). Iron deficiency was associated with an impaired lymphocyte stimulation response to mitogens and decreased neutrophil bacterial capacity (Krantman g; 11., 1982; Kuvibidila g; 91., 1983). However, excess iron can also disrupt the host's ability to defend itself against infec- tious disease (Sherman, 1984). During the last decade, it has been demonstrated that vitamin E exerts a significant influence on immune re- sponse. Because of the close nutritional and bichemical relatonship between selenium and vitamin E , the involve- ment of dietary selenium in immunity has also been studied in both animals and humans. Decreased bactericidal activity of polymorphonuclear cells has been observed in vitamin E and/or selenium-deficient rabbits (Lafuze g; 51., 1983), man (Arvilommi g; 11., 1983), goats (A212 at 51., 1984), l 2 cattle (Gyang g; a1., 1984) and mice (Boyne and Arthur, 1986). Depressed proliferative response of lymphocytes to various mitogens has been demonstrated in vitamin E and/or selenium-deficient mice (Corwin and Shloss, 1980), pigs (Larsen and Tollersrud, 1981; Jensen g; 51., 1988) and dogs (Langweiler g1, 31., 1981). Vitamin E and/or selenium defi- ciency has also been associated with the reduction of anti- body production against foreign pathogens (Reffet g; 31., 1988a,b; Marsh g; a1., 1981). Some studies have indicated that vitamin E and/or selenium-deficient animals are more susceptible to infectious diseases than vitamin E and/or selenium-adequate animals. Teige g; 51, (1982) reported that pigs fed a vitamin E- and selenium-deficeint diet had decreased resistance to experimentally induced Iggponema hyodysenteriae. The metritis-mastitis-agalactia syndrome of sows has been associated with inadequate dietary vitamin E and selenium (Whitehair et al., 1983). If the infectious aspects (metritis and mastitis) of this complex were mani- festations of impaired immunity, the sow could serve as an animal model for investigations of immunocompetence. It is known that ingestion of mammary secretions by neonates serves two major purposes: to transfer immunity from mother to the offspring before they can produce their own antibodies and to sustain the young nutritionally during the early weeks of life. 3 Recent research interest in the immunobiology of mammary secretions has focused not only upon the secretory immunoglobulin components and their contribution to local intestinal mucosa immunity in the neonate, but also upon the significant immunologic role of those viable leuko- cytes present in the mammary secretions (Schollenberger g; 91., 1986). Drew g; 91. (1983) have reported that human colostral lymphocytes can respond in vitro to various nonspecific mitogens such as phytohemagglutinin (PHA) and concanavalin A (Con A). Megs and Beer (1979) have demonstrated that the phagocytic cells in colostrum and milk of humans have the capacity to phagocytize and kill both E. gg11 and C. albicans. Erskine 9; a1. (1987) suggested that supplementa- tion of 1000 IU vitamin E and 6 mg selenium per cow per day during the dry period reduced somatic cell counts and inci- dence of mastitis in subsequent lactations; however, the immunoresponsiveness of colostral and/or milk leukocytes was not assessed. We therefore, decided to study the influence of vitamin E and/or selenium on the immunoresponsiveness of cellular components of peripheral blood, colostrum and milk of sows. The specific objectives of this study were: (1) to determine the effects of dietary vitamin E and/or selenium on immunoresponsiveness of cellular components of peripher- al blood of gestating and peripartum sows, (2) to charac- terize the cellular components of porcine colostrum and 4 milk and (3) to determine the effects of dietary vitamin E and/or selenium on the immunoresponsiveness of the cellular components of colostrum and milk of these sows. LITERATURE REVIEW Historical Vitamin E: Vitamin E was discovered in 1920 by Matill and Conklin. They observed that rats fed basal semi- purified diets, supplemented with all vitamins known at that time, were able to grow normally but were unable to reproduce. Two years later, Evans and Bishop (1922) found that an unknown dietary factor, first called factor X, in fresh green lettuce leaves and wheat germ prevented steril- ity in rats. The factor X was then known as fat-soluble vitamin E. The findings of Evans and Bishop were supported by independent work of Sure (1924) and Matill _; _1. (1924). Further investigations by Evans and co-workers (1936) led to the isolation from wheat germ oil of an alcohol substance having vitamin E activity which was named alpha tocopherol. The name tocopherol was from the Creek tokos (childbirth) and phero (to bear) and 91 for the alcohol form. Subsequently, 2 other tocopherols were isolated from vegetable oil and were designated as B and \’-tocopherol. These 2 substances have lesser biological activity than a tocopherol (Emerson g; 51., 1937). In the following years, some vitamin E-responsive diseases, such as nutritional muscular dystrophy in 5 6 suckling rats (Olcott, 1938) and rabbits (Mackenzie g; 31., 1940), exudative diathesis in chicks (Dam and Glavind, 1939) and nutritional encephalomalacia (Pappenheimer and Goettsch, 1931) were described. 52123133; Selenium was first recognized in 1934 as a result of its association with the poisoning of farm animals (Franke, 1934). Interest in this element increased considerably following the discovery by Schwarz and Foltz (1957) that selenium was an integral part of the "Factor 3" that protected against dietary liver degeneration occurring in rats fed Torula yeast diets. This finding was supported by Patterson g; 31. (1957) who found the beneficial effect of selenium in preventing exudative diathesis in chicks. Subsequently, selenium has been found to alleviate several vitamin E-responsive animal diseases of practical agricul- tural importance, such as white muscle disease in lambs and calves and hepatosis dietetica in young pigs. Extensive work had been reported concerning the biolog- ical activity of selenium compounds, both inorganic and or- ganic. Sodium selenite was active whereas elemental selenium was inactive. Organic selenium compounds varied widely in their bio-potencies, some being inactive and others more active than sodium selenite against dietary liver necrosis in rats (Schwarz and Foltz, 1958; Schwarz and Fredga, 1969; Schwarz, 1961). Glutathione peroxidase: In the 19503, the enzyme glutathione peroxidase (CSH-Px) was discovered by Mills (1957) and Mills and Randall (1958). They reported that GSH-Px was primarily located in cell cytosol and mitochon- drial matrix where it could protect hemoglobin from ascorbic acid-induced oxidation but only in the presence of glucose. This finding was confirmed by Cohen and Hochstein (1963) who also discovered the existence of GSH-Px in erythrocytes and considered it to be an important enzyme for elimination of hydrogen peroxide (H202). Glutathione peroxidase was also reported to be present in other organs such as liver and kidney (Mills, 1959). In 1973, Flohe g; 51. studied extensively the enzymology and biological importance of glutathione per- oxidase and found that, unlike other peroxidases, GSH-Px did not contain heme. The same year, Rotruck 2; a1. (1973) studied this enzyme from rat erythrocytes and discovered that selenium was a component of GSH-Px. 0h 9; a1. (1974) reported that GSH-Px in most species contained 4 gram-atoms of selenium per mole. These discoveries have explained the nutritional function of selenium and have improved the understanding of the complex interrelationships between selenium and vitamin E. Sougggg Vitamin E: Tocopherols (tocols) and tocotrienols (trienols) are two classes of chemically distinct compounds 8 with vitamin E activity. Each class has four isomers known as a , B , Y , and 6 tocols or (r , a , y , and btrienols which occur naturally in variable amounts. Plants are generally better sources of vitamin E than foods of animal origin. This is because of the inability of animals to synthesize vitamin E in their body. Therefore, the vita- min E concentrations in animal tissues or products are usu- ally low and reflect of dietary vitamin E intake (Machlin, 1984; Pike and Brown, 1984). In plants, the highest concen- trations of vitamin E are found in many of the seed oils like soybean, corn, cottonseed, sunflower and wheat germ oil. Some leafy vegetables such as kale and spinach also contain considerable amounts of vitamin E (Bauernfeind, 1980). Selenium: Many studies point to the fact that the selenium contents in feedstuffs from plant origin tend to be greatly influenced by the selenium concentration in the soil. According to Gissel-Nielsen g; 21. (1984), however, a combination of climate and soil conditiom has a more impor- tant effect on the availability of selenium from soil than the selenium concentration in the soil. Selenium that is released from alkaline and well- aerated soils usually is present in the form of selenate. Selenates are highly soluble in water and therefore are easily transported by ground water. In contrast, selenium that is released from acid and poorly-aerated soils is 9 present in relatively reduced insoluble forms such as selenides and selenites. These types of selenium can form stable adsorption complexes with ferric hydroxide and become unavailable to plants (Allaway, 1972; Allaway, 1973). Soil clay content also significantly influences the utilization of selenium by plants. Selenium uptake by plants is generally better from sandy soil than from loamy soil (Sharma and Singh, 1983). As a consequence of the factors previously mentioned, selenium concentration in plants can vary from 0.005 ppm to 5000 ppm (Nigam and McConnell, 1976; Lorenz, 1978; Tan g; 11., 1986). The selenium content of foods of animal origin depends on the selenium intakes of livestock. Animals that are raised in selenium deficient regions will deposit relative- ly low concentrations of this mineral in their edible tissues and products; animals raised in relatively high selenium nutriture, on the other hand, will have much high- er selenium concentrations (Hazell, 1985). According to Burk (1984), however, the variations in selenium contents of animal tissues are less than in plant tissues because animals can conserve selenium when it is in short supply and excrete excesses. 10 METABOLISM Absorption Vitamin E: Studies with ruminants and monogastrics have indicated that vitamin E is absorbed in the mid region of the small intestine and only 20 to 40% of ingested vita- min E and/or its derivatives are absorbed (Gallo-Torres, 1980). Blomstrand and Forsgren (1968) suggested that un- esterified vitamin E is absorbed less efficiently than any of its derivatives. Absorption of vitamin E is also signif- icantly reduced when a large dose of vitamin E is consumed orally (Schmandke g; 51., 1969). Efficiency of vitamin E absorption from the intestine is facilitated by dietary lipids, especially medium chain triglycerides and phospho- lipids, whereas, polyunsaturated fatty acids (PUFA) reduce the absorption of vitamin E (Schmandke and Schmidt, 1965; Mathias g; 91., 1981). Like other fat-soluble vitamins, the intestinal absorp- tion process of vitamin E closely follows the pathways of lipid absorption. During the digestion process, the dietary vitamin E is released from associated foods either as a result of acidity in the stomach or by the activity of proteolytic enzymes. In the next step, vitamin E is dis- solved in small fat globules which are formed from lipids of the ingested food during mechanical mixing of the chyme. The fat droplets are then emulsified within the lumen of the small intestine by the interaction of bile acid and ll pancreatic juice (Mathias g; 11., 1981). The pancreatic enzymes, especially lipase, efficiently hydrolyze tocopherol esters. Bile salts are responsible for solubilizing tocopherols by the formation of mixed micelles together with monoglycerides, long-chain fatty acids, phospholipids and cholesterol (Gallo-Torres, 1980). The absolute requirement for bile acids for tocopherol absorp- tion is clearly demonstrated in children who do not secrete bile acids into the intestinal lumen. These individuals do not absorb vitamin E, and as a result, will develop periph- eral neurologic abnormalities, primarily areflexia and ataxia. The progression of these disorders can be con— trolled by parenteral administration of vitamin E (Rosenblum g; a1., 1981; Guggenheim g; 1., 1982; Sokol g; a1., 1983). The formation of mixed micelles is an important step which allows vitamin E to traverse to the unstirred water layer covering enterocytes, and, thereby, reaching the ab- sorptive surface in the intestinal mucosa (Gallo-Torres, 1970; Mathias g; 1., 1981). The mechanism by which tocopherol penetrates into the mucosal cells is still unclear, however, it may involve diffusion processes (Gallo-Torres, 1980; Machlin, 1984). Selenium: Absorption of selenium occurs in all segments of the small intestine. In a study on rats by Whanger 9; a1. (1976), using758e, it was demonstrated 12 that the greatest absorption of selenite or seleno- methionine occurred from the duodenum with slightly less absorption from jejunum or ileum. Maximum absorption of selenate, on the other hand, occurred from the ileum (Wolffram g; 1., 1985). Selenium is essentially not absorbed from the stomach of monogastrics or the rumen or abomasum of ruminants (Whanger g; 51., 1976; Wright and Bell, 1966). The intestinal absorption of soluble selenium compounds by rats appears to be more efficient than by humans. It has been shown that rats absorbed 92, 91 and 81% of doses of selenite, selenomethionine and selenocystine, respectively (Thomson and Stewart, 1973; Thomson g; a1., 1975). In humans, the absorption of soluble selenium was found to range from 44 to 70% (Thomson and Stewart, 1974). The inorganic form of selenium is absorbed from the gastrointestinal tract to a greater extent by monogastric animals than by ruminants. This difference may be due to the reduction of the ingested selenite to insoluble or un- available forms by rumen microorganisms (Wright and Bell, 1966). Little is known regarding the physiological processes regulating the absorption of selenium compounds. It has been shown in hamsters that selenomethionine is actively absorbed from the mucosal to the serosal side of small intestine and is inhibited by L-methionine, whereas, selenite and selenocystine are neither transported against 13 a concentration gradient nor inhibited by L-methionine (McConnell and Cho, 1965; Spencer and Blau, 1961). Wolffram £1 a1, (1985) have found that selenate is absorbed by a carrier-mediated process and that the absorption mechanism is not inhibited by selenite but is inhibited by sulfate. According to Li and Vallee (1973), the absorption of selenium is dependent upon the solubility of the selenium compound ingested and also on the dietary ratio of selenium to sulfur. Tpansport V amin E: Following absorption, vitamin E is carried away from the intestine by the chylomicrons. These then enter the lacteals and the intestinal lymphatics through the lamina propria and finally reach the systemic circula- tion via the thoracic duct. Movement of chylomicrons across the lymphatic endothelium occurs both by passive diffussion and active transport within pinocytotic vesicles (Kayden and Traber, 1986). The necessity of chylomicrons in vitamin E metabolism is demonstrated in patients with abetalipoproteinemia. This is a condition in which B-apolipoprotein is not synthe- sized, therefore, the chylomicrons, the very low density ipoprotein (VLDL) or the low density lipoprotein (LDL) are not secreted and vitamin E can't be transported. Pigmentary retinopathy and ataxic neuropathy which develop in this disease are due to vitamin E deficiency (Brin g; 31., 1986; l4 Runge 3; p1,, 1986). In the circulation, tocopherols are incorporated into lipoproteins, primarily the LDL fractions (Machlin, 1984). There are indications that a small amount of vitamin E is also associated with VLDL, high density lipoprotein (HDL) and erythrocytes (Bjornson et al., 1976; Kayden and Bjornson, 1972; Kayden, 1978). Selenium: Once absorbed, selenium is transported in the blood where it is apparently associated with the plasma proteins. In dogs, selenium is mainly transported by alpha- 2 and beta-l-globulins (Schwarz and Foltz, 1957), whereas, albumin seems to be the selenium-carrier protein in mice (Sandholm, 1975). Lee £5 a1. (1969) reported that, besides gamma-globulin, selenium is also carried by plasma lipo- protein. In more recent studies, Motsenbocker and Tappel (1982) have proposed that, in rats, a particular seleno- cysteine-containing protein, selenoprotein P, serves as a selenium transport protein. From the plasma proteins, selenium is rapidly distributed to tissues, including hair and bones, and to erythrocytes and leukocytes (Cousins and Cairney, 1961). T1§sue distribution Vitamin E: Studies in animals and humans have con- firmed that vitamin E is distributed throughout the body. 15 Some tissues like testis, adipose tissue, adrenal glands, pituitary glands and platelets have relatively high con- centrations of vitamin E (Gallo-Torres, 1980; Machlin, 1984). Tissue uptake of tocopherols has a positive cor- relation with the logarithm of the dose administered. This linear relationship has been shown in lung, heart,testis and plasma (Behrens ,5; 51,, 1982). According to Igarashi and his colleagues (1986), concentrations of tocopherols in kidney, lung and heart are influenced by some nutritional factors such as dietary fats, proteins and status of other vitamins. Selenium: Results of animal studies indicated that kidneys have by far the highest concentration of selenium and lesser amounts are in the liver and pancreas (Jones and Godwin, 1962; Schamberger, 1983). Similar results were also reported by Schroeder 55 51. (1970) who examined the selenium concentration in various human internal organs from autopsy samples and found the following descending order for selenium concentration in tissues: kidney>liver> spleen>pancreas>testis>heart>muscles>1ung>brain. The dis- tribution of selenium among tissues appears to depend on the form of selenium ingested and the selenium status of the animals at the time of administration but is relatively independent of the route of administration. Studies in chicks (Jensen 55 51., 1963), rats (Burk 5; 51., 1968) and sheep (Lopez 55 1., 1968; Wright and Bell, 16 1964) have shown that selenium was more efficiently re- tained in selenium-deficient animals than in selenium- adequate animals. This increased retention is probably due to greater tissue demand for selenium. Selenomethionine is preferentially taken up in the pancreas and selenite is mostly taken up in the liver, regardless of whether seleno- methionine or selenite is given orally or parenterally (Osman and Latshaw, 1976). Metabolic conversion Vit5pin E: Metabolism of vitamin E in animal tissue is relatively minimal. The absorbed alpha tocopherol remains in an unesterified form in tissue membranes where its biochemical functions occur (Machlin, 1984). However, there is some evidence for the occurrance of tocopheryl quinone (TQ) in animal tissues (Gallo-Torres, 1980; Hughes and Tove, 1980). Studies with chickens (Scott and Desai, 1964) and rats (Mackenzie and Mackenzie, 1959) indicated that d-alpha— tocopheryl quinone can protect against experimental muscular dystrophy due to vitamin E deficiency. Chow 55 51. (1967) found that, in rat liver, TQ was reduced to tocopheryl hydroquinone which was then excreted as a conjugate of hydroquinone in feces and tocophrenoic acid in urine. - Two other vitamin E metabolites, designated as toco- pheronolactone and tocopheronic acid have been isolated 17 from the urine of rabbits and humans after consuming large doses of alpha-tocopherol. These compounds are also re- ferred to as Simon's metabolites (Simon 5; 51., l956a,b; Green _5 51., 1961). Even though the mechanism for the for- mation of Simon's metabolites is not clear, it has been postulated that the quinone is an intermediate in their formation (Simon 5; 51., l956a,b). ele um: The main pathway of selenium metabolism in animals involves a reduction process (Levander, 1976). Selenite first reacts with glutathione or protein sulfhydryls to form selenotrisulfides (Jenkins and Hidiroglou, 1971). By enzymatic action, selenotrisulfide is reduced to selenide, a common intermediate metabolite. Under normal intake of selenium, selenide is methylated to trimethylselenonium which is then excreted through the urinary tract (Burk 5p 51., 1973; Hsieh and Ganther, 1977; Foster and Ganther, 1984). In cases of excessive selenium intake, the pathway described above is overloaded. As a result, dimethyl— selenide is formed instead of trimethylselenonium. Di- methylselenide is a detoxified form of selenium which is excreted via the lungs, and is responsible for the typical "garlicky odor" in animals consuming toxic amounts of selenium (Schamberger, 1983). 18 Egpretgon Vipppip E: Three different routes of vitamin E excre- tion have been identified. The primary route is via the feces, even though there is a great deal of variation, ranging from 10 to 75% of an administered dose. Approxi- mately 1% of vitamin E in the form of Simon's metabolites is excreted via urine (Hidiroglou 5p 51., 1970). It has also been reported that up to 8% of vitamin E administered is excreted in the bile as an unidentified metabolite (MacMahon 55 51., 1975). Selenium: Excretion of selenium from the body can occur through three different routes: the urinary tract, the intestinal tract and the respiratory tract. The amounts and proportions of selenium excreted are affected by many factors such as dietary selenium level, the route of admin- istration, the form of selenium ingested and the composi- tion of the diet (Schamberger, 1983). It has been demonstrated in rats (Burk 55 51., 1973) and in humans (Griffiths 5p 51., 1976) that the urinary tract is the main route of selenium excretion when organic or inorganic selenium intake is limited, whereas equal proportions of selenium were excreted in the urine and feces when adequate amounts of selenium were consumed. The effect of route of administration of selenium on selenium excretion was investigated by Lopez 5; 51. (1968). In ruminant and some nonruminant species, the urinary tract 19 was the major pathway of excretion when selenium was given orally. The increased fecal excretion by ruminants has been associated with poor selenium absorption rather than eleva- ted endogenous excretion. Excretion via the lungs only oc- curs significantly under condition of very high selenium exposure (Olson 55 51., 1963). Nahapetian 5p 51. (1983) administered three different forms of selenium to rats to study the effect of the form of selenium on mode of selenium excretion. They found that more selenocystine or selenite than selenomethionine was excreted in the urine. Urinary excretion in humans also was significantly higher when they consumed a combination of high-protein, low-phosphorus and high-protein, high phosphorus diets than a combination of low-protein, low-phosphorus and low- protein, high-phosphorus diets (Greger and Marcus, 1981). Functiop Vitamin E; The principal function of vitamin E 1_ vivo is as an effective antioxidant. This role is very important to protect the intracellular membrane against lipid peroxidation. Lipid peroxidation is a process that may be induced by either endogenously produced metabolic by products such as free radicals and other oxidants, or by exposure to such environmental agents as photochemical air pollutants, pesticides and tobacco smoke. (Freeman and Crapo, 1982; Mason and Chignell, 1982). 20 To carry out its function, vitamin E may have several actions. Firstly, vitamin E both quenches and reacts with singlet oxygen (Foote, 1968). Secondly, vitamin E reacts with superoxide radicals (02'). This reaction, however, is not considered to be importence since the reaction is very slow and 02’ does not initiate lipid peroxidation. Except in the present of iron (Fe+3), it can interact with peroxide to form the very reactive hydroxyl radical (Fong 5p 51., 1973). Thirdly and probably the most impor- tant action is the effectiveness of vitamin E as chain terminator. In this situation, vitamin E will react with lipid peroxy radicals to form vitamin E radicals and, even- tually, tocopheryl quinone in order to remove H’ from the membrane lipid. The vitamin E radicals produced are fairly stable because the unpaired electron on the oxygen atom can be delocalized into the aromatic ring structure of the quinone (Burton and Ingold, 1981; Burton 55 51., 1983). It was first suggested that the formation of tocopheryl quinone is irreversible so that molecules of tocopherol were thereby lost from the system. According to Tappel (1962) and Diliberto 5p 51. (1982), however, the vitamin E radical can be regenerated back to vitamin E with ascorbic acid, glutathione and NADPH. This may be a further mecha- nism by which high concentrations of vitamin C protect against peroxidation. In addition to its role as an antioxidant, vitamin E has also been implicated in the modulation of arachidonic 21 acid and prostaglandin (PG) metabolism (Hope 55_ 51., 1975). A vitamin E deficiency in the rat has been known to cause both elevation of serum PGEZ and PGFZQ concen- trations (Hope 55 51., 1975) and enhancement of platelet aggregation in response to collagen (Machlin 55 51.1975). The synthesis of PC in testis or muscle of vitamin E- deficient animals is also increased (Carpenter, 1981; Chan 55 51., 1980). The mechanism by which vitamin E alters the serum PG level has not been established. Several early studies suggested that vitamin E modulated serum PG level through enhanced cyclooxygenase activity (Lands 5; 51., 1972; Nugteren 5p 51., 1966). On the other hand, Karpen 55 51. (1981) has reported that platelets from vitamin E-deficient rats produced more thromboxane A2 (TXAZ) and prosta- cyclin (PGI2) than did platelets from vitamin E— supplemented rats. Therefore, Karpen 5p 51. (1981) suggest- ed that vitamin E deficiency raised serum PG level via en- hanced activity of phospholipase A2. Inhibition of PG synthesis by vitamin E might account for its involvement in the immune system (Tengerdy 55 51., 1978) and in the inflammatory response (Vane, 1971; Jarrat, 1976). fiplgpipg: Since a number of investigators found that selenium could prevent some of the symptoms of vitamin E deficiency, the function of selenium has always been associated with that of vitamin E and, hence, with anti- 22 oxidant property. However, the mechanism of action of selenium was not established until Rotruck 55 51. (1973) discovered that selenium is an essential constituent of the enzyme GSH-Px. In the tissues, GSH-Px catalyses the reduction of hydrogen peroxide and of fatty acid hydroperoxides by using reduced glutathione (GSH) as a hydrogen donor to produce water and hydroxy acid and oxidized glutathione (GSSG) (Ganther _5 _1., 1976). It is clear that both vitamin E and selenium play separate but related roles in the cellular defense mechanism against oxidative damage (see Figure 1). Bryant 55 51. (1982) reported that GSH-Px also cataly- ses the conversion of 12 hydroperoxy eicosatetraenoic acid (HPETE) to 12 hydroxy eicosatetraenoic acid (HETE) in platelets. They later found that platelets from selenium- depleted rats accumulated more HPETE than did platelets from selenium-supplemented rats (Bryant 55 51., 1983). Accumulation of HPETE in platelets causes an accumulation of PGI2 and TXA2 and eventually increases platelet aggregation and vasoconstriction. Similar results were reported by Schiavon 55 51, (1984) who used cultured endothelial cells from pig thoracic aorta and by Levander 55 51. (1985) who used rat aortas and found a significant reduction in the production of platelet aggregation when GSH-Px was added to the incubation medium. From the fore- going evidence, it is likely that vitamin E and selenium, as an essential part of GSH-px, modulate the biosynthesis van—04 02.24. "Sufism >m<...w.o . u . . e .333. mmSDDONC ImG .Amnmav muuwxmom Eoum pervade: .m awe—made, mo ceauocsm ucmudxowucm ecu ow dwnmo«ueawp new new Eswcmaem mo :oduocsm 23.2m4mm >m<...w.o m0<2xO¢O>I . w0<2..(h<0 “Exocmu 29:33.0 -Q mm morn: .. Omh<¢3h40a .H wuswwm 24 synthesis pathways in arachidonic acid and PG metabolism at a number of different points (see Figure 2). Another metabolic role of selenium that has been reviewed is its role on reproduction. Studies in rat (McCoy and Weswig, 1969), mouse (Wallace 55_ 1., 1981), boar (Liu 55 51., 1982) and bull (Palleni and Bacci, 1979) have indi- cated that selenium is important for normal spermatogene- sis. Wu .22 51. (1973) have reported that male rats born to dams fed selenium-deficient diets have abnormal sperm morphology and impaired sperm motility. They also found that supplementation of the diet with a high level of vita- min E and other antioxidants could not prevent these selenium deficiency effects. It is known that selenium can't prevent fetal resorp- tion in vitamin E-deficient rats (Harris 55 51., 1958) nor can it improve the reproductive performance in vitamin E- deficient chickens or turkeys (Creger 55 51., 1960; Jensen and McGinnis, 1960). However, selenium supplementation had beneficial effects on both egg production and hatchability of fertile eggs in chickens (Cantor and Scott, 1974) and in turkeys (Cantor 55 51., 1978). The selenium role in hatch- ability could involve integrity of the special muscles nec- essary for the chick or poultry to break out of the shells. The mechanism by which dietary selenium could maintain the integrity of sperm morphology in the rat remains unex- plained. .nmwmwv Hamzcuoo use mHmEmcwmcmm Eoum cwwmwcoz .cwucmawaumouq "um .wuhnwcamcoame u mo muowwww one .~ wuswwm 398352.: “Giza—0039.1 m... :=u>uO.uO._n_ «.330: to (a! mhmI m u e um. ImU 25 1m0< U_C0qv_p—U°h< «é 33:233.... «1.... .2335. 25380.2 26 Deficiency Vitamin E: The vitamin E deficiency diseases in most species of animals are now well established. Unlike other vitamins, vitamin E deficiency can result in a wide diver- sity of clinical signs and pathological changes in differ- ent species of animals. The lesions can occur in the repro- ductive tract, nervous tissues, muscle tissues (skeletal, cardiac and smooth), liver, alimentary tract, fat depots or even outer integument. The lesions of deficiency usually are degenerative in nature due to instability of biological membranes. According to Horwitt (1965) and Machlin (1984) the tissues affected appear to depend on the species and other nutrients, especially PUFA and selenium. For example, young bullocks develop nutritional degenerative myopathy, where- as, young pigs develop microangiopathy when they are fed a low vitamin E diet. Gershoff and Norkin (1962) showed ex- perimentally that in cats, vitamin E deficiency, concurrent with a low level of dietary PUFA, produced chronic degener- ative myositis. In addition, vitamin E deficiency with normal or excess dietary PUFA caused steatitis which is the commonly recognized clinical symptom often seen in the overweight cats on inappropriate diets. Similarly, in chickens, a combination of vitamin E-deficiency and high PUFA caused encephalomalacia. Selenium can't prevent this disorder. A combination of vitamin E and selenium deficien- cy diet, on the other hand, produced exudative diathesis, 27 which was preventable by either vitamin E or selenium sup- plementation (Machlin, 1984; Scott, 1980). Sglenipm: The vitamin E-related, selenium-responsive deficiency diseases in several domestic animals have been well documented for years. A summary of those conditions is given in Table l. A specific selenium deficiency disease was first pro- duced experimentally in chicks by Thompson and Scott (1969) and in rats by McCoy and Wesarch 55 l., 1973). The most significant abnormalities during selenium deficiency in the chicks were poor growth, poor feathering and pancreatic degeneration followed by mortality. Attempts to diminish the disorders by supplemneting the diet with a high dose of vitamin E failed (Thompson and Scott, 1969). Subsequent in- vestigations by Thompson and Scott (1970) showed that pro- longed depletion of selenium in chicks caused significant impairment of fat hydrolysis and a reduction of vitamin E level. They also reported that pancreatic fibrosis occurred before the chicks became deficient in vitamin E. Similarly, rats fed a low selenium ration containing Torula yeast and adequate vitamin E, had a selenium-responsive disease with the characteristic signs: poor growth and coat, sterility and depigmentation of the iris. These signs of deficiency were different from those of the liver necrosis syndrome in rats caused by a deficiency of both vitamin E and selenium (McCoy and Weswig, 1969). Selenium-responsive disease also 28 Table 1. Vitamin E and/or selenium responsive diseases in animals. Tissue/ Responsive to PUFA Disease organ Animals Vit. E Se influence affected Abortion fetus/ cattle, x x - placenta pig,horse, sheep Embryonic vascular pig,rat, x ? x degeneration syatem of mouse embryo Encephalo- cerebellum chicken x - x malacia Exudative vascular chicken, x x - diathesis system turkey Gastric stomach pig x - - ulcers Hepatic liver rat x x - necrosis Hepatosis liver pig x x - dietetica Impaired spermatozoa rat,pig, ? x - spermato- mouse genesis Kidney kidney rat,mouse, x - - degeneration tubules monkey Nutritional skeletal all x x - muscular &smooth species dystrOphy muscles Pancreatic pancreas chicken - x ? fibrosis Poor egg turkey, x x x hatchability embryo chicken Retained uterus/ cattle ? x - placenta placenta Steatitis adipose cat,mink, x ? x 5 tissue horse Testicular testis calf,pig, - x - degeneration mouse Modified from Machlin (1984). 29 occurs naturally. Multiple myocardial necrosis and low selenium status are observed in pigs fed grain and vegeta- bles grown in the areas in which Keshan disease is endemic in China (Zhu and Lu, 1981). onicity Vitamin E: Vitamin E is considered to be relatively nontoxic in both animals and human beings (Anonymous, 1975; Hillman, 1957). Hypervitaminosis E in chicks can be induced by feeding 2200 IU of vitamin E/kg of diet (March 55 51., 1973). According to Martin and Hurley (1977) the LDSO for all-rac-alpha-tocopherol or all-rac-alpha-tocopheryl acetate is 2000 mg/kg body weight for rats, mice and rabbits. Selenium: Historically, cases of selenium poisoning of animals had been recognized long before the causative agents were even defined (Franke, 1934; Schwarz and Foltz, 1957). The clinical manifestations of selenium toxicosis in domestic animals were described by Rosenfeld and Beath (1964) who classified the syndromes into 3 different catagories: (1) acute; (2) subacute, blind staggers type; (3) chronic, alkali disease type. Acute selenosis occurs under condition in which large quantities of selenium accumulator (seleniferous) plants are consumed by animals within a short period of time. Acute selenosis can also be produced experimentally by 30 giving a lethal dose of selenite or selenate to animals by any route (Morrow, 1968; MacDonald 55 51 , 1980; Carvaggi 55 51., 1970). It is generally known that acute lethality of selenium compounds is greater when given parenterally than orally. The average oral LDSO is about 5.9 mg selenium/kg of body weight, whereas, the average parenteral LDSO value is about 2.0 mg selenium/kg of body weight (Ammar and Couri, 1981; Palmer 55 51., 1973; Ostadalova 55 51., 1978). The clinical signs of selenium toxicity include anorexia, elevated body temperature and pulse rate , abnor- mal movement and posture, diarrhea, and "garlicky" breath odor. Labored breathing and death from respiratory failure often follows within a few hours. The incidence of acute selenium toxicity among livestock under field conditions is low, since grazing animals usually avoid seleniferous plants, except in times of pasture shortage (Underwood, 1981; Hopper 55 1., 1985). Subacute selenosis or the blind staggers syndrome usu- ally occurs as a result of ingestion of limited amounts of seleniferous plants over a period of weeks to months. The affected animals have ataxia, disorientation, impaired vision and respiratory distress (Rosenfeld and Beath, 1964). This type of selenium toxicity can be produced ex- perimentally by administration of water extracts of selenium accumulator plants. However, it has been reported to be almost impossible to reproduce the syndromes by the administration of pure selenium. Factors other than 31 selenium are thought to contribute to the development of blind staggers (Maag and Glenn, 1967; Van Kampen and James, 1978). Alkali disease is the chronic type of selenium toxicity associated with prolonged ingestion of feeds containing 5 to 40 mg selenium/kg. Most common signs are emaciation, rough hair coat, hair loss, hoof malformations and lameness. Death usually results from starvation, since affected animals are unable to graze (Rosenfeld and Beath, 1964). Toxicity of selenium in animals is influenced by many factors. Among these are the chemical form of selenium, type of diet, duration of intake, species of animals and individual acclimation (Jaffe and Mondragon, 1975; Halverson and Monty, 1960; Palmer and Olson, 1974). MATERIALS AND METHODS Animals and diets Twenty four multiparous sows, consisting of purebred Yorkshires, Duroc/Yorkshire crossbreds and Landrace/ Yorkshire crossbreds, averaging approximately 225 kg in body weight, served as experimental animals. These sows were serviced at the first postweaning estrus, equalized for parity and allotted to 4 groups. In actuality, because the analytical aspects of the planned experiment were labor intensive and could not be conducted on stored samples, 4 sows (2 controls and 2 treated) were assigned to experiment each month (Table 2), creating a split plot design. Assign- ment to diet was by random numbers. The experimental diets (Table 3) were corn/soybean meal based in which the corn was previously ensiled, high- moisture corn (for destruction of vitamin E) dried before use to approximately 13% moisture. This diet, without supplemental Se, constituted the -vitamin E-Se treatment; with 0.3 mg Se/kg, constituted the -vitamin E+Se diet; with 60 IU vitamin E/kg and without supplemental Se, constituted the +vitamin E-Se diet; and with both 0.3 mg Se/kg and 60 IU vitamin E/kg, constituted the +vitamin E +Se or control diet. By analyses, the basal diet contained 0.29 IU vitamin 32 33 masses as“: u z madame eauunoaoo u o madame pecan u m 2.0.m a n m m N wm+ m+ o 2.0.m m a m m N mm: m: m z.o.m a m a m N wm+ m+ c 2.0.m m m m m N mm: m: m z.o.m m m m N wm+ m+ q 2.0.m m m m N mm: m+ m 2.0.m m m N em+ m+ q 2.0.m m m N mm! m+ m z.o.m m N mm+ m+ N z.u.m m N wm+ m: H z.o.m m N mm+ m+ N z.o.m m N wm+ m: H am.coz aw.amm mm.cmh ww.owo mm.>oz mw.uoo ww.nwm mw.w=< ww.~=~ ww.:=H macaw weapon coaum>ummno esouw\: gown pawEumwuH anewuwe amuseENuwnxm .N wanna 34 .Auewv mx\wm we m.o hfiqnam cu coucwswaddsmv wxxmm we CON pmcwsucoo xNEoum o .Auwfiv mx\m .ua> 9H om hannsm cu pwucmeansmv wx\m .uw> DH ooo.o0m vocwmucoo xNEwum a .mE mm.o .mcvaN “we NH .uwddoo “we m.< .omwcemcme “wme.HN .coua .wE ca .ocuu “we ~.NmH .weduoHno mcwaono «ms cc.N .u :NEMuN> «m1 MN . m :«Eau«> «we NH.~N .caomua “we cm.m~ .pwom owcwzuoucmnrp .wE oa.m .:N>m~monwu 12H Nah .ma enaaus> “aH comm .< aseeus> "Hmwe we we nae massoasom an» emfifisasm a oo.ooo~ oo.oco~ oo.ooo. oo.ooo~ om.~ oo.o om.H oo.o coo esgamfiwm ~H.° ~H.¢ oo.o oc.o assas>om Nam m essaus> om.~ om.~ om.~ om.~ Auoov wesuoaso seaflono om.~ om.~ om.~ ow.~ «useaue zs> am: oo.m oo.m oo.m oo.m use» uefiswmm om.ks cm.kfi om.kH om.k~ manganese essoseofleuoeoz om.- om.~. om.~s cm.~H ensconumo assuage oo.oo~ oo.oo~ oo.oo. oo.oo~ Ancev Hews cmmnsom mm.mmm ww.amm cm.mmm oo.oew 53 messages an“: emwua mm+ u+ mm- m+ mm+ m- mm- m- acesewuwcH muwwc mo :oNuwmoquo .m «same 35 E/kg and 0.089 mg selenium/kg whereas, the supplemented diets contained 55.31 IU vitamin E/kg and/or 0.35 mg selenium/kg. The bred sows were fed 2.5 kg of diet/day and tap water was available ad libitum. At approximately 7 days before farrowing the sows were moved into a facility equipped with metal farrowing stalls. Sample collections Blood samples from the jugular vein of the sows were 1 tubes for cell collected into heparinized-vacutainer isolation and into red top vacutainer1 tubes for vitamin E, selenium, glutathione peroxidase and cholesterol analyses at the beginning of experiment, at the end of each trimester of pregnancy and at parturition. Colostrum samples were obtained during parturition and milk samples were obtained on day 4 postpartum by adminis- tering 40 USP of oxytocin intramuscularly. Subsequently, the udder was washed with warm water, disinfected with 70% ethanol, dried with paper towelling and hand-milked. Ap- proximately 50 ml of colostrum or milk were collected into 2 sterile polypropylene centrifuge tubes at each sampling. 1Beckton Dickinson, Rutherford, New Jersey. 2Corning Glass Works, Corning, New York. 36 Tubes were capped and immediately placed in an ice-bath and transported to the laboratory. Blood samples were allowed to clot at room temperature for at least 3 hours and were centrifuged at 400xg for 15 minutes. Serum from each sample was withdrawn with a Pasteur pipette, placed into a 5 ml plastic vial and frozen until assays were conducted. Isolation of cells Peripheral blood lymphocytes: Approximately 20 m1 of heparinized blood were diluted 1:4 with sterile 0.9% NaCl solution. Ten milliliters of diluted blood were then layered carefully onto 3 m1 Ficoll-hypaque (1.3570) density gradient in a 17x100 mm sterile plastic tube with cap1 and centrifuged at 400xg for 20 min. After centrifugation, the lymphocyte-rich layer was withdrawn from the buffy inter- face with a sterile Pasteur pipette, placed into another sterile 17x100 mm plastic tube, diluted with 10 m1 of sterile EDTA phosphate buffered saline (EPS) solution and centrifuged at 275xg for 6 min. The supernatant was then discarded and the cell pellet was examined for red blood cell (RBC) contamination. If RBC were present, the cell pellet was subjected to RBC decontamination (described below) before continuing. After RBC decontamination, the cell pellet was washed with sterile saline two times; the first followed by centrifugation at 225xg for 6 min and the 37 second followed by centrifugation at 275xg for 6 min. Washed lymphocytes were suspended in 2 ml of Rosewell Park Memorial Institute (RPMI) 1640 culture media3 which was supplemented with fungizone (6 ul/ml), Hepes (12 ul/ml), NaHCO3 (20 ul/ml), gentamycin sulfate (0.32 ul/ml) and heat-inactivated porcine serum (0.1 ml/ml). A Neubauer hemacytometer was used for counting the lymphocytes under light microscope and viability was assessed by trypan blue exclusion. Dilution with RPMI media was done to obtain a final concentration of l to 2x106 viable cells per ml. RBC decontamination: Cell pellets with evidence of RBC contamination were resuspended in 5 ml sterile, doubly distilled water (dd H20) and mixed gently for 15 seconds to lyse the RBC. An equal volume of sterile, double isotonic strength EDTA phosphate-buffered saline (2xEPS) (1.8% NaCl, 0.7% EDTA-Na3, 0.2% KHZPOA, ph 7.4) was added to the cell suspension to prevent lymphocyte lysis. The mixture was then centrifuged at lSOxg for 8 min. mor o u le r N : After the lymphocyte layer had been removed for lymphocyte isolation, the plasma layer and ficoll layer were withdrawn with a Pasteur pi- pette. The RBC-granulocyte pellet was then suspended in 3Gibco Laboratories, Grand Island, New York. 38 5 ml of dd H20 and mixed vigorously for 5 seconds to lyse RBC. Five milliliters of 2xEPS were immediately added, mixed well and centrifuged at lSOxg for 8 min. After cen- trifugation, the supernatant was carefully removed and dis- carded. The RBC lysis steps were repeated until the cell pellet was clean and slightly greenish in color. The granulocytes were counted on a Neubauer hemacytometer and the cell count was adjusted to 1x106 cells/ml in Hanks' 3 balanced salt solution (HBSS) without phenol red. Colostrum and milk cell isolation: The 50 ml colos- trum and milk samples were diluted 1:5 and 1:2, respective- ly with cold, sterile, phosphate-buffered saline (PBS). The diluted colostrum or milk was then defatted by centrifuga- tion at 400 xg for 20 min. The supernatant with the fat layer was discarded and the cell pellet was washed twice in cold PBS by centrifugation at 215xg for 15 min. For differential cell counts, the washed cells were suspended in 1 ml PBS containing 50% inactivated bovine serum and smeared on 3x1 inch glass slides for staining and counting. For lymphocyte and granulocyte isolation, the washed cells were reconstituted with EPS. Ten milliliters of the cell suspension were then layered onto 3 ml 1.3570 specific gravity Ficoll-hypaque and centrifuged at 400xg for 40 min. After centrifugation, the lymphocyte layer was isolated from the buffy interface and the granulocytes were isolated 39 from the cell pellet lying at the bottom of the tubes. The number of lymphocytes and granulocytes was adjusted to 1x106 cells per ml in media. Lymphocyte blastogenesis assay One hundred microliters of cell suspension containing 1 to 2x105 cells isolated from colostrum, milk or periph- eral blood were added to each of 12 wells in a 96 well, round-bottom, tissue culture plateh. The first three wells were unstimulated cells. To each of the three remaining wells, 10 ul of the appropriate mitogen were added: Phytohemaglutinin (50 ug/ml), Concanavalin A5 (50 ug/ml) and Pokeweed Mitogen5 (10 ug/ml). A dose response curve was previously established to determine the right concentration of mitogen which provided optimal stimulation of swine lymphocytes in this experiment. The plates were 6 and incubated for 72 then covered with mylar sealers hours at 37°C in a 5% C02 incubator. After 72 hours, 1 uCi 3H-thymidine7 in 20 ul of sterile saline was added to each well and the plates were then incubated for an aFlow Laboratories, Inc., McLean, Virginia. 5Sigma Chemical Co., St. Louis, Missouri. 6Dynatech Laboratories Inc., Chantilly, Virginia. 7DuPont, Boston, Massachusetts. 40 additional 18 to 24 hours. After incubation, the cells were harvested, by using a semiautomatic harvesters. This process included aspirating the lymphocytes onto filter paper and washing with distilled water and 5% trichloro acetic acid (TCA). Each individual filter disc was then punched out into a scintillation vial by using forceps. One hundred milliliters of Soluene-3509 were then pipetted into each vial and each vial allowed to stand at room temperature for 30 min. Following this step, 5 ml of scintillation fluid were added to each vial, and each vial was capped and labelled properly. Radioactivity was counted in a Beta Trac 689510 counter. Data were expressed as mean counts per minute. Yeast phagocytic assay The phagocytic and microbicidal activities of the PMN cells were quantified by using the method described by Simpson 55 51, (1979). 8Titertek Cell Harvester 550, Flow Laboratories Ltd., Irvine, Scotland. 9Packard Instrument Co., Downer's Grove, Illinois. 10TM Analytic Inc., Elk Grove Village, Illinois. 41 Ereparation 9f ygasp gpigpipp: Dried baker's yeast11 was used in this study. Two grams of yeast dis- solved in 100 m1 of saline created a stock solution of ap- proximately 5-7 x 108 yeast particles/ml. An evenly sus- pended solution was obtained by stirring for at least 30 min on a magnetic mixer. The yeast cell-concentration was then adjusted to 1x106 per m1. est ocedure: Equal volumes (200 pl) of yeast sus- pension (1x106 cells/ml), PMN cell suspension (1x106 cells/ml), autologous blood plasma and HBSS were pipetted into a sterile tube1 with cap. The mixture was then in- cubated at 37°C on a shaking water bath for 60 min. After the incubation, 1 m1 of 0.01% methylene blue was added into the mixture which was then centrifuged at 400xg for 10 min. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 20 pl of H388 and counted using the Neubauer hemacytometer. To quantify the phagocytic activity, the number of PMN cells containing 2 or more yeast particles per 100 cells were counted. The killing ability of PMN cells was deter? mined by counting the number of PMN cells containing 2 or more dead yeast particles per 100 PMN cells. 11Fleischmann's Yeast Inc., Oakland, California. 42 Vitamin E ana es Serum and feeds were assayed for vitamin E by the high pressure liquid chromatography (HPLC) methods modified from that of Bieri 55 51. (1979) and used in the Clinical Nutrition section of the Animal Health Diagnostic Laborato- ry. 55555: One ml of each serum sample was pipetted into a labelled 16x100 mm disposable culture tube. An equal volume of absolute ethanol was added and the mixture was vortexed for 5 seconds on a maximum power to denature the protein in the serum. Two milliliters of ultra violet (U.V) grade hexane were repipetted to the mixture, vortexed for 2 min and then centrifuged at 550xg for 10 min. After cen- trifugation, the hexane layer was withdrawn with a dispos- able Pasteur pipet and passed through a 0.45 micron cellu- lose filter12 in a Swinney-type filter holder. One hundred microliters of the hexane extract were injected into the chromatographic system automatically13. Separa- tion was isocratic in a Waters microporasil13 column (3.9x15 cm long) with a 85:15 mixture of degassed hexane: chloroform. The mixture was pumped through the HPLC unit at 12Millipore Corp., Bedford, Massachusetts. 13Waters Intelligent Sample Processor (WISP), Waters Associated Inc., Milford, Massachusetts. 43 1.1 ml/min at an intended pressure of 1500 psi and detec- tion was with U.V. spectrometry14 at wavelength of 280 nm. In this system the peaks of alpha-tocopheryl acetate and alpha-tocopherol occurred at 2.45 and 4.59 min, respec- tively, after sample injection. E555; Approximately 500 mg of milled feed were trans- ferred into a labelled 16xl25 mm disposable culture tube. Two milliliters of milliporre water were added to each tube and allowed to stand in the dark for at least 1 hour. Two milliliters of absolute ethanol were repipetted and vortexed for 10 min on a maximum power. Two milliliters of U.V. grade hexane were then added to the mixture, vortexed for 10 min and centrifuged at 550xg for 10 min. After cen- trifugation, the hexane layer was removed and the subse- quent steps were as described above for serum samples. The final concentration of vitamin E in feed was quan- tified on a dry weight basis. To determine dry weight of feed, approximately 2 g of milled feed were placed in an aluminium pan and dried in an oven at 56°C. After 24 hours the dried feed was weighed and the wet weight to dry weight ratio was calculated. 14Waters Associates Model 440 Absorbance Detector, Waters Associated Inc., Milford, Massachusetts. 44 gholgstepol 5na1y§is Serum cholesterol was determined spectrophotometrically by using a commercially available kit from Sigmas. Ten microliters of standards and serum samples were pipetted into labelled 12x75 mm disposable borosilicate tubes. Cholesterol standards used in this analysis had concentra- tions of 0, 0.5, 1.0 and 2.0 mg/ml. One milliliter of cholesterol reagent was then added to each standard and sample and the resulting mixtures were vortexed and then incubated in a water bath at 37°C for 10 min. Absorbance of the standards and samples was read in a spectrophoto- meterls set at a wavelength of 500 nm. The cholesterol values were calculated using a curvilinear standard regression line. Selenium analyges §erum: Selenium concentration in serum was assayed by using a modification of the phosphoric/nitric acid diges- tion process (Reamer and Veillon, 1983) followed by fluoro- metric detection procedure described by Whetter and Ullrey (1978). One milliliter of serum sample was digested in 4 m1 HNO3 and 3 ml H3P04 in 50 ml Erlenmeyer flasks on a 15Model 920, Gilford Instruments, Oberlin, Ohio. 45 hot plate (230°C) until brown fumes disappeared. The samples was cooled down to approximately 150°C and 3 ml of 30% hydrogen peroxide (H202) were added to each flask and samples were reheated again at 230°C until bubbles were fine and flask content reached the critical weight (5.2-6.5 g). The samples were cooled down and the remaining HNO3 was removed by adding 6 ml of 50% hydrochloric acid (HCl) and 2 m1 of formic acid. The mixture was reheated for 10 min and then cooled down by turning the hot plate to low. Five milliliters of masking agent, prepared by dissolving 10 g of EDTA and 25 g hydroxylamine HCl in 1 liter of distilled water, were added and the pH of the mixture was adjusted to between 2.3-2.8 as indicated by a golden yellow amber color with cresol red. Five milliliters of 2,3-diaminonaphthaline (DAN) were then added to each sample to form diazoselenol, a light sensitive complex. This complex was then extracted with 5 ml of cyclohexane on a rotary shaker16 for 5 minutes. The cyclohexane extract was floated to the neck of the Erlenmeyer flask with H20 and then transferred into a cuvette. The fluorescence was measured with a spectro- l7 fluorometer at excitation and emission wavelengths of 16Lab-line Instruments, Inc., Melrose Park, Illinois. 17Model LS-3B, Perkin-Elmer, Norwalk, Connecticut. 46 376 and 510 nm, respectively. Selenium concentration was calculated by using a curvilinear regression. E555: Feed selenium concentration was determined by using a modification of the microwave digestion technique (Kingston and Jassie, 1986) followed by the fluorometric detection procedure described by Whetter and Ullrey (1978). Five hundred milligrams of milled feed were put in a 200 m1 teflon digestion vessel and 5 ml HNO3 and 3 ml H3P04 were added. A relief valve and cap were put on each vessel and a venting tube was put into each cap. Twelve vessels were torqued closed and positioned in the microwave carousel (turntable). The samples were then digested simultaneously in a microwave oven18 using 100% power for l min followed by 35% power for 13 min. The power was then reduced to 0% for 5 min and the vessels were vented. The second phase of digestion was done at 100% power for 1 min and 45% power for 13 min. The power was reduced to 0% for 5 min and the vessels were again vented. The last phase of digestion was done on 100% power for 6 min and 0% power for 5 min. The microwave was turned off, vessels were vented and the samples were cooled down for 60 min. The cooled samples were transferred into a 50 ml Erlenmeyer flask and 6 ml of 50% HCl and 2 ml of formic 18CEM Corporation, Indian Trail, North Carolina. 47 acid were added. The subsequent steps were done in the same manner as described for serum samples. Glutathione perozidase activity Serum glutathione peroxidase activity was determined by the coupled assay described by Paglia and Valentine (1967) and Lawrence g; 9;. (1974). Forty microliters of serum were pipetted into a cuvette to which was added 50 pl of reduced glutathione5 (GSH) and 0.9 m1 of reagent mixture containing 100 mM potassium phosphate buffer (pH 7), 3 mM EDTA, 1 mM NaN3 (sodium azide), 0.1 mM nicotinamide adenine dinucleotide phosphate (NADPH)S and l enzyme unit (EU) of glutathione reductases. Ten microliters of 30% H202 were then added for initiating the reaction. Rate of conversion of NADPH to NADH over time was monitored at 340 nm in a 15 spectrophotometer equipped with an automatic cuvette changer for enzyme kinetics and recorded on a Varian chart recorderlg. 19Varian model 9176, Gilford Instruments Laboratories Inc., Honeywell Ft.Washington, Philladelphia. 48 Stat stical an es The variance selenium follows: Yijk’ (i-' where y - M - T - A - p - TP - E - data were analyzed by split-plot analysis of with the main effect of dietary vitamin E and/or and time over treatment. The linear model is as 't, j-1'°'n per i, k-1"'p) the individual variable the overall (grand) mean effect of treatment effect of animal within treatment effect of period interaction between treatment and period residual error Source of variation Degree of freedom T - -E+Se vs +E+Se (Exp. 1) +E-Se vs +E+Se (Exp. 2) (t-1)- (2-1)- 1 -E-Se vs +E+Se (Exp. 3) E(l)- animals/T (Aijk) t(n-l)- 2(4-1)- 6 P - observation period (p-1)- (5-1)- 4 TP - dietary treatment x period (t-1)(p-1)- (2-l)(5-1)- 4 E(2)- residual error t(n-1)(p-1)- 2(2-1)(5-1)- 24 49 Significant difference between treatments (within period) was determined by using Student T test. Pearson correlation coefficients were used to determine the correlation between serum selenium concentration and glutathione peroxidase activity. In this study a difference was considered signif- icant at the level of P<0.05 (Gill, 1978). All statistical analyses were performed by an IBM 4381 computer using the SAS20 program. 2oStatistical Analytical System, Cary, North Carolina. RESULTS Serum vitamin E The effects of vitamin E and/or selenium depletion on sow serum vitamin E are presented in figures 3a, b and c and appendix table 10. Starting at 30 days of experiment and continuing through parturition, sows fed the vitamin E- deficient diet (group 1) and sows fed the diet deficient in both vitamin E and selenium (group 5) had significantly lower (p<0.05) serum vitamin E concentrations than sows fed the control diet. Serum vitamin E concentrations of the control sows increased gradually during gestation. Serum selenium The effects of vitamin E and/or selenium depletion on sow serum selenium are presented in figures 4a, b and c and appendix table 11. Within 30 days of initiating the experi- mental diets, serum selenium concentrations in sows fed the +E-Se diet (group 3) and the -E-Se diet (group 5) were sig- nificantly lower (p<0.05) than serum selenium concentra- tions in sows fed the control diet. These differences con- tinued for the remainder of the experiment. With the excep- tion of control group 6, serum selenium concentrations of the control sows increased gradually during gestation. 50 Figure 3. 51 Serum vitamin E concentrations in sows fed: (a) -E+Se («-) and +E+Se (Cr) diets; (b) +E-Se (4-) and +E+Se (O~) diets; (c) -E-Se (a-) and +E+Se (<>) diets from conception to parturition. Each point represents a mean i SD. *Significantly different from +E+Se (p<0.05). uglal E: «mail oglnt 52 Vitamin I 2.5 - g D- Lb *- 1 , u b 0 0 l 8 3 6 loath: on experiment "" -t+8¢ (group I) + 68+“ (group 8) Vitamin E T 2 D- 1.5 > parturition 1 r- We 0.5 '- 0 1 J 1 .l 1 L 0 l 2 3 4 Months on experiment —- +E-Se (group 3) + +E+Se (group 4) Vitamin I t r 1.5 r- ' F m 0.3 . ' put-1th. o l l J l A j 0 I 2 8 4 "‘ 41-S- (troup 8) + on“ (group 6) Honths on experiment Figure 4. 53 Serum selenium concentrations in sows fed: (a) -E+Se (a-) and +E+Se (<>) diets; (b) +E-Se (~p) and +E+Se (<>) diets; (c) -E-Se (q-) and +E+Se (O~) diets from conception to parturition. Each point represents a mean 1 SD. *Significantly different from +E+Se (p<0.05). 54 Selenium 260 - 220 - 200 - i :00 ~ \ O , :00 ~ 140 ~ ‘20 _ "In“. t” J .l 1 I i I 4 1 l J o i 8 3 6 Months on experiment "“"' ~E+Se (group i) + §l+So (group 2) Selenium 2 .- 180 - I 160 L 3 no - 3 ‘ 120 - eonoepuon 100 '- p tion w l A l L l 1 J l L I o i z a 4 Months on experiment + +E-Se (group 3) 4' +E+Se (group 4) E O 8 1'0 4 1 1 j 1 J L L l j 0 I 8 8 6 Iontha on experiment F: '“" 4'80 incur 5) "' “+80 (Ir-up fl 9 . 4 55 §erum glutathione peroxidase The effects of vitamin E and/or selenium depletion on sow serum GSH-Px are presented in and figures 5a, b and c and appendix table 12. By 60 days on experimental diet, and continuing through parturition, serum GSH-Px concentrations of selenium-deficient sows (group 3) were significantly lower (p<0.05) than the selenium-adequate sows (group 4). Serum GSH-Px in sows fed diets deficient in both vitamin E and selenium (group 5) was significantly lower (p<0.05) than in sows fed the vitamin E and selenium-adequate diet (group 6). These differences were observed from 30 days on trial through parturition. The mean serum GSH-Px concentrations were positively correlated with serum selenium concentrations in sows fed the vitamin E-deficient diet (r-0.62; p<0.05); in sows fed the selenium-deficient diet (r-0.68; p<0.05);and in sows fed the vitamin E and selenium-deficient diet (r-0.83; p<0.05). Serum cholesterol The effects of vitamin E and/or selenium depletion on sow serum cholesterol are presented in figures 6a, b and c and appendix table 13. Serum cholesterol values for sows in this study were quite variable. No significant difference (p>0.05) was ob- served between the mean serum cholesterol concentrations of any groups of the sows or at any of the sampling periods during feeding. Figure 5. 56 Serum glutathione peroxidase activity in sows fed: (a) -E+Se (q-) and +E+Se (Cr) diets; (b) +E-Se (q-) and +E+Se (<>) diets; (c) -E-Se (-») and +E+Se (<>) diets from conception to parturi- tion. Each point represents a mean i SD. Significantly different from +E+Se (p<0.05). EU- Enzyme Unit. Fig.5 cum! EUIMI IUIQI 2.5 0.5 0.5 57 3 Glutathione Peroxidaae . T 1 1 l l 1 L 1 1 0 i 2 3 Months on experiment “" -B+8e (group i) + etefi (group 2) Glutathione Peroxidase parturition T T .. i eonoeption O 0 b pm tion 4 1 1 J. 1 L 1 i L 0 i 2 3 4 Months on experiment "" #E-Se (group 3) + +E+Se (group 4) ‘ Glutathione Peroxidaae 15'- 8:- ”a 8i- . -‘ .4 "b H I I. j. l- m 05> 0 1 a 1 J 4 x 1 L _L 0 i 8 3 6 Months on experiment "‘ -l-So (group 8) '9' 0898- (group O) Figure 6. 58 Serum cholesterol concentrations in sows fed: (a) -E+Se (4-) and +E+Se ((r) diets; (b) +E-Se (4-) and +E+Se () diets from conception to parturition. Each point represents a mean i SD. 59 Cholesterol wig/ll o l 2 a 4 Months on experiment "- -£+Se (group i) + eEOSe (group 2) Cholesterol :70 ‘r- iso'r 130‘- HO- Moldl 90 __ parturition w l J 1 L l 1 L l 1 l 0 l 2 3 4 Months on experiment "" +E-Se (group 3) ""- +E+Se (group 4) Cholesterol up!“ _ m L 0 l 8 3 4 Months on experiment . o “'- 4'3‘ (1'9“! 5) + 034's. (group 0) . F l9. 6 60 Ipmunoresponsiveness pf peripheral blood coppopepts ho t t u a n The effects of vitamin E and/or selenium depletion on 3H-thymidine uptake of unstimulated lymphocyte are pre- sented in figures 7a, b and c and appendix table 14. No significant differences in 3H-thymidine uptake of un- stimulated lymphocyte were observed between treatments. PHA mitpgen: The effects of vitamin E and/or selenium depletion diet for sows on the PHA stimulation of their PBL are presented in figures 8a, b and c and appendix table 15. At 90 days on experimental diets and at parturition, the response of the PBL to PHA stimulation was significant- ly lower (p<0.05) for sows fed the vitamin E-deficient diet (group 1) than for sows fed the control diet (group 2). A significant reduction in the response of PBL to PHA was also observed for sows fed the -E-Se diet (group 5) com- pared to the response of PBL to PHA stimulation in sows fed the +E+Se diet (group 6), as early as 60 days on experi- ment. PW mitogep: The effects of vitamin E and/or selenium depletion diet for sows on the PWM stimulation of their PBL are presented in figures 9a, b and c and appendix table 16. Pokeweed mitogen stimulation of PBL from sows fed the -E+Se diet was significantly lower (p<0.05) compared to PBL from sows fed the +E+Se diet, at 90 days on experimental diet and at parturition. The PWM stimulation of PBL from sows Figure 7. 61 3H uptakes of unstimulated peripheral blood lymphocytes from sows fed: (a) ~E+Se (4-) and +E+Se (<>) diets; (b) +E-Se (4-) and +E+Se (or) diets; (c) -E-Se (4-) and +E+Se (<>) diets from conception to parturition. Each point represents a mean i SD. Fig.7 b C CPM Hop '0.) CPM i '0. I0, 3 5 ll-thymidino uptake 62 Months on experiment “F -E*Se (group i) 3 H-Lhymidine uptake eonéeptlon l L L. ‘ ‘ 0 l ""“' #E-Se (group 3) 8 ‘ l-thymldine uptake 2 Months on experiment + +E¢So (group 2) + +E+Se (group 4) 3. 3 .. 2.5 ~ T L I I 3 . Ii 'MUOI 1.5 4 . J 4 i 1 , . J 0 l 3 4 .. [/1 n‘ u I w s - I I 7‘3 '1‘ Wh- m ' b o A 4 J .l i A J 1 4% o l 8 8 4 Months on experiment "'" -l-8e (group 5) + +t¢8e (group 0) Figure 8. PHA stimulatory lymphocytes +E+Se (-<>) diets; (c) conception to parturition. *Significantly different a mean i SD. (p<0.05). 63 responses sows diets; peripheral blood ~E+Se (q-) and (b) +E—Se (4-) and -E—Se (a-) and +E+Se (<>) Each point +E+Se (-<>) diets from represents from +E+Se 64 3 ‘ l-thymidine uptake _. 5 . T 2 to 2 ~— 0 z ‘ ’ ‘ O U 3 omooptioa parturition 2 1 1 1 1 .1 1 1 1 1 1 0 l 2 8 4 Months on experiment “‘" 4380 ("our 1) + Hirs- (1’9“? 2) 3 6 H-thymidine uptake ‘2 a 3 I ‘ U 3.5 #- parturition 3 1 1 1 1 1 1 J 1 1 0 l 2 3 4 Months on experiment “" *E-Se (group 3) + +E+Se (group 4) 3 . l-thymltline uptake 8.5 r- .2 o 5 ’ .0 ' . 3 4s - u 0 m 0 '- m 3.5 4 I 1 1 A 1 l 1 l o l s s 4 Months on experiment "‘ 44- tenor 5) *- rlflo (eronr 0) Fig.8 Figure 9. 65 PWM stimulatory responses of peripheral blood lymphocytes from sows fed: (a) -E+Se (a-) and +E+Se (<>) diets; (b) +E-Se (q-) and +E+Se (Cr) diets; (c) -E-Se (-p) and +E+Se (<>) diets from conception to parturition. Each point represents a mean i SD. *Significantly different from +E+Se (p<0.05). 66 5 E-thymidlne uptake 5.5 [ 5 ‘2 a 4.5 > .9 x ‘ ’ ‘ U 3.5 - 3 D 2.5 '- 2 1 1 J 1 J l 1 1 A l 0 I 2 5 4 Months on experiment """' ~Eese (group i) + 0£+Se (group 2) 3 6 H-thymidine uptake 5.5 >- "o a 5 '- 3 I 4.5 '- .- U ‘ _ conception parturition 5.5 '- 3 l 1 1 1 1 l 1 l 1 1 0 i 2 3 4 Months on experiment """' +E-Se (group 3) + +E+Se (group 4) 5 ‘ l-thymidlno uptake .. 5.5 - o 6' “r I 3 _I .2 _. z ‘ . I . N a. u D b h 4.5 a. W ‘ A I l J j l l I l l s 3 Months on experiment "*' ~t-Ie (group 5) + olole (group C) 67 fed the -E-Se diet, was significantly lower (p<0.05) than for PBL from sows fed the +E+Se diet (group 6), at 60 and 90 days of gestation and parturition. Concanavalin A mitogen: Dietary treatments caused no significant difference in the responses of PBL to Con A ( Figures 10a, b and c and Appendix Table 17). Phagocytic activity by polymorphonuclear cells The phagocytic activities of polymorphonuclear cells of peripheral blood are summarized in appendix table 18. The ability of PMN cells from -E+Se (group 1) and +E-Se (group 3) sows to phagocytize yeast cells was significantly decreased (p<0.05) at 90 days on experiment and at parturi- tion (Figures 11a, b). The phagocytic ability of PMN from sows fed the -E-Se diet, however, was significantly de- creased (p<0.05) by 60 days on experiment (Figure 11c). Microbicidal activity by polymorphonuclegr cells The effects of vitamin E‘and/or selenium depletion of sows on the microbicidal activities of peripheral blood polymorphonuclear cells are presented in figures 12a, b and c and appendix table 19. At 90 days and at parturition, vitamin E deficiency (group 1), selenium deficiency (group 3) and the combined vitamin E and selenium deficiency (group 5) resulted in decreased ability of polymorphonuclear cells to kill the Figure 10. 68 Con A stimulatory responses of peripheral blood lymphocytes from sows fed: (a) -E+Se (4-) and +E+Se (<>) diets; (b) +E-Se (4-) and +E+Se (-O) diets; (c) -E-Se (q-) and +E+Se (<>) diets from conception and parturition. Each point repre- sents a mean 1 SD. 69 5 B-thymidine uptake 5.5 - A ‘.5 b I 12% 2 1 I D parturition 5 3.: > I ‘ U 2.5 )- ouooptloo 1.5 '- 0'5 1 1 l 1 L 1 l 1 1 l 0 l 2 5 4 Months on experiment "' -E+Se (group i) + ¢E+Se (group 2) a 51 li-thymidine uptake ”L .. 4.7 +- 2 3 4.5 P T E 4.3 r- I. U "l '- oonoeption 5.9 >- parturition 3.7 '- 3': A 1 1 1 1 1 J L l l 0 l 2 3 4 Months on experiment *“- +E-Se (group 5) + +E+Se (group 4) 5 I-thymldine u take 5.5 p .. 5 ' 2 a .2 2 4.5 '- ‘ U Q r u l j l l I l l l l 0 I 8 8 4 Months on experiment "" -l-So (group 5) + elOSe (group 4) Fig.10 Figure 11. 70 Phagocytic activity of blood polymorphonuclear cells from sows fed: (a) -E+Se (a-) and +E+Se (<>); (b) +E-Se (a-) and +E+Se (<>); (C) -E-Se (q-) and +E+Se (<>) diets from conception to parturition. Each point represents a mean i SD. *Significantly different from +E+Se (p<0.05). a 71 Phagocytic Activity too .5 .- T i w I- 1 v J I .5 b o n J» . )- omooptioa 75 L parturition 7o 1 1 1 1 1 1 1 1 L o i 2 3 4 Months on experiment "" -E¢Se (group i) + +E+Se (group 2) Pha oc ie Activity 05 I ,1 F 95 b as - x 75 — 65 - 55 L 1 1 1 41b 1 1 1 1 o l 2 s 4 Months on experiment -‘— rE-Se (group 3) + +E+Se (group 4) 35 Phagocytic Activity so - I as - f x .0 _ n i- 7. 1 1 1 1 1 1 1 1 o l 2 3 Months on experiment Fig .1] "'" -l-8e (group 3) '9' +E+Se (group O) Figure 12. 72 Microbicidal activity of blood polymorphonuclear cells from sows fed: (a) -E+Se (u-) and +E+Se (<>); (b) +E-Se (4-) and +E+Se (<>); (c) -E-Se (q-) and +E+Se (<>) diets from conception to parturition. Each point represents a mean 1 SD. *Significantly different from +E+Se (p<0.05). 73 Microhicidal Activity 7° r go .- 50 i- I 20 J 1 1 J1 1_ 1 1 1 o l a a 4 Months on experiment "" 4:084 (troop 1) + +308- (erour 2) Microbicidal ActiVity 60 '- T 50 *- I ‘0 .- ouoepuon 30 - . . 'MDOII 2° 1 1 1 1 1 L 1 1 1 0 l 2 3 4 Months on experiment -"- +E-Se (group 3) + 92+Se (group 4) Mlerobiddal Activity 70 . go . go . l O. r- ” . a 1 1 1 1 1 1 J J1 l o i s s 4 Months on experiment ‘ig ‘2 -" -E-$e (youp 5) + +E+Se (group I) o 74 engulfed yeast cells. Differential cell counts The differential cell counts for sow colostrum and for sow milk are presented in table 4 and table 5, respective- ly. There was no significant treatment effect on the differential cell counts for the mammary secretions (colos- trum and milk) of sows. The polymorphonuclear cell was the predominant cell type (68 Z) in colostrum. Polymorpho- nuclear cells remained prominent (44 Z) in milk even though their percentage was lower than in colostrum. Epithelial cells were higher in sow milk than in sow colostrum. Immunoresponsiveness of colostral components Lymphocyte stimulation The effects of vitamin E and/or selenium depletion of sows on the response of their colostral lymphocytes to mitogens are presented in table 6. Dietary treatments caused significantly lower (p<0.05) responses of colostral lymphocytes to PHA stimulation in sows fed the -E+Se diet (group 1) and sows fed the -E-Se diet (group 5) compared to sows fed the control diets, groups 2 and 6. The pokeweed mitogenic response of colostral lympho- cytes from sows fed the -E-Se diet was also significantly 75 .zmm H cases one oosuo>n .mooauoo Houcoeduoaxo you N canoe oomo as.ouos.a mn.oumo.o mo.nuoo.a~ a~.summ.m mo.sums.~o s om+ m+ o m~.cuoo.a a~.ounm.c om.~un~.om Ho.auoa.a as.muom.am s am- a. m mo.cuca.~ ac.ouo~.c nm.suoo.s~ ca.oumo.s sc.snma.ao s mm+ m+ e ~o.ouns.s nn.ouno.c no.numm.a~ em.summ.q ma.nnoe.mo « mm- m+ n on.ounw.~ os.onoa.o 56.nuos.a~ aq.HHmm.m oo.mH¢m.so e om+ u+ ~ o~.ouns.a cm.onom.H as.eHos.o~ 08.8Hmo.m n-.sfio~.eo c om+ m- a III-1|- IIIIII 4.111111111111144 11111 N IIIIIIIIIIIIIIIIIIIII 11111. 3.26 .suaam 33.353“..— oouhooaniq mommnnouomz 3:33:02 mason» mommy Haoo asoumxc no“: aeoEuoouH .cowuwuouumn cam cowumoocoo :oosuon auouv eouuoaaoo Eswcoaom uo\o:m m cweouw> com msom scum enuumoaoo mo mucsoo Haoo Howucouommaa .c wanes 6 7 .zmm H once: one oosao> n .mooauoa soucoeduonxo you ~ ounce oomo ss.~fimm.n~ n~.cuoq.o ma.~um~.ms aa.~uo~.as ~w.~ucn.ms s ¢m+ m+ o as.HHco.- n~.oum~.o ~o.auna.n~ 6H.~Hoa.aa sa.au~s.am e mm- a- m mo.~uoa.s~ oc.cun~.o H~.nuno.o~ o~.~ucs.ns mo.sums.ss s om+ u+ s ~n.~no~.m~ -.ouos.o oo.aucc.- oc.nnmo.ss 39.nwmm.an e um. m+ n ~o.~uao.m~ as.onmm.o mo.~uom.as nm.~umm.as so.ownm.an s om+ m+ N am.~Hao.n~ a~.cums.o ~5.~Hmm.m~ ~a.~Ho~.n~ oo.numa.on c om+ m- a IIIIIIII|I||I|l|IUIIIIIIIIIIIIIII N llllllllllllllllllll IIII ogoo .nuuam mash—233m 3:00:35.— mowocdouomz mafia—nouns; noon» moans Haoo asoum\c node acomuaoua .cowuwusuumo one :Owunoocoo coosuoa muowu coauosnov Esacoaom uo\ucm m :wEMuw> cow m3Om Eoum xawe mo mucaoo Haoo fiowucohoumua .m canoe 77 .Amo.cUVdv o noon» eonu aconoumno Anucoonm«:mnwo .Amo.ouqu m noon» Eonu neonoumno haucoonmnemnmo .zmm H ocooe ono oosno> a .ooonnon noncoEnnonxo nou ~ onnou oomo ~n.oHnn.s oc.oumo.n oo.ouma.m no.owmn.~ s om+ m+ o uam.ouon.n an.oumn.n umm.oun~.m nn.onnm.~ e om- m- m on.onno.n on.ounn.m mn.ouam.m oo.ouam.~ q um+ o+ s o~.cuno.n a~.cHOm.n .nn.ouno.m an.oumc.N e um. n+ n ne.ouon.n an.ouno.~ nn.on~q.m nn.onn~.~ s om+ o+ ~ an.ouos.~ n~.onnm.n omo.ouao.m nm~.onms.~ q om+ m- n iiiiii uniiianunui Aoamoav zmo inunuuunuiunnuniuunniinii ooosoxom < 5 ~o>ocoocou onenuammosozoizm m H n o o a s o n w annoo oononsanuoicowonnz oouo~52nno== noonw\: nonn acomuoonh .:0nnnn:nnoq vco :Onnnoocoo coosnon muono connoaaoo Eoncoaoo no\o=o m :nEonn> com ozom mo monhooanAH Honnoonoo oouoH55nnmicom0nnE mo ozone: ocnonEhauimm .0 oanoa 78 lower than for colostral lymphocytes from sows fed the +E+Se diet. No colostrum lymphocyte stimulation by Con. A was observed. P a oc tic ac iv t b 0 nor 0 la The effects of vitamin E and/or selenium depletion on the phagocytic activity of colostral and milk polymorpho- nuclear cells is summarized in table 7. No significant difference in the phagocytic activity of colostral polymorphonuclear cells was attributable to either vitamin E or selenium depletion of the sows. How- ever, in the vitamin E- and selenium-deficient sows, the phagocytic activity was significantly lower (p<0.05) than in the vitamin E- and selenium-supplemented sows. Microbicidal activ t b 01 nor hon clear ce 8 The vitamin E and/or selenium-depletion effects on the microbicidal activity of colostral and milk polymorpho- nuclear cells of sows are presented in table 8. The abilities of colostral polymorphonuclear cells, from sows deprived of vitamin E or selenium or both, to kill engulfed yeast cells were significantly lower (p<0.05) than colostral polymorphonuclear cells from sows supple- mented with vitamin E and selenium. 79 .zmm H ucooe.ono noono>n .ooOnnon Honcoannoaxo now N ounon oomo o~.ouso.~ a~.onmo.~ cm.ou~m.~ nn.ounn.~ s om+ m+ o un.cnme.~ Nn.oune.~ nn.cu~s.~ mn.onom.~ e am. a. m Nn.cuan.~ nn.ounn.~ an.ouao.~ mo.oums.~ s om+ m+ s o~.owno.~ c~.ouaa.~ sn.ouan.~ on.oH~m.~ e um. m+ n n~.ouoo.~ an.ouoo.~ an.ouoo.~ on.ou~n.~ s om+ m+ N an.oucs.~ m~.owco.~ n~.oumo.~ ann.ounm.~ s om+ n- n iniiiiiuiiuuiiinuui Aoamonv zmo uuuuuuuuuuuuuuuuuu nun: ooosoxom < 5 ~o>o=oocoo encn nonwmoeocouamm m n a o o o noon» ouuoo oono~56nnoicomonnz oononssnuoca noonm\c you: neoEnoonH .COMHMnaunoo oco cennooocoo coozuon muonu connosdoc Ezncoaoo no\u:o m unsoun> cow mac» mo monsoonnsxn xane oonoassnnoucowOnnE mo oxoudo ocnonahznia .u onnoh m 80 once no N acncnoneoo onsoo zzm mo noses: ocn mo oocnmoo on hnn>nnoo onnhoomosa N och .Amo.ouvav o noon» sonu neonommno haucoonMnewnmo .zwm H ocooa ono oosno>o .oHHoo 22m can non oononnnoa noooh n .ooonnoa noncoEnnoaxo now N onnon oomo nn.~nm~.n~ aq.mumm.nm s om+ u+ o mm.~um~.n~ ona.ouo~.mn s mm. m- m mo.munn.on mm.ouam.es e om+ n+ c os.nuoa.an sn.mu~s.mm s on. o+ n No.nuom.n~ nq.muo~.om s om+ m+ N us.nnnn.n~ nn~.muso.ms s om+ m- n mason» an“: EsnnmoHoo noonmxc non: ncosuoona .=Onnnn:unon oco acnunoocoo :ooznon mnowo Eoncoaoo no\o:o m =«Eou«> com ozoo mo mason nooaoscosanEAHOQ sane oeo HonnooHoo mo Ann>nnoo onnhoomocm .m oHnos 81 Immunoregpongiveness of milk components Lymphocyte stimulation Lymphocytes isolated from sow milk were not stimulated by any mitogen used (Table 9). Phagocytic gctivity by polvmorphonuclear cells No significant effects of diet on the phagocytic activ- ity of milk polymorphonuclear cells were detected (Table 7). Microbicidal activity of polymorphonuclear cells A significant decrease in the ability of milk poly- morphonuclear cells to kill the engulfed yeast cells was associated only with the combined depletion of vitamin E and selenium (group 5 vs group 6) (Table 8). 82 no N oocnoucoo unnoo zzm mo noses: on» no oononsonoo no: Ann>nuoo Hoononnonone N one .Amo.ouvav w anon» Eonm .Amo.cuvov o noon» Eonm .Amo.ouvaV N noon» Eonm neonommno hnucoonuncmnm m neonouuno snucaonunconmo neonouuno nnueoonnnconmo .zwm H ocooe ono noono>o .onnoo zzm con non mononunoa nooo» ooov onoe n .ooonnoa Honcoannoaxo now N oanou oomo n~.onn~.~ on.nuso.nn s um+ m+ o unn.cun~.n noo.~unn.m e um. o- m nn.onon.~ mo.nu-.o s om+ m+ s oe.cumo.n moo.ouoo.~ e um. m+ m on.ounn.~ mw.oumn.o s om+ m+ ~ ac.ouo~.~ o.oom.cumo.m s om+ m- n IIIIIIII. IIIIII ON IIIIIIIIIII meson» xnnz EsnumoHou noonmxe non: acoEnoonH .:0nnnn=nnoa vco connooocoo coozuon muono cannoadoo Esncosoo no\u:o m unsoun> vow m30o mo oHHoo noo~an¢02nno5>~oo xnne oeo HonumoHoo mo >nn>nnoo Hoononnononz .a oHnoe DISCUSSION Results of this research indicate that the vitamin E- deficient diet with or without supplemental selenium sig- nificantly reduced the response of sow PBL to PHA. Phyto- hemagglutinin is known to be a T-lymphocyte mitogen for several animals including pigs (Shimizu and Shimizu, 1979) and T-lymphocytes are primarily involved in cell-mediated immunity. According to Nichols g; 11. (1979), the effect of diminished vitamin E in this situation may relate to its metabolic function as an antioxidant. Vitamin E deficiency enhances lipid peroxidation of cellular membranes and eventually increases membrane fluidity. These changes could alter receptor movement on the lymphocyte membranes and thereby affect the initiation of lymphocyte blastogenesis. The selenium-depletion diet, in comparison to the +E+Se diet, resulted in no significant differences in sow lympho- cyte proliferation responses to PHA mitogen during gesta- tion but tended to have a significant effect (p<0.1) by parturition when serum selenium concentration had declined to about 123 ng/ml. Perhaps if dietary selenium could have been reduced to less than 0.089 mg/kg in the present study, a significant difference in the lymphocyte proliferative 83 84 response to PHA in sows fed +E-Se diet would have been detected. The level of dietary selenium in this study was similar to the previously recommended level of selenium (0.1 mg/kg) for gestating sows by the NRC (1979). On the other hand, it is possible that the certain lymphocyte function tested by PHA stimulation is less affected by selenium status than by vitamin E and that vitamin E deficiency decreases the lymphocyte blastogenesis irre- spective of the selenium status. In this study, pokeweed mitogen stimulation of PBL from sows fed both the -E+Se and -E-Se diets was significantly lower than from sows fed the control diet. There is still a controversy regarding the target cell for PWM; some inves- tigators indicate that PWM stimulates predominantly B-cells (Tizard, 1987) which are believed to be involved primarily in humoral immunity. Other investigators believe PWM stimu- lates both T and B cells (Greaves g; al., 1974; Renshaw pp al., 1977). While the target cell for PWM among swine lymphocytes 'was not established in this study, an effect of vitamin E on humoral immunity has been demonstrated in other species. In chickens and mice, vitamin E supplementa- tion stimulated humoral immunity by increasing production of antibody producing cells (Tengerdy _p 91., 1978). Dietary treatments caused no significant difference in response of PBL to Con A, a mitogen considered to stimulate T cells or to be indicative of cellular immunity. This is consistent with the work of Corwin and Shloss (1980) who found vitamin E ineffective in enhancing lymphocyte 85 proliferation in the presence of optimal concentrations of Con A. The difference between PHA and Con A stimulation of PBL may indicate that Con A affects a different subset of T-lymphocyte than PHA, as suggested by Stobo and Paul (1973). The results of this study indicated a decrease in the abilities of blood PMN cells of sows fed the ~E+Se, +E-Se and -E-Se diets to phagocytize yeast particles and to kill the engulfed yeast. This demonstrated that both vitamin E and selenium are important in phagocytic, as well as microbicidal, activities of sow polymorphonuclear cells. Comparable studies in mice and cattle fed selenium- deficient diets revealed no impairment in the ability of PMN to ingest g. albicans, although microbicidal activity was significantly impaired (Boyne and Arthur, 1986; Gyang g; 11., 1984). According to Aziz g; _1. (1984), reduction in bactericidal activity by PMN from selenium-deficient goats is associated with a reduction in PMN GSH-Px activi- ty. It is known that GSH-Px catalyses the reduction of hydroperoxide and superoxide produced by neutrophils during phagocytosis. The hydrogen peroxide can act through myelo- peroxidase to help destroy ingested particles in neutro- phils (Klebanoff, 1975). Decreased GSH-Px activity, and possible subsequent damage to the neutrophils by toxic oxy- gen metabolites, could therefore contribute to the decrease in PMN microbicidal activity. The data indicate that GSH-Px activity of approximately 1.5 EU/ml and above would be 86 adequate to maintain normal microbicidal activity of PMNs in gestating sows. Selenium intake significantly affected the activity of GSH-Px in sow serum in the current study. The positive correlation between serum selenium and serum GSH-Px ob- served in this study (r-0.62 to 0.83) is in agreement with previous reports (Chavez, 1979; Hakkarainen g; al., 1978; Stowe and Miller, 1985). The initial values for GSH-Px, as well as for selenium, for sows in group 5 (-E-Se) and 6 (+E+Se) were higher than those of sows in group 1, 2, 3 and 4, even though all sows had been similarly fed prior to assignment to this study. It is possible this difference was due to different genetic pools among the sows and the reported genetic influences on selenium metabolism (Atroshi g; 11,, 1981; Jorgensen g; al., 1977; Stowe and Miller, 1985). The interrelationship between plasma GSH-Px activity and vitamin E has also been studied. Yang and Desai (1978) provided evidence that there was a negative linear rela- tionship between the logarithm of vitamin E intake and GSH-Px activity in liver and plasma of rats. However, large amounts of dietary vitamin E were required to show its re- pressive effect on the activity of GSH-Px. Even though such a relationship was not evaluated in this study, the closest relationship between GSH-Px and selenium was observed in sows fed -E-Se diet; this suggests both selenium and vita- min E have a role in GSH-Px homeostasis. 87 It has been suggested that the mechanism of action by which vitamin E alters the phagocytic action of PMN relates to its ability to maintain the integrity of cellular mem- brane by preventing auto-oxidative damage during phago- cytosis (Baehner et al., 1977). The increased formation of malonaldehyde in the vitamin E-deficient rat PMN (Harris g; 91., 1980) is evidence of membrane lipid peroxidation and auto-oxidative damage. Malonaldehyde is a peroxidation product that is used as an index of lipid peroxidation of PUFA (Stossel g; 1., 1979). Results of this study indicated that there is no sig- nificant difference in the lymphocyte function between sows fed +E-Se and +E+Se diets. Other investigators have report- ed a significant enhancing effect of selenium on lymphocyte function when the selenium is given at a rate considerably above the NRC recommendation (Corwin and Shloss, 1980). Therefore, it is necessary to re-evaluate the established recommended intakes for nutrients like vitamin E and selenium that could be classified as "immuno stimulatory". Increased intakes of vitamin E and/or selenium could be beneficial during periods of stress such as weaning, gesta- tion and parturition. It is important to point out that no other clinical evidence of vitamin E-deficiency disease, such as muscular dystrophy or hepatosis dietetica, accompanied the dimin- ished immune response. However, in the present study, a smaller litter size was noted in sows fed the -E+Se diet 88 (x-8.25) and sows fed -E-Se diet (i-9.75) compared to the litter size for sows fed the +E+Se diet (i-12.5) (see Table 20). Since only small numbers of sows were used and the data were recorded only over one gestation and farrowing period, the information associated with the reproductive performance in this study is likely to be biased. The possible effect of vitamin E and selenium on litter size is consistent with the work of Ullrey g; 11. (1971) and Vale (1983) which indicated that supplementation of vitamin E and selenium in sow diets throughout gestation and lacta- tion could, compared to nonsupplemented sows, produce a larger litter size at birth and increase the livability of the pigs at three weeks. Five types of cells, namely neutrophils, macrophages, lymphocytes, eosinophils and epithelial cells were identi- fied in both colostrum and milk of the sows in this study. Similar results were reported by Schollenberger and coworkers (1986), however, they indicated the presence of a sixth cell type (anucleate cell) in sows' milk. As in cows (Lee g; 91., 1980) and sheep (Lee and Outteridge, 1981), PMN cells were the predominant cell type in sow colostrum varying from 55.99 to 71.99 X and remained prominent (range 29.39 to 48.78 1) in sow milk even though their percentage was lower than in colostrum. The decline in the proportion of PMN cells was reported to occur in cow and sheep milk (Lee 3; 11., 1980; Lee and Outteridge, 1981). 89 The concentrations of lymphocytes were similar for both colostrum and milk in this study with ranges of 21.47 to 33.16% for colostrum and 12.47 to 32.35% for milk. These concentrations are, however, higher than the concentrations of lymphocytes reported for other species (see Table 21). It is possible that the relatively higher lymphocyte con- centrations in sow colostrum and milk provide special protection of the sow mammary gland against invading micro organisms, either by enhanced local production of antibody or by enhanced cell-mediated immunity in the gland itself. According to Bourne (1973), 60% of Ig A in colostrum or 90% of Ig A and 70% of Ig G in milk are produced locally in mammary tissues of sows. The persistence of viable leukocytes in the mammary se- cretions throughout the observed lactation period (4 days) suggests that these cells may play a crucial role in de- fending the mammary gland, as well as the gut of the suckling neonate, from infections. The proportion of epithelial cells in sow milk (19.83 to 27.69%) was much higher than in sow colostrum (0.55 to 2.21 2) and apparently higher than in cows' milk (72) (Lee 9; al., 1980). This may be due to the more frequent and aggressive suckling habit of piglets. Few studies involving mitogenic stimulation of colos- tral or milk lymphocytes in animals have been published. Results of the present study indicate a low proliferative response of colostral lymphocytes to mitogens relative to 90 the response of PBL to mitogens. No mitogen-induced prolif- erative activity was observed in milk lymphocytes of sows. Similar results have also been reported with human colostral lymphocytes (Parmely g; 11., 1976) and with bovine and canine milk lymphocytes (Smith and Schultz, 1977). Thus, hyporesponsiveness of mammary gland lympho- cytes (MGL) to mitogen appears to be a general phenomenon. The cause for the diminished functional capacity shown by MGL, however, is not yet clear. It has been suggested that hyporesponsiveness of MGL to the mitogen is due to a relative absence of a certain lymphocyte population in the colostrum and milk but which is prominent in PBL (Parmely 2; al., 1976). According to Brock and Mainou-Fowler (1983), the hyporeactivity of MGL is associated with the action of soluble milk components which may depress lymphocyte pro- liferation by masking surface receptors involved in the mitogen-induced blastogenesis or by affecting nutritional requirements for blastogenesis. While vitamin E and/or selenium supplementation certainly did not improve the responsiveness of sow milk lymphocytes to mitogens in the present study, the -E+Se and -E-Se diets did significantly reduce the response of colostral lymphocytes to PHA and PWM. This is further evidence of the influence of vitamin E on cell-mediated immunity. Cytostatic and cytotoxic factors, which were reported to be present in milk but not in colostrum (Drew g; gl,, 1984), may also contribute to the hyporesponsiveness of 91 MGL. This hyporesponsiveness of MGL could have an important role in preventing an allogeneic reaction in the suckling neonate or in preventing the self-destructive effects of these cells on the mammary gland. Phagocytic capacity of PMN cells of mammary secretions from nonporcine species has been observed i3 vitgo by sev- eral investigators. All the results have pointed to the fact that the phagocytic activity of PMN cells isolated from mammary secretions was lower compared to that of PMN cells isolated from the blood (Wisniowski g; al., 1975; Ho and Lawton, 1979). The reason for this disparity was inves- tigated by Paape g; _1. (1975) who indicated that the fat globules and casein in milk may exert an inhibitory effect t 31. (1975) also found that pre- on phagocytosis. Paape incubation of peritoneal macrophages with fatty acids sig- nificantly reduces their phagocytic activity. This was at- tributed to alterations in membrane viscosity brought about by incorporation of fatty acids into the cell membrane phospholipids. In addition to reducing phagocytosis, fat globules can also reduce the ability of the PMN leukocytes to kill en- gulfed bacteria. This reduction may result from disruption of phagolysosomes by the ingested milk fat and from a sub- sequent depletion of phagolysosome enzymes (Paape and Cuidry, 1977). The mechanism by which casein reduces phago- cytic and killing ability of milk PMN leukocytes was re- ported to involve binding of casein to the surface membrane 92 and degranulation after ingestion of casein by PMN (Russel and Reiter, 1975; Russel g; 51., 1976). Naidu and Newbould (1973) found that PMN cells isolated from milk contained 38% less glycogen than PMN cells iso- lated from blood and suggested that the glycogen content could be a factor affecting phagocytosis by milk PMN cells. Addition of glucose to media containing PMN cells of milk can increase the ability of those cells to ingest bacteria. The reduced phagocytic and microbicidal activity of PMN cells from mammary secretions of sows noted in this study is consistent with the above earlier studies. In addition, the present study provides information that both phagocytic and microbicidal activities of PMN cells of colostrum and milk were significantly affected by nutrition, specifically vitamin E and/or selenium concentrations in the diet. This information could be expected to have application in mastitis control programs among farm animals. Although vitamin E and/or selenium deficiency in this study significantly depressed both functions of T-lympho- cytes and PMN cells, further investigations still need to be done to establish the practical effect of vitamin E and/ or selenium on the susceptibility or resistance to dis- eases . SUMMARY AND CONCLUSIONS Twenty-four multiparous sows were assigned at concep- tion to a split plot experiment to determine the influence of vitamin E and/or selenium deficiencies on the immuno- responsiveness of cellular components of peripheral blood, colostrum and milk. The sows were fed corn/soybean meal- based diets in which dried, high moisture corn was used because of its vitamin E deficient nature. Four groups of sows were established and fed the basal vitamin E and Se-deficient diet (.29 IU vit. E and 0.089 mg Se/kg); a vitamin E deficient-diet (basal supplemented with .3 mg Se/kg); 3 Se- deficient diet (basal supplemented with 60 IU vit. E/kg); and a control diet (basal supplemented with both 60 IU vit. E and .3 mg Se/kg). Cell-mediated and humoral immunity was monitored by mitogen-stimulation studies with lymphocytes obtained from peripheral blood at the onset of, and at monthly intervals during, gestation and at parturition; from colostrum ob- tained at parturition; 'and from milk obtained at day 4 of lactation. The abilities of polymorphonuclear cells from peripheral blood, colostrum and milk to phagocytize and kill yeast cells were also monitored simultaneously with the lymphocyte stimulation studies. 93 94 The vitamin E depletion diet significantly decreased serum vitamin E (range of .27 to .48 ug/ml) and depressed both cell-mediated and humoral immunity as evidenced by decreased 3H-thymidine uptake of the phytohemagglutinin and pokeweed mitogen-stimulated lymphocytes, respectively, from peripheral blood and colostrum. In addition, the vita- min E depletion diet significantly decreased the abilities of PMN cells from both peripheral blood and colostrum to phagocytize and kill yeast cells. No other clinical signs of vitamin E deficiency, such as myopathy, accompanied these manifestations of immunodepression. The selenium depletion diet resulted in a modest de- pression in serum selenium (range 123 to 144 ng/ml). While this depression was not sufficient to alter 3H-thymidine uptake by mitogen-stimulated lymphocytes of any source at any sampling time, it was asssociated with a decreased ability of blood and colostral PMN cells to phagocytize and kill yeast cells. The diet deficient in both vitamin E and selenium sig- nificantly reduced the 3H-thymidine uptake responses of both blood and colostral lymphocytes to PHA and PWM stimu- lation. In addition, this diet was associated with signifi- cant reductions in the phagocytic and microbicidal activi- ties of PMNs from blood and colostrum as well as in the microbicidal activity of PMN cells from milk. It can be concluded that sow diets which maintain serum vitamin E concentrations above 1.4 ug/ml and serum selenium concentrations above 160 ng/ml during late gestation and 95 the peripartum period should be associated with optimal cell-mediated and humoral immune response, other things being equal. However, to attain optimal phagocytic and microbicidal activity of PMN cells (nonspecific immunity) in addition to optimal cell-mediated and humoral immunity, diets are required which will maintain serum selenium concentrations in the 180 to 220 ng/ml range, as well as serum vitamin E higher than 1.4 ug/ml, for sows in late gestation and the peripartum period. These observations on selenium provide justification of the current, and rather recently approved, selenium supplementation rate of .3 mg Se/kg diet for swine. LI ST OF REFERENCES LIST OF REFERENCES Allaway, W. H. 1972. An overview of distribution patterns of trace elements in soils and plants. Ann. NY Acad. Sci.199:l7. Allaway, W. H. 1973. Selenium in the food chain. Cornell Vet.63:151. Ammar, E. M. and D. Couri. 1981. Acute toxicity of sodium selenite and selenomethionine in mice after ICV or IV administration. Neurotoxicol.2:383. Anonymous. 1975. Evaluation of the health aspects of toco- pherols and alpha-tocopheryl acetate as food ingredi- ents. Report No. P8262 653 prepared for the FDA by the Federation of American Societies for Experimental Biology, Bethesda, MD. Arvilommi, H., K. Poikonen, I. Jokinen, 0. Muukkonen, L. Rasanen, J. Foreman and J. K. Huttenen. 1983. Selenium and immune functions in humans. Infect. Immun.4l:l85. Atroshi, F., S. Sankari, S. Osterberg and M. Sandholm. 1981. Variation of erythrocyte glutathione peroxidase activity in Finn sheep. Res. Vet. Sci.3l:267. Aziz, E. S., P. H. Klesius and J. C. Frandsen. 1984. Effects of selenium on polymorphonuclear leukocyte function in goats. Am. J. Vet. Res.45:1715. Baehner, R. L., L. A. Boxer, J. M. Allen and J. Davis. 1977. Autooxidation as a basis for altered function of polymorphonuclear leukocytes. Blood 50:327. Bauernfeind, J. 1980. Sources of vitamin E. In: L. J. Machlin (Ed.) Vitamin E, A Comprehensive Treatise. pp 99-101. Marcel Dekker, Inc.,New York. Behrens, W. A., J. N. Thompson and R. Madere. 1982. Distri- bution of a-tocopherol in human plasma lipoproteins. Am. J. Clin. Nutr.35z691. Beisel, W. R. 1982. Single nutrients and immunity. Am. J. Clin. Nutr.35:4l7. 96 97 Bieri, J. G., T. J. Tolliver and G. L. Catignani. 1979. Simultaneous determination of alpha tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am. J. Clin. Nutr.32z2134. Bjornson, L. K., H. J. Miller and A. N. Moshell. 1976. The transport of a tocopherol and 5 carotene in human blood. J. Lipid Res.17:343. Blomstrand, R. and L. Forsgren. 1968. Labelled tocopherols in man. Intern. Z. Vitaminforsch 38:328. Bourne, F. J. 1973. The immunoglobulin of the suckling pig. Proc. Nutr. Soc.32:305. Boyne, R. and J. R. Arthur. 1986. The response of selenium- deficient mice to Candida albicans infection. J. Nutr. 116:816. Brin, M. F., T. M. Pedley, R. E. Lovelace, R. G. Emerson, P. Gouras, C. MacKay, H. J. Kayden, J. Levy and H. Baker. 1986. Electrophysiological features of abeta- lipoproteinaemia: functional consequences of vitamin E deficiency. Neurology 36:669. Brock, J. H. and T. Mainou-Fowler. 1983. The role of iron and transferrin in lymphocyte transformation. Immunol. Today 12:347. Bryant, R. W., T. C. Simon and J. M. Bailey. 1982. Role of glutathione peroxidase and hexose monophosphate shunt in the platelet lipoxygenase pathway. J. Biol. Chem.257: 4937. Bryant, R. W., T. C. Simon and J. M. Bailey. 1983. Hydro- peroxy fatty acid formation in selenium-deficient rat platelets: coupling of glutathione peroxidase to the lipoxygenase pathway. Biochem. Biophys. Res. Comm. 117:183. Burk, R. F., R. Whitney, H. Frank and N. Pearson. 1968. Tissue selenium levels during the development of dietary liver necrosis in rats fed Torula yeast diets. J. Nutr. 95:420. Burk, R. F., R. J. Saly and K. W. Kiker. 1973. Selenium: dietary threshold for urinary excretion in the rat. Proc. Soc. Exper. Biol. Med.l42:214. Burk, R. F. 1984. Selenium. In: R. E. Olsen (Ed.) Present Knowledge in Nutrition. pp 519-527. The Nutrition Foundation Inc., Washington D.C. 98 Burton, G. M., K. H. Cheeseman, T. Doba, K. U. Ingold and T. F. Slater. 1983. Vitamin E as an antioxidant Lg vitrp and 1p, vivo. In: P. R. Whelan (Ed.) Biology of Vitamin E. pp 4-12. Pitman Books,London. Burton, G. W. and K. U. Ingold. 1981. Autooxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic anti- oxidants Lp vitpo. J. Am. Chem. Soc.103:6472. Cantor, A. H., P. D. Moorhead and K. I. Brown. 1978. Influence of dietary selenium upon reproductive perfor- mance of male and female breeder turkeys. Poultry Sci. 57:1337. Cantor, A. H. and Scott, M. L. 1974. The effect of selenium in the hen's diet on egg production, hatchability, per- formance of progeny, and selenium concentration in eggs. Poultry Sci.53:l870. Carpenter, M. P. 1981. Antioxidant effects on the prostaglandin endoperoxidase synthetase product profile. Fed. Proc.40:189. Carvaggi, C., F. L. Clark and A. R. B. Jackson. 1970. Acute selenium toxicity in lambs following intra- muscular injection of sodium selenite. Res. Vet. Sci.ll:l46. Chan, A. C., C. E. Allen and P. V. J. Hegarty. 1980. The effects of vitamin E depletion and repletion on prostaglandin synthesis in semitendinosus muscle of young rabbits. J. Nutr.110:66. Chavez, E. R. 1979. Effect of dietary selenium on glutathione peroxidase activity in piglets. Can. J. Anim. Sci.59:69. Chow, C K., H. H. Draper, A. S. Csallany and M. Chiu. 1927. The metabolism of CIA-a -tocopheryl quinone and C -a -tocopheryl hydroquinone. Lipids 2:390. Cohen, G. and P. Hochstein. 1963. Glutathione per- oxidase: the primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochem.2:1240. Corwin, L. M. and J. Shloss. 1980. Influence of vitamin E on the mitogenic response of murine lymphoid cells. J. Nutr.110:9l6. Cousins, F. B. and I. M. Cairney. 1961. Some aspects of selenium metabolism in sheep. Aust. J. Agr. Res.12z927. 99 Creger, C. R., R. H. Mitchell, R. L. Atkinson, T. M. Ferguson, B. L. Reid and J. R. Couch. 1960. Vitamin E activity of selenium in turkey hatchability. Poultry Sci.39z59. Dam, H. and J. Glavind. 1939. Alimentary exudative diathesis, a consequence of vitamin E avitaminosis. Nature 143: 810. Diliberto, E. J., G. Dean, C. Carter and P. L. Allen. 1982. Tissue, subcellular, and submitochondrial distributions of semidehydroascorbate reductase: possible role of semidehydroascorbate reductase in cofactor regenera- tion. J. Neurochem.39:563. Drew, P. A., O. M. Petrucco and D. J. C. Shearman. 1983. Inhibition by colostrum of the responses of peripheral blood mononuclear cells to mitogens. Aust. J. Exp. Biol. Med. Sci.61:451. Drew, P. A., 0. M. Petrucco and D. J. C. Shearman. 1984. A factor present in human milk, but not colostrum, which is cytotoxic for human lymphocytes. Clin. Exp. Immunol.55:437. Emerson, O. H., G. A. Emerson, A. Mohammad and H. M. Evans. 1937. Tocopherols from various sources. J. Biol. Chem. 122:99. Erskine, R. J., R. J. Eberhart, L. J. Hutchinsm and R. W. Scholz. 1987. Blood selenium concentrations and glutathione peroxidase activities in dairy herds with high and low somatic cell counts. J. A. V. M. A.190: 1417. Evans, H. M. and K. S. Bishop. 1922. On the existance of a hitherto unrecognized dietary factor essential for reproduction. Science 56:650. Evans, H. M., O. H. Emerson and G. A. Emerson. 1936. The isolation from wheat germ oil of an alcohol, a -tocopherol, having the properties of vitamin E. J. Biol. Chem.ll3:3l9. Flohe, L., W. A. Gunzler and H. W. Shock. 1973. Glutathione peroxidase: a selenoenzyme. FEBS Letts.32: 132. Fong, K. L., P. B. McCay, J. L. Poyer, B. B. Keele and H. Misra. 1973. Evidence that peroxidation of lysosomal membranes is initiated by hydroxyl free radicals pro- duced during flavin enzyme activity. J. Biol. Chem. 248:7792. 100 Foote, C. 1968. Photosensitized oxygenations and the role of singlet oxygen. Accounts Chem. Res.1:104. FOSte7S S. J. and H. E. Ganther. 1984. Synthesis of ( Se) trimethyl selenoniumiodide from ( 5Se) selenocystine. Anal. Biochem.137:205. Franke, K. W. 1934. A new toxicant occuring naturally in certain samples of plant foodstuffs. I. Results ob- tained in preliminary feeding trials. J. Nutr.8:597. Freeman, B. A. and J. D. Crapo. 1982. Biology of disease free radicals and tissue injury. Lab. Invest.47:412. Gallo-Torres, H. E. 1970. Obligatory role for bile for the intestinal absorption of vitamin E. Lipids 5:379. Gill, J. L. 1978. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol 3. The Iowa State University Press, Ames, Iowa. Gallo-Torres, H. E. 1980. Absorption. In: L. J. Machlin (Ed.) Vitamin E, a Comprehensive Treatise. pp 170. Marcel Dekker, Inc., New York. Ganther, H. E., D. G. Hafeman, R. A. Lawrence, R. E. Serfass and W. G. Hoekstra. 1976. Selenium and glutathione peroxidase in health and disease: a review. In: H. H. Prasad (Ed.) Trace Elements in Human Health and Disease. II. Essential and toxic elements. pp 165. Academic Press, New York. Gershoff, S. N. and S. A. Norkin. 1962. Vitamin E deficien- cy in cats. J. Nutr.77:303. Gissel-Nielsen, G., V. C. Gupta, M. Lamand _and T. Westermack. 1984. Selenium in soils and plants and its importance in livestock and human nutrition. Adv. Agron.37:397. Green, J., A. T. Diplock, J. Bunyan and E. E. Edwin. 1961. Studies on vitamin E. 8. Vitamin E, ubiquinone and ubichromenol in the rabbit. Biochem. J.79:108. Greaves, M. F., G. Janossy and M. Doenhoff. 1974. Activa- tion of human T and B lymphocytes by polyclonal mitogens. Nature 248:698. Greger, J. L. and R. E. Marcus. 1981. Effect of dietary protein, phosphorus and sulfur amino acids on selenium metabolism of adult males. Ann. Nutr. Metab.25:97. 101 Griffiths, N. M., R. D. H. Stewart and M. F. Robinson. 1976. The metabolism of 75Se-selenomethionine in four women. Br. J. Nutr.35z373. Guggenheim, M. A., S. P. Ringel, A. Silverman and B. E. Crabert. 1982. Progressive neuromuscular disease in children with cholestasis and vitamin E deficiency: diagnosis and treatment with alpha tocopherol. J. Pediatr.100:51. Gyang, E. 0., J. B. Stevens, W. G. Olson, S. D. Tsitsamis and E. A. Usenik. 1984. Effects of selenium-vitamin E injection on bovine polymorphonucleated leukocyte phagocytosis and killing of Staphylococcus agrggs. Am. J. Vet. res.45:175. Hakkarainen, J., P. Lindberg, G. Bengtsson and L. Jonsson. 1978. Serum glutathione peroxidase activity and blood selenium in pigs. Acta. vet. Scand.19:269. Halverson, A. W. and K. J. Monty. 1960. An effect of dietary sulfate on selenium poisoning in the rat. J. Nutr.70: 100. Harris, P. L., M. I. Ludwig and K. Schwarz. 1958. Ineffectiveness of factor 3-active selenium compounds in resorption-gestation bioassay for vitamin E. Proc. Soc. Exp. Biol. Med.97:686. Harris, R. E., L. A. Boxer and R. L. Baehner. 1980. Consequences of vitamin E deficiency on the phagocytic and oxidative functions of the rat polymorphonuclear leukocyte. Blood 55:338. Hazell, T. 1985. Chemical forms and bioavailability of dietary minerals. In: G. H. Bourne (Ed.) World Review of Nutrition and Dietetics. pp 1-123. Karger, Basel. Hidiroglou, M., H. E. Jenkins and J. R. Lessard. 1970. Metabolism of vitamin E in sheep. Br. J. Nutr.24:917. Hillman, R. W. 1957. Tocopherol excess in man:creatinuria associated with prolonged ingestion. Am. J. Clin. Nutr. 5:597. Ho, P. C. and J. W. M. Lawton. 1978. Human colostral cells: Phagocytosis and killing of E. cgli and g. albicans. J. Pediat.93:910. Hoekstra, W. G. 1975. Biochemical function of selenium and its relation to vitamin E. Fed. Proc.34:2083. 102 Hope, W. C., C. Dalton, L. J. Machlin, R. J. Filipski and F. M. Vane. 1975. Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood. Prostaglandin 10:557. Hopper, S. A., A. Greig and C. H. McMurray. 1985. Selenium poisoning in lambs. Vet. Rec.ll6:569. Horwitt, M. K. 1965. Role of vitamin E, selenium and polyunsaturated fatty acids in clinical and experimen- tal muscle disease. Fed. Proc.24:68. Hsieh, H. S. and H. E. Ganther. 1977. Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems. Biochim. Biophys. Acta 497: 205. Hughes, P. E. and S. B. Tove. 1980. Synthesis of alpha- tocopherol quinone by the rat and its reduction by mitochondria. J. Biol. Chem.255:7095. Igarashi, O., K. Mouri and L. M. Chen. 1986. Nutritional factors affecting vitamin E levels in tissues. In: 0. Hayaishi and M. Mino (Ed.) Clinical and Nutritional Aspects of Vitamin E. pp 63-72. Elsevier Science Publishers, Amsterdam. Jaffe, W. G. and C. Mondragon. 1975. Effects of ingestion of organic selenium in adapted and non-adapted rats. Br. J. Nutr.33z387. Jarrat, M. 1976. Diagnosis and treatment of epidermolysis bullosa. South. Med. J.69:113. Jenkins, K. J. and M. Hidiroglou. 1971. Comparative uptake of selenium by low cystine and high cystine proteins. Can. J. Biochem.49:468. Jensen, L. S. and J. McGinnis. 1960. Influence of selenium, antioxidants, and type of yeast on vitamin E deficiency in the adult chicken. J. Nutr.72z23. Jensen, L. 8., E. D. Walter and J. S. Dunlap. 1963. Influence of 9 etary vitamin E and selenium distribution of Se in the chick. Proc. Soc. Biol. Med.112:899. Jensen, M., C. Fossum, M. Ederotti and R. V. J. Hakkarainen. 1988. The effect of vitamin E on the cell-mediated immune response in pigs. J. Vet. Med.35: 549. Jones, C. B. and K. O. Godwin. 1962. Distribution of radio- active selenium in mice. Nature 196:1294. 103 Jorgensen, P. F., J. Hyldgaard-Jensen and J. Moustgaard. 1977. Glutathione peroxidase activity in porcine blood. Acta Vet. Scand.18:323. Karpen, C. W., A. J. Merola, R. W. Trewyse, D. G. Cornwell and R. V. Panganamala. 1981. Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E. Prostaglandin 22:651. Kayden, H. J. and M. G. Traber. 1986. Vitamin E absorption, lipoprotein incorporation and transfer from lipo- proteins to tissues. In: 0. Hayaishi and M. Mino (Ed.) Clinical and Nutritional Aspects of Vitamin E. pp 129-138. Elsevier Science Publishers, Amsterdam. Kayden, H. J. and L. Bjornson. 1972. The dynamics of vitamin E transport in the human erythrocyte. Ann. NY Acad. Sci.203:127. Kayden, H. J. 1978. The transport and distribution of alpha-tocopherol in serum lipoproteins and the formed elements of the blood. In: C. de Duve and O. Hayaishi (Ed.) Toc0pherol, Oxygen and Biomembranes. pp 131-142. Elsevier/North Holland Biomedical Press, Amsterdam. Kingston, H. M. and L. B. Jassie. 1986. Microwave energy for acid decomposition at elevated temperatures and pressures using biological and botanical samples. Anal. Chem.58:2534. Klebanoff, S. J. 1975. Antimicrobial mechanisms in neutro- philic polymorphonuclear leukocytes. Semin. Hematol. 12:117. Krantman, H. J., S. R. Young, B. J. Ank, C. M. O'Donnell, G. S. Rachelefsky and E. R. Stiehm. 1982. Immune function in pure iron deficiency. Am. J. Dis. Child.l36:844. Kuvibidila, S., K. M. Nauss, B. S. Balign and R. M. Suskind. 1983. Impairment of blastogenic response of splenic lymphocytes from iron-deficient mice. 1n_vivo repletion. Am. J. Clin. Nutr.37:15. Lafuze, J. E., S. J. Weisman, L. M. Ingraham, C. J. Butterick, L. A. Alpert and R. L. Baehner. 1983. The effect of vitamin E on rabbit neutrophil activation. In: P. R. Whelan (Ed.) Biology of Vitamin E. pp 130-146. Pitman Books, London. Lands, W. E. M., P. R. Letellier, L. H. Rome and J. Y. Vanderhoek. 1972. Inhibition of prostaglandin bio- synthesis. Adv. Biosci.9:15. 104 Langweiler, M., R. D. Schultz and B. E. Sheffy. 1981. Effect of vitamin E deficiency on the proliferative response of canine lymphocytes. Am. J. Vet. Res.42: 1681. Larsen, H. J. and S. Tollersrud. 1981. Effect of dietary vitamin E and selenium on the phytohaemagglutinin response of pig lymphocytes. Res. Vet. Sci.31z301. Lawrence, R. A., R. A. Sunde, Schwartz, G. L. and W. G. Hoekstra. 1974. Glutathione peroxidase activity in rat lens and other tissues in relation to dietary selenium intake. Exp. Eye Res.18:563. Lee, M., A. Dong. and J. Yano. 1969. Metabolism of 758e- selenite by human whole blood in vitro. Can. J. Biochem.47:79l. Lee, C. S., F. B. P. Wooding and P. Kemp. 1980. Identification, properties and differential counts of cell populations using electron microscopy of dry cow secretions, colostrum and milk from normal cows. J. Dairy Res.47z39. Lee, C. S. and P. M. Outteridge. 1981. Leucocytes of sheep colostrum, milk and involution secretion, with particular reference to ultrastructure and lymphocyte sub-populations. J. Dairy Res.48z225. Levander, O. A. 1976. Selected aspects of the comparative metabolism and biochemistry of selenium and sulfur. In: A. S. Prasad (Ed.) Trace Elements in Human Health and Disease. II. Essential and Toxic Elements. pp 135. Academic Press, New York. Levander, O. A., V. C. Morris and M. Mutanen. 1985. Effect of selenium deficiency on rat aortic glutathione peroxidase activity and on the ability of rat aorta to produce prostacyclin-like activity. Fed. Proc.44:l670. Li, T. K. and B. L. Vallee. 1973 The biochemical and nutritional role of trace elements. In: S. Goodhart (Ed.) Modern Nutrition In Health And Disease. Lea and Febiger, Philadelphia. Liu, C. H., Y. M. Zhang, M. Y. Huang, Q. Su, Q. Lu, 2. H. Yin, R. X. Shao, G. 2. Feng and P. L. Zheng. 1982. Preliminary studies on influence of selenium deficieny to the development of genital organs and spermato- genesis of infant boars. Acta Vet. Zootechnica Sinica 13:73. 105 Lopez, P. L., R. L. Preston and W. H. Pfander. 1968. In vitro uptake of selenium-75 by red blood cells from immature ovine during varying selenium intakes. J. Nutr.4:219. Lorenz, 2. K. 1978. Selenium in wheats and commercial wheat flours. Cereal Chem.55z287. Maag, D. D. and M. W. Glenn. 1967. Toxicity of selenium in farm animals. In: 0. H. Muth, J. E. Oldfield and P. H. Weswig (Ed.) Selenium In Biomedicine. pp 127-138. The AVI Publishing Co., Westport, Connecticut. MacDonald, D. W., R. G. Christian, K. I. Stransz and J. Roff. 1980. Acute selenium toxicity in neonatal calves. Can. Vet. J.22:279. Machlin, L. J., R. Filipski, A. L. Willis, D. C. Kuhn and M. Brin. 1975. Influence of vitamin E on platelet aggregation and thrombocythemia in the rat. Proc. Soc. Exp. Biol. Med.149:275. Machlin, L. J. 1984. Vitamin E. In: L. J. Machlin (Ed.) Handbook Of Vitamins: Nutritional, Biochemical And Clinical Aspects. pp 99-145. Marcel Dekker, Inc., New York. MacMahon, M. T., G. Neale and F. Weber. 1975. Metabolism of vitamin E in the rat. Clin. Sci.38:l97. Mackenzie, C. G., M. D. Levine and E. V. McCollum. 1940. The prevention and cure of nutritional muscular dystrophy in the rabbit by alpha-tocopherol in the absence of a water-soluble factor. J. Nutr. 20:399. Mackenzie, J. B. and C. G. Mackenzie. 1959. The effect ofcz -tocophery1 hydroquinone and their esters on experimen- tal muscular dystrophy in the rat. J. Nutr. 67: 223. March, B. E., E. Wong, L. Seier, J. Sim and J. Biely. 1973. Hypervitaminosis E in the chick. J. Nutr.103:377. Marsh, J. A., R. R. Dietert and G. F. Combs. 1981. Influence of dietary selenium and vitamin E on the humoral immune response of the chick. Proc. Soc. Exp. Biol. Med. 166:228. ' Martin, M. M. and L. S. Hurley. 1977. Effect of large amounts of vitamin E during pregnancy and lactation. Am. J. Clin. Nutr.30:l629. Mason, R. P. and C. F. Chignell. 1982. Free radicals in pharmacology and toxicology. Pharmacol. Rev.33:189. 106 Mathias, P. M., J. T. Harres, T. J. Peters and D. P. R. Muller. 1981. Studies on the in vivo absorption of micellar solutions of tocopherol and tocopheryl acetate in the rat: demonstration and partial characterization of a mucosal esterase localized to the endoplasmic reticulum of the enterocyte. J. Lipid Res.22:829. Mattill, H. A. and Conklin, R. E. 1920. The nutritive pro- perties of milk with special reference to reproduction in the albino rat. J. Biol. Chem.44:137. Mattill, H. A., J. S. Carman and M. M. Clayton. 1924. The nutritive properties of milk. III. The effectiveness of the X substance in preventing sterility in rats on milk rations in fat. J. Biol. Chem.6l:729. McConnell, K. P. and G. J. Cho. 1965. Transmucosal movement of selenium. Am. J. Physiol.208:ll9l. McCoy, K. E. M. and P. H. Weswig. 1969. Some selenium re- sponses in the rat related to vitamin E. J. Nutr.98: 383. Meggs, P. D. and A. E. Beer. 1979. Ln vitro stimulation of colostral lymphocytes by cytomegalo virus. Am. J. Obstet. Gynecol.133:703. Mills, G. C. 1957. Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J. Biol. Chem.229: 189. Mills, G. C. 1959. The purification and properties of glutathione peroxidase of erythrocytes. J. Biol. Chem. 234:502. Mills, G. C. and H. P. Randall. 1958. Hemoglobin catabo- lism. II. The protection of hemoglobin from oxidative breakdown of the intact erythrocyte. J. Biol. Chem.232: 589. Morrow, D. A. 1968. Acute selenite toxicosis in lambs. J. Am. Vet. Med. Ass.152:l625. Motsenbocker, M. A. and A. L. Tappel. 1982. A seleno- cysteine-containing selenium transport protein in rat plasma. Biochem. Biophys. Acta 719:147. Nahapetian, A. T., M. Janghorbani and V. R. Young. 1983. Urinary trimethyl selenonium excretion by the rat: effect of level and source of selenium-75. J. Nutr. 113:401. 107 Naidu, T. G. and F. H. S. Newbould. 1973. Glycogen in leukocytes from bovine blood and milk. Can. J. Comp. Med.37:47. Nichols, W., J. Dunlap and L. Hebeprand. 1979. Feline lymphocytes: Observation on surface membrane concanavalin A receptor mobility. Am. J. Vet. Res.40: 959. Nigam5 S. N. and W. B. McConnell. 1976. Metabolism of Na2 SeOA in Astragalus bisulgatus lima bean, and wheat: a comparison study. J. Exp. Bot.27:565. NRC. 1979. Nutrient Requirements of Domestic Animals, No. 2. Nutrient Requirements of Swine. Eighth Revised Ed. National Academy of Sciences-National Research Council. Washington, DC. Nugteren, D. H., R. K. Beerthnis and D. A. Van Dorp. 1966. The enzymatic conversion of all-cis 8,11,14- eicosa- trienoic acid into prostaglandin E1. Recl. Trav. Chim. Pays-Bas.85:405. Oh, S. H., H. E. Ganther and W. G. Hoekstra. 1974. Selenium as a component of glutathione peroxidase isolated from ovine erythrocytes. Biochem.13:1825. Olcott, H. S. 1938. The paralysis in the young of vitamin E deficient female rats. J. Nutr.15z221. Olson, O. E. B. M. Schultze, E. I. Whitehead and A. W. Halverson. 1963. Effect of arsenic on selenium metabo- lism in rats. J. Agric. Food Chem.llz532. Osman, M. and J. D. Latshaw. 1976. Biological potency of selenium‘ from sodium selenite, selenomethionine and selenocystine in the chick. Poultry Sci.55z987. Ostadalova, I., A. Babicky and J. Obenberger. 1978. Cataract induced by administration of a single dose of sodium selenite to suckling rats. Experentia 34:222. Paape, M. J., A. J. Cuidry, S. P. Kirk and . J. Balt. 1975. Measurement of phagocytosis of 3 P-labelled Staphylococcus aureus by bovine leukocytes: lysostaphin digestion and inhibitory effect of cream. Am. J. Vet. Res.36:1737. Paape, M. J. and A. J. Guidry. 1977. Effect of fat and casein on intracellular killing of Staphylococcus aureus by milk leucocytes. Proc. Soc. Exp. Biol. Med.155z550. 108 Paglia, D. E. and W. N. Valentine. 1967. Studies on the quantitative and qualitative characterization of eryth- rocyte glutathione peroxidase. J. Lab. Clin. Med. 70: 158. Palmer, I. S., R. L. Arnold and C. W. Carlson. 1973. Toxicity of various selenium derivatives to chick embryos. Poultry Sci.52zl84l. Palmer, I. S. and O. E. Olson. 1974. Relative toxicities of selenite and selenate in the drinking water of rats. J. Nutr.104z306. Palleni, V. and E. Bacci. 1979. Bull sperm: selenium in bound to a structural protein of mitochondria. J. Submicr. Cytol.ll:l65. Panganamala, R. V. and D. G. Cornwell. 1982. The effects of vitamin E or arachidonic acid metabolism. Ann. NY Acad. Sci.393z376. Pappenheimer, A. M. and M. Goettsch. 1931. A cerebellar disorder in chicks, apparently of nutritional origin. J. Exp. Med.53:ll. Parmely, M. J., A. E. Beer and R. E. Billingham. 1976. In vitro studies on the T-lymphocyte population of human milk. J. Exp. Med.144z358. Patterson, E. L., R. Mostry and E. L. R. Stockstad. 1957. Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exp. Biol. Med.95:617. Pike, R. L. and M. L. Brown. 1984. Vitamin E. Nutrition An Intregrated Approach (3rd Ed.). John Wiley&Sons, New York. Reamer, D. C. and C. Veillon. 1983. Elimination of perchloric acid in digestion of biological fluids for fluorometric determination of selenium. Anal. Chem. 55:1606. Reffet, J. K., J. W. Spears and T. T. Brown. 1988a. Effect of dietary selenium on the primary and secondary immune response in calves challenged with infectious bovine rhinotracheitis virus. J. Nutr.1182229. Reffet, J. K., J. W. Spears and T. T. Brown. 1988b. Effect of dietary selenium and vitamin E on the primary and secondary immune response in lambs challenged with parainfluenza virus. J. Anim. Sci.66:1520. 109 Renshaw, H. W., W. P. Eckblad, D. E. Everson, P. D. Tassinari and D. Amos. 1977. Ontogeny of immuno- competence in cattle: evaluation of phytomitogen- induced in vitro bovine lymphocyte blastogenesis using a whole blood culture technique. Am. J. Vet. Res.38: 1141. Rosenblum, J. L., J. P. Keating, A. L. Prensky and A. L. Nelson. 1981. A progressive neurologic syndrome in children with chronic liver disease. N. Eng. J. Med. 304:503. Rosenfeld, I. and O. A. Beath. 1969. Selenium. Geobotany, bichemistry, toxicity and nutrition. Academic Press, New York. Rotruck, J. T. A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman and W. G. Hoekstra. 1973. Selenium: bio- chemical role as a component of glutathione peroxidase. Science 1792588. Runge, P., D. P. R. Muller, J. McAllister, D. Calver, J. K. Llyod and D. Taylor. 1986. Oral vitamin E supplements can prevent the retinopathy of abetalipoproteinaemia. Brit. J. Opthalmol.70:l66. Russel, M. W. and B. Reiter. 1975. Phagocytic deficiency of bovine milk leukocytes: An effect of casein. J. Retic. Endothel. Soc.l8:l. Russel, M. W., B. E. Brooker and B. Reiter. 1976. Inhibi- tion of the bactericidal activity of bovine polymorpho- nuclear leukocytes and related systems by casein. Res. Vet. Sci. 20:30. Sandholm, M. 1975. Function of erythrocytes in attaching selenite-Se onto specific plasma proteins. Acta Pharmacol. Toxicol.36:321. Sankari, S. 1985. Plasma glutathione peroxidase and tissue selenium response to selenium supplementation in swine. Acta Vet. Scand. supp1.81:1. Schamberger, R. J. 1983. Biochemistry of Selenium. Plenum Press, New York. Schiavon, R., G. E. Freeman, G. C. Guidi, G. Perona, M. Zatti and V. V. Kakkar. 1984. Selenium enhances prostacyclin production by cultured endothelial cells: possible explanation for increased bleeding times in volunteers taking selenium as a dietary supplement. Thrombosis Res.34z389. 110 Schimizu, M. and Y. Schimizu. 1979. Lymphocyte prolifera- tive response to viral antigen in pigs infected with transmissible gastroenteritis virus. Infect. Immun.23: 239. Schmandke, H. and G. Schmidt. 1965. Absorption of a- tocopherol from oily and aqueous solution. Int. 2. Vitaminforsch 35:128. Schmandke, H., C. Sima and R. Manne. 1969. Die resoption von a-tokopherol blemm menschen. Int. 2. Vitaminforsch 39: 796. Schollenberger, A., A. Degorski, T. Frymus and A. Schollenberger. 1986. Cells of sow mammary secretions. I. Morphology and differential counts during lactation. J. Vet. Med.33z3l. Schroeder, H. A., D. V. Frost and J. J. Balassa. 1970. Essential trace metals in man: selenium. J. Chron. Dis. 23:227. Schwarz, K. 1961. Development and status of experimental work on Factor 3 selenium. Fed. Proc.20:666. Schwarz, K. and C. M. Foltz. 1957. Selenium as an integral part of Factor 3 against dietary necrotic liver genera- tion. J. Amer. Chem. Soc.79:3292. Schwarz, K. and C. M. Foltz. 1958. Factor 3 activity of selenium compounds. J. Biol. Chem.233:245. Schwarz, K. and A. Fredga. 1969. Biological potency of organic selenium compounds. Aliphatic monoseleno- and diseleno-dicarboxylic acids. J. Biol. Chem.244:2103. Scott, M. L. and I. D. Desai. 1964. The relative anti- muscular dystrophy activity of the d- and l-epimers of a -tocopherol and of other tocopherols in the chick. J. Nutr.83:39. Scott, M. L. 1980. Advances in our understanding of vitamin E. Fed. Proc.39:2736. Sharma, S. and R. Singh. 1983. Selenium in soil, plant, and animal systems. Crit. Rev. Environ. Control 13:23. Sherman, A. R. 1984. Iron, infection, and immunity. In: R. R. Watson (Ed.) Nutrition, disease resistance, and immune function. pp 23-45. Marcel Dekker, Inc., New York. 111 Simon, E. J., L. S. Gross and A. T. Milhorat. 1956a. The metabolism of vitamin E. I. The absizption and excre- tion of d-a -tocophery1-5-methy1-C succinate. J. Biol. Chem.2212797. Simon, E. J., A. Eisengart, L. Sundheim and A. T. Milhorat. l956b. The metabolism of vitamin E. II. Purification and characterization of urinary metabolites of a-tocopherol. J. Biol. Chem.221z807. Simpson, D. W., R. Roth and L. D. Loose. 1979. A rapid, in- expensive and easily quantified assay for phagocytosis and microbicidal activity of macrophages and neutro- phils. J. Immunol. Met.29:221. Smith, J. W. and R. D. Schultz. 1977. Mitogen and antigen- responsive milk lymphocytes. Cell Immunol.29:165. Sokol, R. J., J. E. Heubi, S. T. Iannaccone, K. E. Bove and W. F. Balistreri. 1983. Mechanism causing vitamin E deficiency during chronic childhood cholestasis. Gastroenterology 85:1172. Spencer, R. P. and M. Blau. 1961. Intestinal transport of selenium-75 selenomethionine. Science 136:155. Stobo, J. D. and W. E. Paul. 1973. Functional heterogeneity of murine lymphoid cells. III. Differential responsive- ness of T cells to phytohemagglutinin and concanavalin A as a probe for T cell subsets. J. Immunol.110:362. Stossel, T. P., R. J. Mason and A. L. Smith. 1979. Lipid peroxidation by human blood phagocytes. J. Clin. Invest.54:638. Stowe, H. D. and E. R. Miller.1985. Genetic predisposition of pigs to hypo- and hyperselenemia. J. Anim. Sci. 60: 200. Sure, B. 1924. Dietary requirements for reproduction. II. The existence of a specific vitamin for reproduction. J. Biol. Chem.58z693. Tan, J., D. Zheng, S. Hov, W. Zhu, R. Li, Z. Zhu and W. Wang. 1986. Selenium ecological and chemico-geography and endemic Keshan disease and Kasschin Bech's disease in child. In: G. F. Combs, J. E. Spallholz, O. A. Levander and J. E. Oldfield (Ed.) Proceedings of the Third International Symposium on Selenium in Biology and Medicine. pp 12-24. AVI Publishing Co., Westport, Connecticut. Tappel, A. L. 1962. Vitamin E as the biological lipid anti- oxidant. Vitam. Horm.20:493. 112 Teige, J., S. Tollersrud, A. Lund and H. J. Larsen. 1982. Swine dysentery: the influence of dietary vitamin E and selenium on the clinical and pathological effects of Ireponema byogysentgriag infection in pigs. Res. Vet. Sci.35z95. Tengerdy, R. P., R. H. Heinzerling and M. M. Mathias. 1978. Effect of vitamin E on disease resistance and immune responses. In: C. de Duve and O. Hayaishi (Ed.) Tocopherol, Oxygen and Biomembranes. pp 191-196. Elsevier/North. Holland Biomedical Press, Amsterdam. Thompson, J. N. and M. L. Scott. 1969. Role of selenium in the nutrition of the chick. J. Nutr.97z335. Thompson, J. N. and M. L. Scott. 1970. Impaired lipid and vitamin E absorption related to atrophy of the pancreas in selenium-deficient chicks. J. Nutr.100:797. Thomson, C. D., B. A. Robinson, R. D. H. Steygrt and M. F. Robinson. 19757 Metabolic studies of ( Se) seleno- cystine and ( Se) selenomethionine in the rat. Br. J. Nutr.43z501. Thomson, 95 D and R. D. H. Stewart. 19737 Metabolic studies of ( Se) selenomethionine and ( 5Se) selenite in the rat. Br. J. Nutr.30:l39. Thomsgg, C. D. and R. D. H. Stewart. 1974. The metabolism of ( Se) selenite in young men. Br. J. Nutr.32z47. Tizard, I. 1987. Veterinary Immunology. An Introduction (3rd. Ed.) W. B. Saunders Co., Philadelphia. Ullrey, D. E., E. R. Miller, D. J. Ellis, D. E. Orr, J. P. Hitchcock, K. K. Keahey and A. L. Trapp. 1971. Vitamin E (selenium and choline), reproduction and MMA. Michigan Agr. Res. Sta. Rep.148:48. Underwood, E. J. 1981. Selenium. In: The Mineral Nutrition Of Livestock (2nd. ed). pp 149-167. Commonwealth Agricultural Bureaux, London. Vale, O. E. 1983. Mastitis, Metritis, Agalactia in swine: Role of vitamin E and selenium. M. S. Thesis. Michigan State University, East Lansing. Vane, J. R. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature New Biol.231:232. 113 Van Kampen, K. R. and L. F. James. 1978. Manifestations of intoxication by selenium-accumulating plants. In: R. F. Kuler, K. R. Van Kampen and L.F. James (Ed.) Effects of Poisonous Plants on Livestock. pp 135-148. Academic Press, New York. Wallace, E., H. 1. Calvin and G. W. Cooper. 1981. Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency. Gamete. Res.4:377. Whanger, P. D., N. D. Pedersen, J. Hatfield and P. H. Weswig. 1976. Absorption of selenite and seleno- methionine from ligated digestive tract segments in rats. Proc. Soc. Exper. Biol. Med.153:295. Whetter, P. A. and D. E. Ullrey. 1978. Improved fluoro- metric method for determining selenium. J. Assoc. Official Anal. Chem.612927. Whitehair, C. K., O. E. Vale, M. Loudenslager and E. R. Miller. 1983. MMA in sows- a vitamin E-selenium defi- ciency. Michigan Agr. Sta. Rep.456:9. Wisniowski, J., K. Romaniukowa and H. Grajewski. 1975. Phagocytosis phenomena in the mammary gland of cows. 1. Opsoning factor and phagocytic activity of leukocytes in milk and blood. Bull. Vet. Inst. Pulavy 9:140. Wolffram, S., F. Arduser and E. Scharrer. 1985. In vivo intestinal absorption of selenate and selenite in rats. J. Nutr.1151454. Wright, P. L. and M. C. Bell. 1966. Comparative metabolism of selenium and tellurium in sheep and swine. Am. J. Physiol.211:6. Wright, P. L. and M. C. Bell. 1964. Selenium-75 metabolism in the gestating ewe and fetal lamb. J. Nutr.84z49. Wu, S. H., J. E. Oldfield and P. D. Whanger. 1973. Effects of selenium, vitamin E and antioxidants on testicular function in rats. Biol. Reprod.8:625. Yang, N. Y. J. and I. D. Desai. 1978. Glutathione peroxidase and vitamin E interrelationship. In: C. de Duve and O. Hayaishi (Ed.) Tocopherol, Oxygen and Biomembranes. pp 233-238. Elsevier/North Holland Biomedical Press, Amsterdam. 114 Zhu, L. and 2. Lu. 1981. Effects of pigs fed the crops grown in Keshan disease affected province of China. In: J. M. Howell, J. M. Gowthone and C. L. White (Ed.) Trace Element Metabolism in Man and Animals. pp 360-364. Springer-Verlag, New York. APPENDIX 5 1 1|. .Amo.o v av o asoum scum acouomwuu sensuouuacuamv .Amo.o v nv N nsoum sown acupuMMHv haucaoauacmamo .zmm H «can: one no=~a> a .mnoauoo Hayseeduooxo pom N mans» coma oo.oumm.a oc.owmn.~ oo.cum~.a Ho.ouno.a mo.cuaa.o a am+ m+ o emc.oume.o u~o.cuhm.c eao.cumm.o u~o.cuom.o m~.ouno.~ s am. a. m ha.ouao.a ao.ouam.a ea.ouos.a no.ouma.a nc.cuno.a q om+ m+ e Ha.ouao.~ no.ouom.a ~c.cwae.a mo.ouks.a wo.cua~.a e um. m+ n an.ouc~.~ a~.cuam.~ o~.ousm.a ma.oH~h.~ -.oums.a s om+ m+ N ooo.ouom.o oha.ouo~.c om~.ouoa.o oec.ouoH.a awa.ouoa.~ s om+ m- a InIIunuluuulnlluutunnulunsnl ~E\m1 uuuuuuuuuuuuuuuuuu taunts: oNH ca cm on o mason» ovum :o Amvsmo osoum\c uofin unmanamuh .:0wuwu=upma use eofiunmocoo cmmzuoa nuoflu coauwaawu Eswcwawm uo\ucm m :«Emaw> cow mzom mo m:0wumuucwocoo m swamuu> Eauom .oH oHnuH 116 .Amo.c v Av w anon» scum acououuuv saucnoauacuamv .Amc.o v_av q asoun scum acououuav knucuouuucuamo .zww H usual ops nosaa>a .nvoauoa unnameduoaxo you N can.» coma $63.22 caéflocafi S.~H8.o2 $64862 Sagas: a 8+ m+ o ennéuooéfi ciauonafl esnéuooé: com.~wooé: $.auomé: a 2.... m- n 3.3:.“2 oeéuflaz SGHSKS 56890: Rfiuooéfl a 5+ u+ a ooéumfinfi unnaufiéfl omméufigz "magmas: 3.2Hom.m3 .V on- m+ n Singh.; Eiufiafi SJNHSAS «SSNNAB SSHSAS a 8+ u+ ~ giufldfi H~.:Hom.~2 3.3Hooé: 8.:HRGS n3.~.~ufi.n2 a 3+ u- H u---------u--------------- He\mc ................ -u----- o- as cm on a sauna comm co Aswan: nsoumxc and: aconuooua coauoanou Esucmamm uo\v:a m cfismuw> com mzom mo acoauuuusoocoo Esqcouou asuom .cowuausuuaa use coauooouoo consume uuuav .~H canoe .Amc.c vow e asonm eonm noonoMMNo huncoonunomnmo .Amo.o vav q coon» Eonm noonoMMNo hancoonmacunmo .zmm H cocoa ono oosuo>o .moonnoa Nonooannooxo now N oHaou oomo 117 an.oumo.~ nn.ousm.~ no.cuon.~ no.oHHn.~ n~.onmo.~ a om+ o+ o u-.ouno.n us~.oune.a uc~.cwnn.n eao.ounm.n Hm.ouem.~ e um- u- m an.ouoo.~ oo.ouon.n so.ounn.n NH.oHso.n on.ousm.n om+ m+ on~.cumc.n onc.ouom.n oa°.cusm.a an.ouam.a an.ouna.n um- m+ o~.cumn.~ an.ouan.~ an.cucc.~ mn.oumo.~ o~.ouce.~ om+ m+ cs.cusn.~ n~.cunn.~ s~.cuno.~ s~.ou~n.~ can.ouaa.n om+ m- I nnnnn nauuuuunuuu Ne\ou«:= Enucm uuuuuuuuuuuuuuuuuuuuuu ONN co co an o noon» ooom so onxoa ozonm\c noun nooMnoonH .sonnnnsunoa oco cownaoooo cooznon muono connosooo Esasoaoo no\o:o m onsonn> vow mzom mo unw>nnoo omoonxonoa oconznousaw.asnom .NH oHnoH 118 .zmm H ocoos ono oosuo> a .mooanoo Honcosnnooxo now N onnou oomo N.N No.0» o.onHo.ao o.nnun.oa ~.snum.onn m.n Hm.~on a om+ m+ o o.c Hn.ao o.m Hm.no m.nnun.moa ~.n Hn.mm m.m No.5» e mm. m- m o.onuo.o~n «.0 Hm.no o.nnuo.so m.~num.non m.o Hm.~a s om+ m+ e n.nnua.~nn o.n Hm.mo o.» Ho.mn s.m Hm.oo a.» Ho.na e um- m+ n ~.m Ho.ncn a.e Ho.mo n.s Hm.~o m.m Hm.om o.m Hm.aa s um+ o+ N ~.nnnn.ao n.onuo.ma m.namm.ooa s.~ Hm.mn aw.nnun.non s om+ m- n unnnunuuuauuuunauuuu:unlit-i: Sima nu uuuuuuuuuuuuuuuuuuuuuuuuuuu Ii: cNH on so an o anon» ooom :o novnoa asonmxc nod: noomuoona .:0nnnn:nnoo ooo connooocoo cooznon ouono connonooo Esnooaom noxoco m onsonn> oom ozom mo uncanonnoooooo Nononoouono esnom .nH ounop 119 I..." Ltlrllirr. -. in .zwm H ocooe ono oosno>o .ooonnoo Noncoannonxo nou N oNaon oowo an.oumn.~ mo.oHnN.~ H~.ounn.~ sm.ouam.~ oc.ouem.~ c om+ u+ m~.cnoo.~ ne.oums.~ ao.ouss.~ mn.onsn.~ mm.ouen.~ e um. m- mo.ounn.~ an.oune.~ oo.ouno.~ mn.ou~m.~ n~.cuoo.~ s om+ o+ so.ouse.~ an.ouon.~ on.cumn.~ mo.ou~s.~ ~n.ons.~ e um. m+ ao.ouns.~ NH.oHns.~ on.ouom.~ ma.ounn.~ o~.ouoo.~ e om+ m+ mn.ounm.~ mo.cume.~ ec.cumm.~ o~.ouom.~ amn.oumn.~ a am+ w- n nununuunnnuununnuuuuuu nofimoav :mo nnnnnnnnnnnnnnnnnnn nu oNn co cm on o mason» ooou :o novsoa anonmxc nonn nooEnoonH .connnnsnnoa woo conuooocoo sooznoa muowo connoaaoo Eonsofloo no\v=o m caaonw> com ozoo mo monsoonnExN ooona Honocannoa oonoNsEnnmcs mo oxouo: ocaanhnuumn .cH oHnoH 120 .Amo.o v av e anon» sonu noonommno huncoonunomnmo .nmo.o v av N anon» sonm noonoumno hauooonmnomumo .zmm H ocooa ono oo=No>o .ooonnoo NouooEnnooxo now N oNnou oomo mc.owmn.m no.ouo~.m on.oun~.m ~n.oHso.m an.ouoo.c a om+ m+ o un~.oueo.e uen.ouoa.e onc.cuso.s n~.oun~.m ~o.oH~n.m a «mu m- m oo.ou~n.m oo.cuma.m n~.ouom.s Hm.ouno.s a~.onmm.s s um+ u+ a on.ouom.s an.cnc~.m sc.cunn.m Nm.cuam.s m~.ouna.e a om- m+ n ao.onnn.m os.cuoa.s n~.ouan.s a~.ounm.s os.oun~.e s om+ m+ N con.ouna.n oo~.onsm.s on.ouam.s mn.ouao.m can.ouaa.s s. mm+ m- n nunuuuuunuuatuuunnnunn Aoamoav zoo uuuuuuuuuuuuuuu :nuuu oNn as as on c guano ooou :o Amvxon noonm\: noun nooMnoonH .connnnsnnoa oco connaoocoo :oosuon muono :0nnonooo EsnooNoo no\o:o m onEona> vow ozoo mo monsoozoesfi oooaa Hononannoo oouo~=Ennmno .moonnoo NonooEnnooxo nom N ounon oomo mc.oum~.m mo.cuo~.m no.cue~.m mo.oHaH.m mn.ouno.e e om+ o+ o c-.ouoa.e coo.ouno.m eso.ouc~.m mo.oumn.m m~.cu~n.m mm- m- so.cunn.m no.cuo~.m on.ouoo.m an.ow~o.s sq.onnn.e am+ m+ nq.ounmuq. so.ouon.m on.oumn.m an.oHoo.m ns.oune.s om- o+ no.cnsa.m an.oHon.m on.oumo.m an.oueo.s os.cu~s.s am+ n+ can.ouan.n omo.ouso.s ac.ounc.m wo.oun~.m smm.ouas.e am+ m- unnnununuuausuuuuunuuuu Aofiwoav :mo uuuuuuuuuuuuuuuuu nun: own as cm on c ooou co Amvhoo noonmxc non: noomnoons .oownnnsunoo oco noduooocoo :oo3uon muowo connonaoo Eonooaoo no\o:o w oneonn> vow ozoo mo oouxoozaEAH oooan Honochnoa oonoNsswnouzzm mo oxonas ocno«5hcuum .oH oanoh 122 .zum H ocoos ono oooao>o .moonnoo Nouooannooxo now N ounon oomo «N.ouno.e No.onnm.s oo.ousm.s fin.ousn.s Nn.oHNn.e s um+ m+ o so.cHNo.e on.ounn.s HN.onN.q on.ousm.s oN.onN.e s cm- on m nn.cumo.< NN.oHNN.s mN.oHaN.q mo.ouon.q ao.oHNN.e c om+ m+ o oN.oHNm.s nN.cunm.e an.oNNo.s NN.onN.q No.9Hom.e e on. u+ N mn.ouon.s NN.oHos.s No.ouom.s oN.cHNN.s as.ouoo.n e om+ m+ N on.oHNo.e en.ouno.s an.oumn.s NN.oHnm.s nqo.oHoN.n a om+ m- n Inuuuunuusunuuuunuunu AonoNv zoo uuuuuuuuuuuuuuuuuu nun: oNN co as on o mason» ooom :o onaon asonm\: noun nooEnoona .conunnsnnoo coo connaoocoo :ooSnon mnowo sonuoaaoo Enuooaoo no\v:o m :weoua> vow meow mo monsoocoEAN ooosn Nononannoa oonoH=Enumu<.oou mo oxonas ocaonahnnumm .NH oHaoH .Amo.o v .Amo.o V av w noon» Av e noon» .Amc.c V AV N anon» Eonu noonoumao unuooouunomumu Bonn noonouuno haucoonmnomumo Eonm noonommao hunoooamnomnmo .zmm H noooe ono oo=No>o .oNNoo zzm OOH non nooo» onoe no N moncnoucoo oNNoo zzm mo nonesc ocn mo coonmoo ow huu>anoo oanhoomoso N ocen .oooanoo NoncoEnnooxo now N oNoon oomo 3 co.oHNm.Na ca.nHNN.mm om.oums.ow mN.aHNq.om Nm.NHos.Nw a om+ m+ o n NNN.nHms.mN non.NHoN.nm oNN.oHoo.2 mo.nums.eo oo.NHNN.oo e mm. m- m eo.NHo~.oo ca.nunm.Na AN.mHoa.am 0N.NHNN.NN NN.mHoe.mm q om+ m+ a oNn.anN.NN oNo.NHNm.oo NN.NHNN.NN oN.NHsa.oa mm.muco.mo s cm. m+ n mo.nuon.cs nm.oHcN.oa ms.numa.am sm.nwmn.Nm Nm.NHaN.oo s am+ m+ N ona.NHom.mo eeo.nuoa.em cN.sHmo.ow Na.nHNN.mN ONm.Nunm.mo s wm+ m- n 'IIIIIIIIIIIIIIIIII'IIII'IIIIII ON IIIII I lllllllllll IIIIII oNn cm as on o anon» ooom oo Amvnoa osonw\o noac noomnoona m cweonn> vow msom mo mNHoo nooNosooconoahaoo oooNn mo hnw>unoo onuhoomonm .cOannsnnoa woo connaoocoo sooSnon muono cannoanoo esncoaoo no\o=o .QN oNan 124 .Amo.ouvav o asonm Eonw noonowwwo huncoowwncmwm w .Amc.cu.ov q noon» Eonw noonowwno huusoowwncmwmo .Amo.ouvav N anonm Eonw noonowwno hnuooonwncmwm v .zmm H ocooe ono nosNo>o .oNHoo 22m ooN non oououunoo noooh oooo onoe no N oocnoncoo oNNoo zzm wo noneso ozu mo oonoasonoo no: hau>wnoo Hooaownonowe N ones .ooonnoo HoncoENnooxo now N ouoou oomo nN.NHmm.mm mN.nHNN.Ns mm.NHNN.Ns Ho.NHNN.Ns mo.NHca.os s um+ m+ o mm.ouno.Nn wan.ousa.Ns no.oHN~.ms oN.NHmn.om as.NHmm.Hm s mm. m- m nm.NHaN.om oe.NHNo.nm Nc.eHNm.sm NN.mHam.ms as.NHme.ms s om+ m+ s one.nHNN.qn com.Nums.mn an.nuan.am mm.NHos.Ns oo.~HNe.Ns s cm. m+ n on.nHNN.Nm aN.NHNG.Nm cN.NHNn.Nm sm.auma.~s mm.nuom.an s um+ m+ N usc.nuoo.oe ueo.nHmN.Ns oo.suon.om ac.sHoN.mm on.HHoN.on s om+ m- N IIIIIIIIIII'IIIIII'IIIIII'III DN IIIIIIIIIIIIIIIIIIIII IIUIII oNn co so an a mason» ooow :0 onnon ozonm\: yoga noosnoona .:0nnwn=nnoa oco cownaoocoo :ooznon muowo :ownoaaoo Eswooaoo no\o=o m :wsonw> oow msom wo mHNoo nooaoococonoewaoa oooHn wo huw>wnoo Hoofiownonowz .mH oanoa 125 .Amo.o v av o anon» Eonw nnonowwwo nwnnoowwwnwwm U .Amo.o v av N anonw Eonw noonowwno hannoonwwcmwmo .zmm H mnoos ono monno> n .moownoa Nounoswnoaxo now N oanon oomo mn.oHNe.n Nm.oHom.Nn a mm+ m+ o mn.owmm.n emo.numn.m s mm. m- m mn.cHaN.n mm.onN.m s wm+ m+ a on.ounm.n mo.nHmN.nn a om- m+ m mn.oHNm.n na.ouoo.nn s mm+ m+ N sn.oumm.n o.nas.nHmN.w a wm+ m- n wav unmwos nonnnw\cnon oanonw mwa Honon>wocw mwwa Nouoe anonm\n now: unosnoone .:0nnwnnnnoa ono scanaoonoo noozuoc ouowo cowuosaoo ennnoaom no\ono m swEonn> now ozom wo oocoEnownoa o>wnonoonaom .oN owaoe .Awwawv owownonuno oco ooA Eonw onoao .Aomanv www.mm «on sonw «page .Aomanv .n Mm ummnonconnonom eonw mama n .nnwono>wns ouonm cowwcowz .nnouonoaoa awowonuoa Noownwao onn Eonw ouono 126 oo.n mN.o . oo.N - . Nm.eN so.an . annoo nannmcunam - . om.o . - om.o . - oq.o mnnnaommm . co.n oo.m oo.o~ om.m oo.a om.N No.n oo.N mnnzaoanmom oo.mn om.» oo.No oo.on mN.m oo.mm na.nn mm.on oo.os mounooeaena oo.sw oo.aN om.N oo.No mN.mN oo.q NN.N om.o os.e mommcaonomz oo.N mN.oo oo.om oo.nn om.no oo.wN na.om oN.No oo.me mnncaonunoz IIIIIIIIIIIII lllllllllllllllllllll N lllllllllllllllllllllllllllllllll on no «a o: oo mm a: no an 83m. 38 meow moan... Sou .msoo wo sane ono EnnnmoNoo .ooown Nonocawnoa wo . mwuaw UCM @300 wnoocoasoo noNnHHoo Noenoz .NN oHnoH TQTE UNIV. LIBRQRIES II RIHIWIIIHHIIHE iii?“ 31293007 H! “Mm 1W ll I! Ill? 88 1075