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Iron and selenium supplementation of sheep Dubeski, Paula Leone 1983

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IRON AND SELENIUM SUPPLEMENTATION OF SHEEP by PAULA LEONE DUBESKI B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1983 © Paula Leone Dubeski, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f 6 c ^ ^ ? 3 ^ e?£(£s/?& The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Iron d e f i c i e n c y and a p o s s i b l e i n t e r a c t i o n between i r o n and selenium were i n v e s t i g a t e d i n lambs r a i s e d under an i n t e n s i v e management system. T r i a l 1 compared 2 l e v e l s of i r o n dextran treatment, 0 and 500 mg Fe, using 35 lambs i n j e c t e d once at b i r t h . T r i a l 2 involved 66 lambs and 3 l e v e l s of i r o n : 0, 250, and 500 mg. A t h i r d t r i a l was r e p l i c a t e d 3 times, using a t o t a l of 121 lambs, i n order to determine i f the i r o n treatment response was l i m i t e d by the marginal Se status of the lambs. Treatments were con-t r o l , +1.5 mg Se, +500 mg Fe, and 1.5 mg Se + 500 mg Fe. The parameters measured included hemoglobin, hematocrit, weight, plasma i r o n , and a plasma p r o f i l e (Ca, P j , glucose, BUN, t o t a l p r o t e i n , albumin, AP, LDH and AT). A d d i t i o n a l l y , plasma Se, plasma p r o t e i n f r a c -t i o n s and disease r e s i s t a n c e were measured i n T r i a l 3. I n j e c t i o n of 500 mg Fe s i g n i f i c a n t l y (P<0.05) increased hemoglobin from 2 to 11 weeks of age i n T r i a l 1, and from 1 to 8 weeks i n T r i a l 3. While i r o n dosages of e i t h e r 250 or 500 mg prevented the depression of hemoglobin from b i r t h to 30 days, plasma i r o n and hemoglobin (P<0.05) were s i g n i f i c a n t l y higher at 4 weeks i n lambs r e c e i v i n g 500 mg Fe. In a l l s t u d i e s , a s i g n i f i c a n t proportion of c o n t r o l lambs were anemic at 3-4 weeks of age. P r e l i m i n a r y information was provided on the e f f e c t of i r o n d e f i -ciency and other f a c t o r s (breed, sex, r e a r i n g , b i r t h weight and growth rate) on the lamb plasma p r o f i l e at 4 weeks. The data i n d i c a t e that i r o n d e f i c i e n c y a f f e c t s plasma metabolites s i m i l a r l y i n lambs and humans. P^, glucose, c h o l e s t e r o l , t o t a l p r o t e i n , a l k a l i n e phosphatase and aspartate - i i i -transaminase responded l i n e a r l y to i r o n dosage. Many parameters were a l s o s i g n i f i c a n t l y c o r r e l a t e d with plasma i r o n . The i n t e r a c t i o n of i r o n with selenium was s i g n i f i c a n t (P<0.05) only f o r plasma selenium l e v e l s . Plasma selenium at 4 weeks was increased i n lambs i n j e c t e d with selenium and not i n j e c t e d with i r o n . Means were 0.085 ppm ( c o n t r o l ) , 0.086 ppm (+Fe), 0.107 ppm (+Se) and 0.088 ppm Se(+Fe+Se), with 18 to 20 lambs per treatment. Disease r e s i s t a n c e was assessed by s u s c e p t i b i l i t y of lambs to sore-mouth; hemagglutination t i t e r to a chicken RBC antigen; and gamma g l o b u l i n l e v e l s from 2 to 6 weeks. Selenium but not i r o n treatment i n f l u e n c e d s u s c e p t i b i l i t y of lambs to soremouth. The response of lambs to a n t i g e n i c challenge from chicken RBC's was a l s o increased (P<0.05) by Se treatment a t b i r t h , even though by the time of i n i t i a l challenge at k weeks, plasma Se was only s l i g h t l y higher i n the S e - i n j e c t e d lambs (0.098 ppm vs. 0.086 ppm). Iron had l i t t l e e f f e c t on t i t e r , except i n selenium-treated lambs. Although i r o n treatment enhanced gamma g l o b u l i n production at 6 weeks of age, i r o n may be more c r u c i a l to c e l l u l a r rather than humoral immunity. This study c o n s i s t e n t l y demonstrated a dramatic response of blood hemoglobin to i r o n treatment, but a l s o i n d i c a t e d that other aspects of i r o n d e f i c i e n c y may be more important than anemia. Marginal d e f i c i e n c i e s of both i r o n and selenium may a f f e c t lamb heal t h , and thus have an economic impact on i n t e n s i v e sheep production systems. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 LITERATURE REVIEW 5 Iron 5 Selenium 29 Iron and Selenium I n t e r a c t i o n s 43 MATERIALS AND METHODS 47 Experimental Design 47 S t a t i s t i c a l A n a l y s i s 51 Animal Management 53 A n a l y t i c a l Procedures 54 RESULTS AND DISCUSSION 60 T r i a l 1 Iron Supplementation 60 Hb, PCV, Plasma Iron 60 Plasma P r o f i l e 65 Weight 72 T r i a l 2 Lev e l of Iron Supplementation 73 Hb, PCV, Plasma Iron 73 Plasma P r o f i l e 77 Weight 85 T r i a l 3 Iron and/or Selenium Supplementation 86 Hb, PCV 86 Plasma Selenium 96 Plasma P r o f i l e 100 Weight 104 Plasma P r o t e i n E l e c t r o p h o r e s i s 107 E f f e c t of Selenium on Epidemiology of Soremouth 115 Hemagglutination Results 118 CONCLUSIONS 125 REFERENCES CITED 129 APPENDICES 147 V LIST OF TABLES Page Table I. E f f e c t of i r o n treatment on plasma p r o f i l e at 24-25 days of age ( T r i a l 1) 66 Table II. E f f e c t of three l e v e l s of i r o n supplementation on plasma p r o f i l e at 30-31 days of age ( T r i a l 2) 79 Table III. E f f e c t of r e p l i c a t e on plasma p r o f i l e at 4 weeks ( T r i a l 3) 101 Table IV. E f f e c t of i r o n and selenium supplementation on plasma p r o f i l e ( T r i a l 3) 102 Table V. Plasma p r o t e i n e l e c t r o p h o r e s i s r e s u l t s compared to l i t e r a t u r e values 110 Table VI. E f f e c t of age and treatment on plasma p r o t e i n s ( T r i a l 3, R e p l i c a t e 3) 111 Table VII. E f f e c t of treatment on plasma p r o t e i n s at 4 weeks ( T r i a l 3, R e p l i c a t e 2) 112 Table VIII. E f f e c t s of selenium treatment on epidemiology of soremouth i n f e c t i o n 117 vi LIST OF FIGURES Page Figure 1. E f f e c t of i r o n supplementation on hemoglobin ( T r i a l 1) 62 Figure 2. E f f e c t of i r o n supplementation on packed c e l l volume ( T r i a l 1) 63 Figure 3. E f f e c t of i r o n l e v e l on hemoglobin ( T r i a l 2) 74 Figure 4. E f f e c t of i r o n l e v e l on packed c e l l volume ( T r i a l 2) 75 Figure 5. E f f e c t of i r o n and selenium supplementation on hemoglobin ( T r i a l 3) 87 Figure 6. E f f e c t of i r o n and selenium supplementation on packed c e l l volume ( T r i a l 3) 88 Figure 7. Hemoglobin versus PCV at 2 days ( T r i a l 3) 91 Figure 8. Hemoglobin versus PCV at 4 weeks ( T r i a l 3) 92 Figure 9. Hemoglobin versus PCV at 8 weeks ( T r i a l 3) 93 Figure 10. Hemoglobin versus PCV for a l l T r i a l 3 data, 5% o u t l i e r e x c l u s i o n 94 Figure 11. Hemoglobin versus PCV f o r a l l T r i a l 3 data 95 Figure 12. E f f e c t of selenium treatment at b i r t h on plasma selenium at 4 weeks 98 Figure 13. E f f e c t of i r o n and selenium supplementation on lamb weight 105 Figure 14. Densitometric t r a c i n g s of plasma p r o t e i n s 109 Figure 15. E f f e c t of i r o n on HA t i t e r 119 Figure 16. E f f e c t of selenium on HA t i t e r 119 Figure 17. E f f e c t of sex on HA t i t e r 119 Figure 18. E f f e c t of selenium X sex i n t e r a c t i o n on hemagglutination t i t e r 121 Figure 19. E f f e c t of i r o n X selenium i n t e r a c t i o n on hemagglutination t i t e r 122 v i i ACKNOWLEDGMENTS Many people have been instrumental i n the development of t h i s t h e s i s . My t h e s i s s u p e r v i s o r , Dr. Malcolm T a i t , spent hundreds of hours catching and blood sampling sheep with me, and a l s o i n such chores as bleeding Dr. Fitzsimmon's chickens (with a g u i l t y eye on the egg production sheets). However, the f r i e n d s h i p developed through t h i s teamwork i s at l e a s t as hig h l y valued as the help and advice generously given. The other members of the committee - Dr. Dick Beames, Dr. Wayne Buckley, Dr. Bob Fitzsimmons, Dr. Bruce Owen, and Dr. Oim She l f o r d - co n t r i b u t e d both g u i -dance and encouragement at many stages. My s i s t e r Glo helped out when I could no longer t e l l a "button" from a "blob" of red blood c e l l s i n the hemagglutination t e s t s . G i l l e s Galzy came to the rescue i n various emer-gencies. Doug Loney and the other s t a f f of MacMillan L i b r a r y were always f r i e n d l y , h e l p f u l and t o l e r a n t . And 3an Howe spent many hours doing a great job typing up the f i n a l r e s u l t . - 1 -INTRODUCTION Iron deficiency is not considered to be of practical importance in ruminants (Ammerman and Goodrich, 1983). This assumption should be reeval-uated as iron deficiency may be a more serious problem under intensive management systems of sheep production. Research i s lacking on the effects of iron deficiency in livestock, other than on hemoglobin production, which may be the most visible but least important aspect of iron deficiency. Various studies have documented the development of iron deficiency anemia in suckling lambs (Carlson jet al., Wi*}; Holman and Dew, 1966; Holz et a l . , 1961; Hibbs et a l . , 1963; Ricketts et a l . , 1965; Ullrey et ^ 1 . , 1965) dairy calves (Matrone et al., 1957; Mollerberg, 1975; Mollerberg et a l . , 1975), and even in beef calves on range (Raleigh and Wallace, 1962). Iron deficiency anemia of pre-natal origin has also been demonstrated in as many as 30% of dairy calves at birth, even though the dams have normal hemoglobin levels (Hibbs jet _al., 1963; Tennant ^ t _al., 1975b). Most of these studies looked only at changes in hemoglobin and red c e l l indices in blood, reflecting the prevalent attitude of physicians and veterinarians: "that iron deficiency had no symptomatology, no morbidity, and no mortality; that i t was for a l l practical purposes c l i n i c a l l y irrelevant, except to the extent that a l i t t l e bit of anemia was good for most people" (Fielding, 1975). Recently, interest in the prevalence of iron deficiency in.livestock and possible effects on health and mortality has been stimulated by current findings on the role of iron in disease resistance, tissue enzymes, tissue morphology, and nutrient absorption. Most research on these subjects concerns humans, as iron deficiency is the leading worldwide nutritional - 2 -problem. Iron d e f i c i e n c y has occurred i n humans for thousands of years, p a r t i c u l a r l y i n peoples with low-meat d i e t s . While the e a r l y Greeks be l i e v e d that i r o n was able to impart strength and f o r c e to persons s u f f e r i n g from weakness (Moore and Dubach, 1960), the connection between d i e t a r y i r o n and i r o n - d e f i c i e n c y anemia was only appreciated since 1895 ( W i t t s , 1969, p. 4 ) . The h i s t o r y of i r o n d e f i c i e n c y i n l i v e s t o c k i s much more recent, and l e s s w e l l understood. Iron d e f i c i e n c y i n l i v e s t o c k was f i r s t i d e n t i f i e d i n the 1920's i n p i g l e t s farrowed and r a i s e d i n confinement ( W i t t s , 1969, p. 4 ) . Since then i r o n i n j e c t i o n of pigs has become a standard p r a c t i c e . Iron d e f i c i e n c y was subsequently i d e n t i f i e d i n milk fed veal c a l v e s , but i s otherwise believed to be unusual i n ruminants. Nonetheless, i r o n d e f i -ciency may develop i n the r a p i d l y growing young of almost any mammalian species, i n c l u d i n g r a b b i t s , monkeys, and elephants (Morgan, 1980; Klos and Lang, 1982). Lambs may be s u s c e p t i b l e to i r o n d e f i c i e n c y f o r three reasons: low p l a c e n t a l i r o n t r a n s f e r (Hoskins and Hansard, 1964), low milk i r o n content (Underwood, 1966) and high growth r a t e . Iron d e f i c i e n c y i s p r i m a r i l y a production disease, as high growth rat e i s the most s i g n i f i c a n t v a r i a b l e i n the e t i o l o g y of the disease. Thus co n d i t i o n s conducive to maximum growth are most l i k e l y to be associated with anemia (Silverman et a l . , 1970). The i n c r e a s i n g trend towards confinement housing of sheep, and improved manage-ment p r a c t i c e s such as a c c e l e r a t e d lambing schedules, may i n t e n s i f y i r o n d e f i c i e n c y problems i n lambs. This study was undertaken to i n v e s t i g a t e i r o n d e f i c i e n c y i n lambs r a i s e d under an i n t e n s i v e management system. In the f i r s t two t r i a l s , - 3 -c o n t r o l lambs were compared with lambs i n j e c t e d with i r o n at b i r t h . Hemo-g l o b i n , hematocrit, and weight were measured at b i r t h and weekly u n t i l weaning, and plasma i r o n was measured at 3-4 weeks. The impact of i r o n d e f i c i e n c y on o v e r a l l metabolism of the lamb was assessed by measuring a p r o f i l e of plasma metabolites. No information was p r e v i o u s l y a v a i l a b l e on the e f f e c t of i r o n s t a tus on the plasma p r o f i l e . These experiments a l s o i n v e s t i g a t e d on the i n f l u e n c e of f a c t o r s such as growth r a t e , sex, breed and r e a r i n g . The f i r s t t r i a l compared treatment e f f e c t s f o r 2 l e v e l s of i r o n , 0 and 500 mg, using 35 lambs. The second t r i a l i nvolved 66 lambs, which were d i v i d e d i n t o 3 treatment groups: 0, 250, and 500 mg Fe. A t h i r d t r i a l , which was r e p l i c a t e d three times, i n v e s t i g a t e d the e f f e c t s of i n j e c t i n g lambs with 500 mg i r o n , with or without 1.5 mg supplementary selenium. As the experimental f l o c k was marginal i n selenium s t a t u s , as shown by the chronic but low incidence of white muscle disease (WMD), the v a l i d -i t y of the i r o n treatment r e s u l t s was questioned f o r se v e r a l reasons. F i r s t l y , selenium and/or vitamin E may be required f o r e r y t h r o p o i e s i s , or may a f f e c t hemolysis and consequently red c e l l turnover. Secondly, the incidence of WMD appeared to be increased by i r o n supplementation i n the f i r s t two experiments. Since i r o n i n j e c t i o n may cause t o x i c i t y i n margin-a l l y S e - d e f i c i e n t p i g s , the question arose - was i r o n i n j e c t i o n causing muscle damage s i m i l a r to and mistaken f or WMD? F i n a l l y , the response to i r o n may have been l i m i t e d by a d e f i c i e n c y i n selenium, and th e r e f o r e an a d d i t i v e or s y n e r g i s t i c response to both i r o n and selenium might be expected. In order to answer these questions, the t h i r d t r i a l t e s t e d the e f f e c t s of i r o n and selenium, separately and combined, on a v a r i e t y of - k -parameters: hemoglobin, PCV, plasma i r o n , selenium and metabolites, plasma p r o t e i n s , and disease r e s i s t a n c e as measured by the hemagglutination t e s t and observations on soremouth. - 5 -LITERATURE REVIEW IRON FUNCTIONS OF IRON I r o n p r o t e i n s evolved out of the ne c e s s i t y to u t i l i z e oxygen e f f e c -t i v e l y , and to c o n t r o l i t s t o x i c i t y . Iron i s e s s e n t i a l to the o x i d a t i o n of organic substances by molecular oxygen, and consequently, to the energy metabolism of a l l l i v i n g c e l l s (Frieden, 1974). The i r o n p r o t e i n s are a di v e r s e group, s t r u c t u r a l l y and fu n c t i o n -a l l y . For convenience, they are u s u a l l y c l a s s i f i e d as heme and non-heme i r o n compounds (H a r r i s o n , 1969). Q u a n t i t a t i v e l y , the heme group of i r o n p r o t e i n s dominates i r o n metabolism. The heme molecule contains an i r o n atom i n the center of a porphyrin r i n g , chelated to the p y r o l l e nitrogen atoms. Heme can be synthesized by a l l aerobic mammalian c e l l s except normal RBC's ( H a r r i s and Kellermeyer, 1970, p.3). Hemoglobin and myoglobin f u n c t i o n as oxygen c a r r y i n g p r o t e i n s . Hemo-g l o b i n increases the oxygen c a r r y i n g c a p a c i t y of the blood about seventy times that c a r r i e d by d i f f u s i o n (Harrison, 1969). Myoglobin i s s i m i l a r s t r u c t u r a l l y to hemoglobin, but has only one heme group instead of four, and i s located i n the sarcoplasm of s k e l e t a l and heart muscle. As myoglo-b i n has a higher a f f i n i t y f o r oxygen than does hemoglobin, i t accepts oxygen released from hemoglobin and acts as a t i s s u e r e s e r v o i r (Moore and Dubach, 1960). Heme pro t e i n s i n c l u d e the cytochromes. These redox enzymes c a t a l y z e e l e c t r o n t r a n s f e r r e a c t i o n s through the a b i l i t y of the heme i r o n to undergo r e v e r s i b l e o x i d a t i o n . They transport hydrogen to molecular oxygen as part of the c e l l u l a r e l e c t r o n t r a nsport system (Malstrom, 1970; Wrigglesworth - 6 -and Baum, 1980). Cytochromes^ (cytochrome oxidase), b, and c, are located i n the mitochondria c r i s t a e . Cytochrome P i + 5 0 i s located i n microsomal membranes, and f u n c t i o n s i n the o x i d a t i v e degradation of drugs and other metabolites, e s p e c i a l l y i n the l i v e r (NRC, 1979, p. 120). The c a t a l a s e s and peroxidases are important heme enzymes which destroy t o x i c oxygen d e r i v a t i v e s such as peroxides. They are found i n the cytoplasm of most animal c e l l s , but c a t a l a s e i s a l s o found i n organelles such as peroxisomes. Peroxidases are involved i n b i o l o g i c a l defence mech-anisms. For example, myeloperoxidase i n blood c e l l s and i n t e s t i n a l mucosa i s important i n disease r e s i s t a n c e , and lactoperoxidase i n milk and s a l i v a has a n t i b a c t e r i a l a c t i v i t y (Wrigglesworth and Baum, 1980). The non-heme i r o n enzymes c a t a l y z e a wide v a r i e t y of metabolic reac-t i o n s . The m e t a l l o f l a v o p r o t e i n s i n c l u d e succinate dehydrogenase, «-glycer-ophosphate dehydrogenase, and NADA-dehydrogenase, a l l located i n mitochon-d r i a , and monoamine oxidase, xanthine oxidase, and aldehyde dehydrogenase i n the cytoplasm (NRC, 1979, p. 120). Most of these are i r o n - s u l p h u r p r o t e i n s . Among the major iron-dependent, non-heme i r o n enzymes which do not c o n t a i n sulphur are the p r o l y l and l y s y l hydroxylases ( c o l l a g e n s y n t h e s i s ) , phenylalanine hydroxylase (catabolism of phenylalanine), t y r o s i n e hydroxy-l a s e (melanin and epinephrine s y n t h e s i s ) , and tryptophan hydroxylase (sero-tonin s y n t h e s i s ) . Ribonucleotide reductase c a t a l y z e s an e s s e n t i a l , con-t r o l l e d step of DNA s y n t h e s i s and thus may a f f e c t the a c t i v i t y of non-heme enzyme systems (Wrigglesworth and Baum, 1980). - 7 -IRON METABOLISM I r o n homeostasis i s achieved through c o n t r o l of i r o n absorption as i r o n e x c r e t i o n i s l i m i t e d . Very l i t t l e i r o n i s excreted through the usual routes of u r i n e , s k i n , h a i r and endogenous s e c r e t i o n s i n t o the gastro-i n t e s t i n a l t r a c t , as i r o n i s t e n a c i o u s l y conserved and r e u t i l i z e d . Quanti-t a t i v e l y , the d a i l y intake of i r o n represents only a f r a c t i o n of the amount of i r o n c i r c u l a t e d through the plasma d a i l y and used i n synthesis of i r o n compounds. Maximal absorption of i r o n occurs when the i r o n requirement i s hig h , as i n young and ge s t a t i n g animals. Percentage absorption g e n e r a l l y r i s e s with decreasing i r o n content of the feed, but t o t a l absorption decreases. Many f a c t o r s i n a d d i t i o n to age and d i e t a f f e c t i r o n absorption i n the ruminant. They include i r o n s t a t u s ; health; g a s t r o i n t e s t i n a l condi-t i o n s such as m o t i l i t y , pH, mucosal turnover and p a r a s i t i s m ; hypoxia; and blood l o s s . Any co n d i t i o n s s t i m u l a t i n g e r y t h r o p o i e s i s normally i n c r e a s e i r o n absorption (Kolb, 1963; Underwood, 1971). Conversely, i n many disease s t a t e s , the release of a leukocyte endogenous mediator from white blood c e l l s l i m i t s a bsorption r e g a r d l e s s of i r o n s t a t u s (Kampschmidt et, a l . , 1973) The most important s i t e of i r o n absorption i s the duodenum and jejunum, i n both man and animals. Some i r o n can a l s o be absorbed through the stomach, ileum, and colon (Bothwell jet al_. , 1979, p. 269). The proximal i n t e s t i n e , where most i r o n absorption occurs, contains s p e c i f i c , g l y c o p r o t e i n , receptor s i t e s i n the brush border. More receptor s i t e s are produced during i r o n d e f i c i e n c y , a f t e r a lag phase r e l a t e d to turnover of mucosal c e l l s . - 8 -The process of i r o n absorption can be d i v i d e d i n t o two steps: uptake from the i n t e s t i n a l lumen i n t o the mucosal c e l l , and t r a n s f e r from the s e r o s a l surface of the c e l l to the plasma. The uptake step of i r o n absorption i n v o l v e s e i t h e r an energy-depen-dent a c t i v e t r a n s p o r t process, as reviewed by Linder and Munro (1977), or a passive d i f f u s i o n process as described by May and Williams (1980). According to the a c t i v e t r a n s p o r t theory, i o n i c d i v a l e n t i r o n absorbed by receptors i n the brush borders i s moved i n t o the c e l l i n a process r e q u i r i n g energy and i n t a c t p r o t e i n synthesis (Linder and Munro, 1977). Consequently, cycloheximide, t e t r a c y c l i n e , and other a n t i b i o t i c s impair i r o n absorption by i n h i b i t i o n of p r o t e i n s y n t h e s i s ( F o r t h , 1974). While the a c t i v e transport mechanism has been the subject of numerous r e p o r t s , i t has not been unequivocally e s t a b l i s h e d . Much of the research may be better explained i n terms of simple, passive d i f f u s i o n of chelated i r o n i n e q u i l i b r i u m with various i n t e r a c t i n g pools of i r o n (May and W i l l i a m s , 1980). The passive d i f f u s i o n theory proposes that i r o n i s absorbed as chelated complexes. The importance of c h e l a t i o n to i r o n absorption has been g r a d u a l l y recognized (Thomas, 1970). Low molecular weight, l i p o p h i l i c complexes of i r o n i n e i t h e r o x i d a t i o n s t a t e are equally w e l l absorbed. Contrary to e a r l i e r work, F e + 3 i s absorbed as w e l l as F e + 2 (Christopher et a l . , 1974). The more l i p o p h i l i c the i r o n complex, the greater the amount of i r o n t r a n s f e r r e d through the membrane (May and W i l l i a m s , 1980). Probably the many f a c t o r s which i n f l u e n c e i r o n absorption are not mediated by a s i n g l e rate-determining step (May and W i l l i a m s , 1980). In the mucosal c e l l , e q u i l i b r i u m i s e s t a b l i s h e d between a r a p i d l y exchangeable - 9 -i r o n pool, the slowly exchangeable f e r r i t i n i r o n pool, and the t r a n s f e r r i n i n plasma. The r a p i d l y exchangeable i r o n pool, p o s s i b l y a p r o t e i n or pol y p e p t i d e , has a major i n f l u e n c e on i r o n metabolism. The s i z e and degree of s a t u r a t i o n of t h i s pool regulate how incoming i r o n i s proportioned between c e l l storage and plasma t r a n s f e r r i n . When the l a b i l e i r o n pool i s c l o s e to s a t u r a t i o n , most i r o n i s d i v e r t e d to f e r r i t i n s y n t h e s i s . As t h i s pool i s only slowly exchangeable, excess i r o n i s sequestered i n t h i s form and l o s t during normal c e l l e x f o l i -a t i o n , preventing i r o n overload. In i r o n d e f i c i e n c y , l a b i l e binding to t h i s pool d i r e c t s most incoming i r o n immediately to the c i r c u l a t i o n . A c a r r i e r p r o t e i n may be inv o l v e d i n the t r a n s p o r t of i r o n across the mucosal c e l l , a distance 10,000 to 20,000 times the diameter of the i r o n atom (Linder and Munro, 1977). The c a r r i e r may be a t r a n s f e r r i n - l i k e pro-t e i n . The concentration of t h i s p r o t e i n i s increased i n mucosal c e l l s of i r o n d e f i c i e n t mice, but not i n i r o n d e f i c i e n t s l a mice, which are gene t i -c a l l y incapable of t r a n s f e r r i n g adequate i r o n . S l a i s the gene i n v o l v e d (Bothwell et a l . , 1979, p. 274). The release of i r o n from the mucosal c e l l to the plasma i s the f i n a l step of i r o n absorption. During the i n i t i a l , r a p i d phase of i r o n r elease 60 to 80% of the eventual t o t a l may be t r a n s f e r r e d w i t h i n 30 minutes. I r o n released i n t h i s phase o r i g i n a t e s from the r a p i d l y exchangeable i r o n p o o l . The slow phase l a s t s 12-24 hours, as i r o n i s gr a d u a l l y released from f e r r i t i n (Bothwell et al^. , 1979, p. 272). The importance of i n d i v i d u a l d i e t a r y f a c t o r s to i r o n absorption i s u n c e r t a i n , even f o r monogastrics. Anions forming i n s o l u b l e or only weakly s o l u b l e s a l t s with i r o n l i m i t i t s absorption, as i r o n must be i n a s o l u b l e - 10 -complex. C h e l a t i n g agents can improve or depress i r o n a b s o r p t i o n . As pre-v i o u s l y mentioned, weaker c h e l a t i n g agents are necessary f o r i r o n absorp-t i o n . They may improve i r o n a v a i l a b i l i t y by preventing the formation of i n s o l u b l e i r o n phosphates and hydroxides, and a l s o by maintaining the i r o n i n a s o l u b l e , absorbable s t a t e (Conrad, 1970). Organic a c i d s and reducing agents such as ascorbate, c i t r a t e , l a c t a t e , pyruvate, su c c i n a t e , c y s t e i n e , h i s t i d i n e , l y s i n e , and some sugars thus f a c i l i t a t e absorption (Thomas, 1970). HC1 i s of major importance as a complexing agent, i n a d u l t humans, i n f a n t s , and pigs (Beutler and Fairbanks, 1980; Bothwell _et ^1_., 1979, p. 267; and Hannan, 1971). Strong i n t r a l u m i n a l c h e l a t i n g agents depress i r o n absorption by competing with the c e l l u l a r acceptor s i t e f o r i r o n . Phytates, endotoxins, a l k a l i n i z i n g agents, p a n c r e a t i c s e c r e t i o n s , phosphates, o x a l a t e s , and other endogenous and exogenous c h e l a t i n g agents depress i r o n absorption i n t h i s way (Thomas, 1970). Iron i s transported by t r a n s f e r r i n between s i t e s of absorption, s t o r -age, u t i l i z a t i o n , and e x c r e t i o n , and i n both plasma and e x t r a v a s c u l a r spaces (Aisen, 1980). T r a n s f e r r i n minimizes the l o s s of i r o n from the body, by d e p o s i t i n g surplus i r o n i n t i s s u e s adapted f o r i r o n storage. T r a n s f e r r i n i s a l s o important i n d i s t r i b u t i n g i r o n i n proportion to need (Bothwell et _ a l . , 1979, p. 293). T r a n s f e r r i n , or s i d e r o p h i l i n , i s a f?-globulin containing a carbohy-drate f r a c t i o n . The l i v e r i s the major s i t e of s y n t h e s i s . T r a n s f e r r i n has two metal binding s i t e s which are capable of binding a wide v a r i e t y of b i v a l e n t and t r i v a l e n t metals, but have the highest a f f i n i t y f o r F e + 3 (Brown, 1977). - 11 -Normally, t r a n s f e r r i n i s one-third saturated with i r o n . The d e l i v e r y of i r o n i s a f f e c t e d by the degree of s a t u r a t i o n of t r a n s f e r r i n , as uptake of i r o n i s highest from d i f e r r i c t r a n s f e r r i n f o r a l l t i s s u e s (Bothwell e_t a l . , 1979, p. 293). The two i r o n - b i n d i n g s i t e s of t r a n s f e r r i n appear f u n c t i o n a l l y s i m i l a r . However, one s i t e of d i f f e r r i c t r a n s f e r r i n may pre-f e r e n t i a l l y donate i r o n to developing red blood c e l l s and the placenta (Jacobs, 1977a). Najean et _al_. (1970) suggested a l a b i l e i r o n pool may be i n s t r u m e n t a l i n maintaining a d e s i r a b l e , s t a b l e l e v e l of'plasma i r o n . The t r a n s i t pool may be a c y s t e i n e - c o n t a i n i n g non-heme pro t e i n (Najean et jal_, 1970), or a low molecular weight complex (Jacobs, 1977b). Evidence f o r an i n t r a c e l l u -l a r t r a n s i t i r o n pool has been obtained f o r r e t i c u l o e n d o t h e l i a l c e l l s , red c e l l p recursors, c u l t u r e d Chang c e l l s and l i v e r . Iron may enter the tran-s i t pool from endogenous heme breakdown, m o b i l i z a t i o n of f e r r i t i n , and exchange with t r a n s f e r r i n (Jacobs, 1977b). The main storage forms of i r o n are f e r r i t i n and hemosiderin. F e r r i t i n s t o r e s t r i v a l e n t i r o n i n a s o l u b l e form which can be mobilized as r e q u i r e d , whereas hemosiderin i r o n i s l e s s a v a i l a b l e ( C r i c h t o n , 1975). F e r r i t i n has a l a r g e capacity f o r i r o n , eg. 4500 Fe atoms per molecule, but u s u a l l y maintains a reserve capacity of one t h i r d of t h i s (Harrison et al_. , 1980). F e r r i t i n contains on average 21% i r o n , stored as a f e r r i c - h y d r o x i d e -phosphate core i n s i d e of a s p h e r i c a l p r o t e i n s h e l l . Iron passes f r e e l y i n and out of the s h e l l through s i x channels; the p r o t e i n s h e l l i s thought to have enzyme a c t i v i t y . F e r r i t i n i s formed i n a l l c e l l s i n response to enlargement of the l a b i l e p r o t e i n pool. The major s i t e of s y n t h e s i s i s the l i v e r , and a l s o spleen and bone marrow (Kaneko, 1980). - 12 -H e m o s i d e r i n i s i n s o l u b l e , c o n t a i n s 2 5 - 3 3 % F e + 3 i n a d d i t i o n t o F e + 2 , a n d i s much l a r g e r t h a n f e r r i t i n ( K a n e k o , 1 9 8 0 ) . H e m o s i d e r i n i s c o n s i d e r e d t o be a b r e a k d o w n p r o d u c t o f f e r r i t i n ( H a r r i s o n et^ j a l _ . , 1 9 8 0 ) . IRON INTERACTIONS M o s t t r a c e m i n e r a l s , i n c l u d i n g i r o n , a r e m e t a l s o f t h e f i r s t t r a n s i -t i o n s e r i e s on t h e p e r i o d i c t a b l e . Many e x a m p l e s o f i r o n i n t e r a c t i o n s w i t h o t h e r m i n e r a l s i n v o l v e s u b s t i t u t i o n o r a n t a g o n i s m b e t w e e n m i n e r a l s o f s i m i l a r s i z e a n d e l e c t r o n s t r u c t u r e . I n t e r a c t i o n s c a n o c c u r i n t h e l u m e n o f t h e g u t , a t a b s o r p t i v e s i t e s , a n d a t v a r i o u s m e t a b o l i c l e v e l s . T h e i r o n r e q u i r e m e n t i s i n c r e a s e d by h i g h d i e t a r y l e v e l s o f z i n c , c a d m i u m , c o p p e r a n d m a n g a n e s e . T h e s e m i n e r a l s c a n c o m p e t e w i t h i r o n f o r t h e i r o n b i n d i n g s i t e s i n t h e i n t e s t i n a l m u c o s a ( U n d e r w o o d , 1 9 7 1 ) . C o n v e r s e l y , h i g h l e v e l s o f i r o n c a n i n d u c e C o , C u , Z n , Mn a n d Se d e f i c i e n -c i e s ( P u i s , 1 9 8 1 ) . I r o n d e f i c i e n c y r e s u l t s i n i n c r e a s e d a b s o r p t i o n o f h e a v y m e t a l s , m a i n l y F e , M n , Zn a n d N i , b u t n o t Cu ( B o t h w e l l e t j d . , 1 9 7 9 , p . 2 7 3 ) . P o s s i b l y t h e s i t e o f i n t e r a c t i o n i n t h i s c a s e i s n o t t h e i r o n -b i n d i n g s i t e , b u t t r a n s f e r r i n , w h i c h i s r e s p o n s i b l e f o r i r o n u p t a k e f r o m t h e m u c o s a . T h e f a c t t h a t t h e e l e c t r o n s t r u c t u r e o f M n + 2 a n d F e + 3 a r e f u n c t i o n a l l y i d e n t i c a l ( T h o m a s , 1 9 7 0 ) a n d t h a t t r a n s f e r r i n i s a n i m p o r t a n t p r o t e i n f o r t h e t r a n s p o r t o f Zn ( B r o w n , 1 9 7 7 ) s u p p o r t s t h i s i n t e r p r e t a -t i o n . F u r t h e r m o r e , Zn may r e d u c e i r o n a b s o r p t i o n by i n t e r f e r i n g w i t h i r o n i n c o r p o r a t i o n i n t o , o r r e l e a s e f r o m , f e r r i t i n ( U n d e r w o o d , 1 9 7 1 ) . B i o l o g i c a l s u b s t i t u t i o n o f i r o n w i t h o t h e r m i n e r a l s i s m o s t l i k e l y t o o c c u r b e t w e e n M n + 2 a n d F e + 3 , a n d F e + 2 a n d C o + 3 . T h e s e i o n p a i r s s h a r e s i m i l a r e l e c t r o n s t r u c t u r e s . When d i e t a r y m a n g a n e s e i s h i g h , o r - 13 -d u r i n g anemia, the degree of b i o l o g i c a l s u b s t i t u t i o n of manganese fo r i r o n i s g r e a t l y increased. Mn i s incorporated i n t o the heme molecule, with the same rate of synthesis and turnover as the i r o n porphyrin (Thomas, 1970). The a v a i l a b i l i t y of i r o n at the metabolic l e v e l i s dependent on copper enzymes (Frieden, 1974). Nearly a l l metabolic processes i n v o l v i n g i r o n depend on the i n t e r c o n v e r s i o n of ferrous and f e r r i c i r o n . Two copper c o n t a i n i n g enzymes, or f e r r o x i d a s e s , are known to c a t a l y z e the o x i d a t i o n of ferrous to f e r r i c i r o n . One of these i s ceruloplasmin. D i f f e r e n t f e r r o x i d a s e s occur i n various species. Iron accumulates i n the l i v e r very r a p i d l y i n response to even a mild copper d e f i c i e n c y , as a shortage of c eruloplasmin impedes i t s m o b i l i z a t i o n (Grassman and Kirchgessner, 1974). On the other hand, the copper content of the l i v e r increases g r e a t l y as a r e s u l t of i r o n d e f i c i e n c y , i n d i c a t i n g that i r o n i s required for copper u t i l i z a t i o n . Iron i n t e r a c t i o n s with phosphorus are important at high d i e t a r y l e v e l s of i r o n . Too much i r o n i n the d i e t i n t e r f e r e s with phosphorus absorpton by forming an i n s o l u b l e phosphate. R i c k e t s may then r e s u l t on an otherwise adequate d i e t with a good phosphorus content. Studies with p i g l e t s fed d i f f e r e n t l e v e l s of i r o n as ferrous s u l f a t e i n d i c a t e that the amont of i r o n required to produce a t o x i c i t y depends on the amount and source of phosphorus i n the d i e t . In one experiment, feeding 5000 ppm i r o n reduced growth r a t e , serum inorganic phosphorus, and femur ash w i t h i n 5 weeks, (0*Donovan ejt al_. , 1962). However, ruminants appear to be s e n s i t i v e to much lower l e v e l s of i r o n than are p i g l e t s , and 500 ppm should be regarded as the maximum l e v e l t o l e r a b l e by ruminants (ARC, 1980, p. 242). - 14 -DIETARY IRON Most feeds contain generous amounts of i r o n . There i s u s u a l l y more than 20 tonnes of i r o n per acre i n the top 15 cm of s o i l , so s o i l contamin-a t i o n can g r e a t l y i n f l u e n c e the i r o n content of feeds ( W r e t l i n d , 1968). In the Fraser V a l l e y of B r i t i s h Columbia, mean and ranges of i r o n i n common feeds were: grass hay, 540 ppm (130-1370); a l f a l f a hay, 580 ppm (40-2185); corn s i l a g e , 384 ppm (40-1490); grass s i l a g e 1373 ppm (40-5550); and pasture 1076 ppm (40-5370), (Cathcart et a l . , 1980). Cereal g r a i n s are poor sources of i r o n , c o n taining 30-60 ppm i r o n ; o i l s e e d meals f r e q u e n t l y c o n t a i n 100-200 ppm i r o n (Underwood, 1981). S o l u b i l i t y of ino r g a n i c i r o n sources appears to be a primary, but not e x c l u s i v e , determinant of t h e i r a v a i l a b i l i t y f o r ruminants. Ammerman et^ a l . (1967) tested the a v a i l a b i l i t y of four inorganic i r o n sources f o r ruminants. On the basis of t i s s u e F e 5 9 r e t e n t i o n , f e r r o u s sulphate, ferrous carbonate and f e r r i c c h l o r i d e were ranked i n decreasing order of a v a i l a b i l i t y , but d i f f e r e n c e s were not s i g n i f i c a n t . Iron i n f e r r i c c h l o r i d e was 3 to 4 times as a v a i l a b l e as that i n f e r r i c oxide ( F e 2 0 3 ) to ir o n - d e p l e t e d c a l v e s . Ferrous sulphate and f e r r i c c h l o r i d e are very s o l u b l e , and ferrous carbonate and f e r r i c oxide are s l i g h t l y or non-s o l u b l e . The absorption c o e f f i c i e n t of sol u b l e i r o n as f e r r i c c h l o r i d e was 0.60 for young calves when the d i e t provided 30 mg i r o n d a i l y , and 0.30 when the d i e t provided 60 mg i r o n d a i l y (Matrone et a l _ . , 1957). Hemoglobin s y n t h e s i s was used as the c r i t e r i o n of a v a i l a b i l i t y i n t h i s study. Iron i n plant products appears to be l e s s a v a i l a b l e than i r o n i n s o l u b l e i r o n s a l t s . Most of the i r o n i n plants i s i n the f e r r i c ( F e + 3 ) - 15 -form i n organic complexes (NRC, 1980, p. 243). Iron i n grass was 48 to 63% as e f f e c t i v e , and i r o n i n legumes 47 to 57% as e f f e c t i v e , compared to f e r r i c c h l o r i d e f o r improvement of hemoglobin (Raven and Thompson, 1959; Thompson and Raven, 1959). Hoskins and Hansard (1964) estimated that the true a v a i l a b i l i t y of i r o n from a d i e t of maize, soybean meal, and cottonseed h u l l s was 0.29 f o r pregnant ewes. The d i e t provided 19 ppm i r o n . M i l k i r o n a v a i l a b i l i t y was found to be 26% for calves and 30% f o r p i g l e t s (ARC, 1972). A s i m i l a r value should p r e v a i l f o r the pre-ruminant lamb. These values r e f l e c t the low i r o n content of milk and the l a r g e demand f o r i r o n . IRON REQUIREMENTS The i r o n requirements of sheep and c a t t l e are not w e l l defined. Few experiments comparing l e v e l s of i r o n have been conducted on a long-term b a s i s ; the requirement for maintenance and/or d e p o s i t i o n of i r o n s t o r e s i s gen e r a l l y ignored; and the source of i r o n i s t y p i c a l l y a s o l u b l e i r o n s a l t more a v a i l a b l e than feed i r o n . Work with calves i n d i c a t e d that the average d a i l y gain during growth, r a t h e r than body weight of the animal, i s the major f a c t o r determining the amount of i r o n r e q u i r e d . The requirement f o r maintenance i s low compared to that f or growth (Matrone _et j i l _ . , 1957). Mollerberg ejt al_. (1975a) c a l c u l a t e d that the i r o n requirement for 1 kg growth i n calves was 40-45 mg. Assuming 25% maximum d i e t a r y i r o n r e t e n t i o n , the a c t u a l requirement for 1 kg growth would be at l e a s t 160-180 mg i r o n (Mollerberg et a l . , 1975a). - 16 -Subsequently, S u t t l e (1979) found that the i r o n concentrations of lamb and c a l f carcasses are s i m i l a r . Iron concentration ranged from 52.6 -75.1 mg/kg fre s h carcass weight for lambs weighing 18 - 69 kg, decreasing s l i g h t l y with age at s l a u g h t e r . The value of 55 mg iron/kg carcass gain was taken to represent the approximate net growth requirement, e x c l u s i v e of i r o n storage. The value f o r calves was s i m i l a r . S u t t l e (1979) concluded that the t o t a l net requirement of ruminants for i r o n should be defined i n terms of d i e t a r y intake rather than concentration because of the r e l a t i v e l y l a r g e and constant c o n t r i b u t i o n of the growth component. Most st u d i e s set the i r o n requirement as the minimal l e v e l f o r hemo-g l o b i n maintenance, and thus' may underestimate the t o t a l i r o n requirement. Demands on d i e t a r y i r o n f o r hemoglobin synthesis supersede demands f o r myoglobin maintenance i n the c a l f . For example, s i g n i f i c a n t increases i n myoglobin occurred when d i e t a r y i r o n fed to d a i r y calves was increased from 24 mg/kg to 44 and 104 mg/kg d i e t (Bremner _et j i l _ . , 1976). Other s t u d i e s with d a i r y calves i n d i c a t e a minimum i r o n requirement as high as 100 mg/kg dry matter (ARC, 1980, p. 236). This f i g u r e i s i n l i n e with a p o s s i b l e requirement of 100-125 ppm i r o n for milk-fed baby pigs (Hitchcock et^ a l . , 1974; U l l r e y _et a l . , 1960), but much higher than the g e n e r a l l y accepted requirement of 30-60 ppm based on e a r l i e r work (Matrone et a_l., 1957). The i r o n requirements of the m i l k - f e d lamb have not been studied experimentally. In view of the work with c a t t l e (Matrone et aj_., 1957; Mollerberg _et a l _ . , 1975) and sheep and c a t t l e ( S u t t l e , 1979), i t i s l i k e l y that the i r o n requirement of the s u c k l i n g lamb i s higher on a dry matter b a s i s than that of the o l d e r , weaned lamb. - 17 -Two experiments on the i r o n requirement of weaned lambs were c a r r i e d out by Lawlor ejt a_l. (1965). A d i e t a r y l e v e l of 70 ppm i r o n was adequate f o r g r o w i n g - f i n i s h i n g lambs fed a s e m i - p u r i f i e d d i e t . In the second t r i a l , 40 ppm i r o n met the requirements of weaned lambs, but feed conversion e f f i c i e n c y was poor compared to the d i e t with 70 ppm i r o n . The i r o n requirement of adult sheep i s stated to be 30-50 ppm, or mg/kg, of d i e t dry matter (NRC, 1975, p. 47). Iron d e f i c i e n c y i s not believed to be a common problem of mature ruminants. However, the lower range of i r o n l e v e l s i n feeds were marginal f or two Canadian surveys (Peterson and Waldern, 1977; Cathcart et j a l . , 1980). While i r o n d e f i c i e n c y i s improbable, ruminant reproduction could be adversely a f f e c t e d by low i r o n a v a i l a b i l i t y i n some roughages, as a high c o r r e l a t i o n i s observed between i r o n i n body f l u i d s and f e r t i l i t y ( H i d i r o g l o u , 1979). METHODS OF IRON SUPPLEMENTATION O r a l , p a r e n t e r a l and intravenous methods of i r o n therapy are used. Intravenous i r o n therapy has l i t t l e advantage i n rate and magnitude of response compared to par e n t e r a l i n j e c t i o n , and cannot be j u s t i f i e d even i n acutely d e f i c i e n t human p a t i e n t s , e s p e c i a l l y as more r i s k i s in v o l v e d . This leaves the o r a l and pa r e n t e r a l routes of i r o n a d m i n i s t r a t i o n . O r a l l y , ferrous fumarate, sulphate, succinate and gluconate are among the i r o n s a l t s used s u c c e s s f u l l y to t r e a t i r o n d e f i c i e n c y i n man and animals. The t o l e r a b l e dose i s l i m i t e d , as large amounts of i r o n s a l t s cause diarrhea and damage the i n t e s t i n a l mucosa. The treatment must be repeated at short i n t e r v a l s . Iron supplementation of the feed of s u c k l i n g animals i s i n e f f e c t i v e , due to low consumption of s o l i d feed f o r the f i r s t weeks of l i f e . - 18 -P a r e n t e r a l i r o n a d m i n i s t r a t i o n has become the method of choice for both commercial and experimental i r o n supplementation of l i v e s t o c k . I t i s convenient and dependable. P i g l e t s are r o u t i n e l y i n j e c t e d with i r o n dextran or i r o n d e x t r i n w i t h i n the f i r s t few days of l i f e . The i r o n i s used e f f i c i e n t l y f or Hb s y n t h e s i s : about 94% of the dose i s found i n the red blood c e l l s two weeks l a t e r (Thoren-Tolling, 175, p. 44) . Due to the high molecular weight of i r o n dextran complexes c i r c u l a t i n g n the plasma, renal clearance i s minimal. Iron dextran, or Imferon, i s the most widely used of the p a r e n t e r a l i r o n products. I t has extensive use world-wide i n human and animal i r o n therapy. Others are i r o n s o r b i t e x and d e x t r i f e r r o n (McCurdy, 1970). The e f f e c t s of i r o n dextran i n j e c t i o n have been i n v e s t i g a t e d ( Kolb, 1963; Beresford _et aL., 1957; Martin et a K , 1955). An acute inflammatory r e a c t i o n develops at the i n j e c t i o n s i t e . Lymphatic absorption of i r o n i s r a p i d , and the lymph nodes may act as temporary i r o n s t o r e s (Thoren-T o l l i n g , 1975). Most i r o n i s removed through the l o c a l inflammatory r e a c t i o n ; but i r o n which d i f f u s e s away from the s i t e i s taken up and retained by t i s s u e macrophages. Most of the i r o n i s absorbed w i t h i n three days, accompanied by a sharp r i s e i n the plasma i r o n content. This g r e a t l y exceeds the i r o n -b i n d i n g c a p a c i t y , but has no t o x i c e f f e c t s due to the high s t a b i l i t y of the i r o n dextran complex. Iron dextran must be processed through the r e t i c u l o - e n d o t h e l i a l system to s p l i t the i r o n from the dextran ( S z i l a g y i and E r s l e v , 1970). As a r e s u l t , i r o n dextran takes three days to measur-ably increase blood Hb, compared to l e s s than a day f o r i r o n s o r b i t e x (McCurdy, 1970). - 19 -Side e f f e c t s of iron dextran i n j e c t i o n are only well known in humans. Reactions such as fever, dealyed a r t h r a l g i a , l o c a l discomfort, and skin staining bear l i t t l e r e l a t i o n s h i p to dosage. Rarely, both f a t a l and non-fatal anaphylactic reactions are caused by a repeated dose l a t e r than 5 days after the i n i t i a l dose in humans (McCurdy, 1970) and in beef c a t t l e (Perry et a l . , 1967). STAGES OF IRON DEFICIENCY Iron deficiency progresses through several stages between iron deple-ti o n and actual iron deficiency anemia. I n i t i a l l y , a negative iron balance i s counteracted by several mechanisms. Iron i s mobilized from the body stores of l i v e r , muscle, spleen, and marrow in order to maintain Hb produc-t i o n , while iron absorption i s increased. Normal serum iron, and serum iron saturation percentage, are maintained (Bothwell et a_l., 1979, pp. 44-45). Iron depletion i s sometimes considered a pathological condition, as the tissue concentration of some iron-containing enzymes i s diminished (Verloop _et JJ1_. , 1970). For example, l i v e r enzyme a c t i v i t y in the pentose phosphate shunt i s affected at an unexpectedly early stage i n iron d e f i -c i e n t rats (Jacobs, 1977a). In latent iron deficiency, the increased iron absorption s t i l l helps maintain normal hemoglobin l e v e l s . The serum iron saturation percentage i s decreased, as serum iron i s reduced and/or t r a n s f e r r i n production i s increased. Iron stores are absent. At th i s stage, growth rate, general well-being or disease resistance may respond to iron supplementation. Iron deficiency anemia, the f i n a l stage of deficency, r e s u l t s when the normal hemoglobin l e v e l cannot be maintained. An equilibrium can be - 20 -reached at any l e v e l of Hb below the norm, or Hb may continue to d e c l i n e u n t i l death occurs. Serum i r o n may f a l l below 35 ug/d£ i n anemic humans, and values near zero are not uncommon. The erythrocyte protoporphyrin content i s elevated, denoting d e f e c t i v e heme synthesis ( H a r r i s and K e l l e r -meyer, 1970, pp. 116-117). H e m a t o l o g i c a l ^ , a we l l - d e f i n e d i r o n d e f i c i e n c y anemia i s character-i z e d by a large proportion of red blood c e l l s which are small i n s i z e ( m i c r o c y t i c ) and poorly f i l l e d with hemoglobin (hypochromic). As new c e l l formation i s r e s t r i c t e d , the r e t i c u l o c y t e count (immature red blood c e l l s ) tends to be low. I n i t i a l l y , the l e v e l of blood hemoglobin may be depressed more than e i t h e r the red blood c e l l count or hematocrit ( H a r r i s and Kellermeyer, 1970, p. 115). Bone marrow examination a l s o demonstrates c h a r a c t e r i s t i c changes during i r o n d e f i c i e n c y , i n c l u d i n g normoblastic hyperplasia (Beutler and Fairbanks, 1980). MEASUREMENTS OF IRON STATUS D i f f e r e n t techniques are used to assess i r o n s t a t u s depending on whether or not anemia i s present. When a m i c r o c y t i c , hypochromic anemia can be demonstrated, the few p o s s i b l e causes other than i r o n l ack can be r e a d i l y eliminated ( i e . inflammation, i n f e c t i o n , copper d e f i c i e n c y , lead poisoning) or are extremely r a r e . Thus, i r o n d e f i c i e n c y anemia can u s u a l l y be e a s i l y i d e n t i f i e d by use of hemoglobin or hematocrit t e s t s , though i t i s d e s i r a b l e to confirm the i r o n d e f i c i e n c y by other measurements. The ult i m a t e proof i s a s p e c i f i c , o r d e r l y response of blood hemoglobin to i r o n therapy ( H a r r i s and Kellermeyer, 1970, p. 120). - 21 -I n i r o n d e f i c i e n c y anemia, the plasma i r o n concentration i s low, t o t a l i r o n binding c a p a c i t y ( t r a n s f e r r i n ) i s high, and percentage s a t u r a t i o n of t r a n s f e r r i n i s very low. These measurements can be used to confirm the presence of i r o n - d e f i c i e n c y anemia. The plasma i r o n concentration i s a f f e c t e d by a wide range of condi-t i o n s , and thus i s not very s p e c i f i c for i r o n s t a t u s . Plasma i r o n i s increased i n a s s o c i a t i o n with decreased e r y t h r o p o i e s i s , increased hemolysis, or increased release of i r o n from body s t o r e s , while reduced l e v e l s of i r o n are c o n s i s t e n t l y seen during acute and chronic i n f e c t i o n s , even i f body stores are high. The decrease i n plasma i r o n l e v e l i s a l a t e development i n i r o n d e f i c i e n c y and may occur only a f t e r the mobile i r o n reserves are completely exhausted ( F i e l d i n g , 1980). The percentage i r o n s a t u r a t i o n of t r a n s f e r r i n best measures the supply of i r o n to the e r y t h r o i d marrow. A s a t u r a t i o n of l e s s than 16% depresses basal e r y t h r o p o i e s i s . Normally, t r a n s f e r r i n i s about one-third saturated. In c o n d i t i o n s associated with impaired p r o t e i n production, the t r a n s f e r r i n l e v e l i s decreased. Consequently, the quantity of Tr, a l s o known as the t o t a l i r o n - b i n d i n g capacity (TIBC) should be measured as w e l l as the percentage s a t u r a t i o n (Bothwell _et a^., 1979, pp. 50-56; Crosby, 1975; F i n c h , 1970). Hemoglobin and hematocrit t e s t s are the most r a p i d , accurate and convenient t e s t s , but cannot assess the f u l l range of i r o n status except f o r obvious anemias. The lower values of normal hemoglobin and hematocrit values a l s o overlap the upper range of anemic values (Garby and K i l l a n d e r , 1968). T r a n s f e r r i n measurements may a l s o be i n e f f e c t i v e i n diagnosing b o r d e r l i n e cases of i r o n overload or d e f i c i e n c y (Beutler et^ a_l., 1954; Crosby, 1975). - 22 -In the pre-anemic stages of iron deficiency, the best tests for iron status assess the l e v e l of iron stores. Marrow aspiration or the l e v e l of absorption of a test dose of iron have been the best methods of evaluating iron depletion. Marrow aspiration i s used to determine the presence of hemosiderin, a storage form of iron, in the r e t i c u l o e n d o t h e l i a l c e l l s (Beutler et a l . , 1954). This test i s laborious and unsuited for screening purposes and n u t r i t i o n a l t r i a l s . The iron absorption test i s also not appropriate for experiments with growing animals, espe c i a l l y as i t i s not v a l i d in cases of increased erythropoiesis. The l e v e l of urinary iron excretion a f t e r a test dose of desferrioxamine i s supposed to be correlated with the l e v e l of body stores, but i s also inappropriate for most animal research purposes. Consequently, the degree of iron depletion i s d i f f i c u l t to assess (Bothwell et al_., 1979, pp. 88-104; Harris and Kellermeyer, 1970, p. 118). A new technique that w i l l receive widespread c l i n i c a l a p p l i c a t i o n i s a rapid, 2-site radioimmune assay for serum f e r r i t i n . Serum f e r r i t i n i s highly correlated with body iron stores in a l l states from iron deficiency to iron overload (Jacobs, 1977c; Powell et a l . , 1975). The role of serum f e r r i t i n in iron transport and metabolism i s uncertain (Worwood ^ t ^1_. , 1975; Worwood, 1980). Should the serum f e r r i t i n test prove fe a s i b l e for n u t r i t i o n a l studies in animals, the measurement of both serum f e r r i t i n and either hemoglobin or hematocrit would provide the optimum picture of iron status. EFFECTS OF IRON DEFICIENCY There i s increasing evidence that symptoms of i l l - h e a l t h , reduced growth rate, and possibly decreased disease resistance can occur even i n - 23 -m i l d cases of i r o n d e f i c i e n c y . The i r r e v e r s i b l e or long-term e f f e c t s of i r o n d e f i c i e n c y on i r o n enzymes and c e l l s t r u c t u r e and f u n c t i o n may be more important than the r e a d i l y r e v e r s i b l e anemia. The s u s c e p t i b i l i t y of c e l l s to i r o n d e f i c i e n c y i s determined by the r a t e of c e l l turnover, and the r a t e of turnover of the i r o n - c o n t a i n i n g compounds w i t h i n the c e l l ( F i e l d i n g , 1975). Rapidly p r o l i f e r a t i n g t i s s u e s , such as the g a s t r o i n t e s t i n a l mucosa, respond most r a p i d l y to changes i n a v a i l a b l e i r o n . On the other hand, the higher the rate of turnover of the i r o n - c o n t a i n i n g enzymes and p r o t e i n s w i t h i n the c e l l , the more r e v e r s i b l e the d e f i c i e n c y c o n d i t i o n . The cytochrome oxidase a c t i v i t y i n the i n t e s -t i n a l mucosa reaches normal 48 hours a f t e r i r o n treatment, whereas s k e l e t a l muscle cytochrome £ a c t i v i t y recovers very slowly (Dallman, 1971). Iron d e f i c i e n c y causes a v a r i e t y of enzymatic and morphological defects i n s o l i d t i s s u e s . Many cytochromes and other enzymes are s i g n i f i -c a n t l y decreased by i r o n d e f i c i e n c y . The enzymes are d i f f e r e n t i a l l y a f f e c t e d w i t h i n the c e l l and between t i s s u e s (Jacobs, 1975; Jacobs, 1977a; B u e t l e r , 1963; B e u t l e r and Fairbanks, 1980). The r o l e of i r o n i n DNA synthesis may be responsible for the h i g h l y s i g n i f i c a n t reduction of various non-iron enzymes during i r o n d e f i c i e n c y . For example, the pentose phosphate shunt enzymes (phosphogluconate dehydro-genase and glucose-6-phosphate dehydrogenase) s u f f e r a major reduction e a r l y i n i r o n d e f i c i e n c y . The disaccharidase a c t i v i t y i n the brush border of the i n t e s t i n a l mucosa i s a l s o a f f e c t e d (Jacobs, 1975), as i s g l u t a t h i o n e peroxidase a c t i v i t y i n the erythrocyte (Macdougall, 1972). Defects are often observed i n i r o n - d e f i c i e n t mitochondria. Many workers have found evidence of increased mitochondrial f r a g i l i t y , - 24 -s w e l l i n g , v a c u o l a t i o n , and membrane breakdown i n lymphocytes, i n t e s t i n e and marrow c e l l s . Abnormal mitochondrial morphology i n i r o n - d e f i c i e n t r a t s was r e v e r s i b l e w i t h i n 5 days of i r o n treatment, much more r a p i d l y than the mitochondrial cytochrome d e f i c i e n c y (Dallman, 1971; Jacobs, 1975; Jacobs, 1977a). E p i t h e l i a l l e s i o n s are widespread i n i r o n d e f i c i e n c y . A l l p r o l i f e r -a t i n g t i s s u e s are a f f e c t e d , e s p e c i a l l y the mucous membranes. Enzyme de-f e c t s have been found i n the buccal mucosa, stomach and small i n t e s t i n e but as yet a cause and e f f e c t r e l a t i o n s h i p between the decreased i r o n enzymes and the e p i t h e l i a l l e s i o n s cannot be i n f e r r e d (Verloop et j a l . , 1970). S p e c i f i c l e s i o n s of i r o n d e f i c i e n c y have been w e l l documented i n humans. Most studies have revealed a high frequency of a t r o p h i c g a s t r i c mucosal changes, i e . 85% (Beutler and Fairbanks, 1980). G a s t r i c atrophy i s of major i n t e r e s t , as i t leads to hypochlorhydria which f u r t h e r depresses i r o n absorption, e s p e c i a l l y i n the young ( W i t t s , 1966; W i t t s , 1969, pp. 39-44). Sores at the corners of the mouth, atrophy of the p a p i l l a e of the tongue, esophageal u l c e r a t i o n , poor h a i r growth, and dry, f i s s u r e d s k i n are other t y p i c a l l e s i o n s (Heilmeyer and Harwerth, 1970; H a r r i s and Kellermeyer, 1970, p. 114). S i m i l a r l e s i o n s , e s p e c i a l l y of the g a s t r o i n -t e s t i n a l mucosa, have been observed i n swine and other monogastrics but have not been studied i n ruminants. In general, i r o n d e f i c i e n c y causes widespread metabolic changes, as might be expected through the many functi o n s of i r o n enzymes. Serum t r i g l y c e r i d e l e v e l s are elevated, f o l i c a cid l e v e l s depressed, and basal metabolic r a t e i s depressed (Bothwell _et a l . , 1979, p. 31). A reduction i n the m i t o c h o n d r i a l enzyme ^-glycerophosphate dehydrogenase i n muscle - 25 -t i s s u e leads to excessive production of l a c t i c a c i d , impairing work capa-c i t y (Saltman et a_L., 1982). This can be demonstrated i n i r o n d e f i c i e n t animals even i n the absence of anemia (Bothwell et jal_., 1979, p. 31). Iron d e f i c i e n t animals v o l u n t a r i l y r e s t r i c t p h y s i c a l a c t i v i t y , and a p p e t i t e i s reduced i n the e a r l y stages of anemia, at l e a s t i n calves (Bremner et a l . , 1976). Consequently, i t i s d i f f i c u l t to d i r e c t l y compare the e f f i c i e n c y of feed u t i l i z a t i o n i n normal and i r o n - d e f i c i e n t animals. IRON AND DISEASE RESISTANCE Disease r e s i s t a n c e i s depressed during i r o n d e f i c i e n c y i n a l l species s t u d i e d . Enzymatic and metabolic defects are found i n many components of the immune system (Pearson and Robinson, 1976). The number of leukocytes and lymphocytes are often s i g n i f i c a n t l y reduced by i r o n d e f i c i e n c y , r e s u l -t i n g i n the impairment of both phagocytic f u n c t i o n and cell-mediated immunity, r e s p e c t i v e l y . The e f f e c t of i r o n status on t i s s u e morphology, r e s i d e n t b a c t e r i a l populations ( F l e t c h e r _et a l . , 1975) and i n a c t i v a t i o n of b a c t e r i a l exotoxins and endotoxins (Weinberg, 1971; Oanoff and Zweifach, 1960) may a l s o be important. M o r t a l i t y i n farm l i v e s t o c k i s increased by i r o n d e f i c i e n c y . In c l i n i c a l l y normal but anemic d a i r y c a l v e s , m o r t a l i t y was 22%, mainly from septicemic and e n t e r i c i n f e c t i o n s (Tennant et a l . , 1975a). The incidence of e n t e r i t i s was s i g n i f i c a n t l y l e s s (P<0.001) i n i r o n - i n j e c t e d calves than i n anemic calves fed a commercial veal c a l f milk replacer containing 19 mg Fe/kg d i e t (Mollerberg jet a l _ . , 1975a). Comparable data i s not a v a i l a b l e fo r lambs, but Holz et al_. (1961) noted that m o r t a l i t y was 30.5%, 38.6%, and 13.5% f o r 72 lambs i n j e c t e d with 0, 150 and 300 mg i r o n at - 26 -b i r t h . Iron treatment of p i g l e t s r e s u l t e d i n a s i g n i f i c a n t r eduction of i n f e c t i o u s diseases, p a r t i c u l a r l y diseases associated with F_. c o l i organ-isms, such as scours. M o r t a l i t y was 13% i n the i r o n - t r e a t e d p i g l e t s compared to 17% i n the c o n t r o l s , out of a t o t a l of 494 p i g l e t s (Hopson and Ashmead, 1976). The s u s c e p t i b i l i t y of i r o n - d e f i c i e n t animals to g a s t r o i n t e s t i n a l i n f e c t i o n s may i n v o l v e a f a i l u r e to produce adequate numbers of myeloper-oxidase-containing c e l l s . N e u t r o p h i l s , the most important phagocytic c e l l s , contain myeloperoxidase, an i r o n - c o n t a i n i n g enzyme which a f f e c t s b a c t e r i c i d a l c a p a c i t y . Iron d e f i c i e n t rats had fewer MPO-cells i n the lamina p r o p r i a and submucosa, and were much more s u s c e p t i b l e to challenge with Salmonella typhimurium (Baggs and M i l l e r , 1973). In another study, the i r o n d e f i c i e n t r a t s responded very slowly compared to c o n t r o l s i n production of i n t e s t i n a l MPO, which was c o r r e l a t e d with s u r v i v a l and r e t e n t i o n of j>. typhimurium i n the gut (Baggs and M i l l e r , 1974). Studies with humans i n d i c a t e d that phagocytosis by n e u t r o p h i l s was unaffected i n i r o n d e f i c i e n c y , but i n t r a c e l l u l a r b a c t e r i a l k i l l i n g was s i g n i f i c a n t l y (P<0.001) l e s s i n i r o n d e f i c i e n t p a t i e n t s (Higashi et _ a l . , 1967; Chandra, 1973). Iron d e f i c i e n c y s i g n i f i c a n t l y a f f e c t s the s i z e , s t r u c t u r e and func-t i o n of lymphoid t i s s u e s . Various s t u d i e s document e f f e c t s such as reduc-t i o n of antibody-forming spleen c e l l s ; reduction i n thymus weight and i n thymus mononuclear c e l l s (Chandra _et a l . , 1977). Iron d e f i c i e n c y may be most important during development of these t i s s u e s . Studies with r a t s i n d i c a t e d that i r o n d e p r i v a t i o n during g e s t a t i o n and l a c t a t i o n was more serious than a f t e r weaning, as the subsequent disease r e s i s t a n c e was - 27 -reduced, even a f t e r a period of n u t r i t i o n a l r e h a b i l i t a t i o n (Baggs and M i l l e r , 1973). While i r o n supplementation of young animals would appear to be bene-f i c i a l i n improving disease r e s i s t a n c e , Knight e£ a_L. (1983) have r e c e n t l y cautioned against oversupplementation, s t a t i n g that: "Although confinement-reared pigs required Fe supplementation to prevent anemia, the data presented here and the la r g e amount of p r e v i o u s l y reported evidence from other species i n d i c a t e p o t e n t i a l d e t r i m e n t a l e f f e c t s from over- as w e l l as undersupplementation. I t appears prudent that the e f f e c t s of s u s c e p t i b i l i t y to i n f e c t i o n be included i n determining the optimum Fe supplementation l e v e l s . " These concerns were based p a r t l y on _in v i t r o s t u d i e s on the growth of two E. c o l i s t r a i n s , i n serum taken from i r o n dextran i n j e c t e d p i g l e t s a t va r i o u s i n t e r v a l s a f t e r i n j e c t i o n . The i r o n treatment s i g n i f i c a n t l y enhanced b a c t e r i a l growth i n v i t r o i n some cases, but not c o n s i s t e n t l y , and not l a t e r than day 3 p o s t - i n j e c t i o n (Knight et a l . , 1983). As i r o n i s a c r u c i a l t r a c e mineral f o r m i c r o b i a l growth, an i n c r e a s e i n plasma i r o n (hyperferremia) i n humans may be associated with s u s c e p t i -b i l i t y to b a c t e r i a l and fungal pathogens (Weinberg, 1974). I t should be recognized, though, that the hyperferremia i s extreme and/or long-term, and i s t y p i c a l l y caused by disease c o n d i t i o n s such as v i r a l h e p a t i t i s , s i c k l e c e l l anemia, malaria and others which could tax the immune system regard-l e s s of i r o n l e v e l . Thus, a comparison between hyperferremia-associated diseases i n humans and the r i s k s of the r a p e u t i c i r o n supplementation i n l i v e s t o c k i s not completely v a l i d . Whether or not i r o n supplementation increases disease s u s c e p t i b i l i t y depends on seve r a l f a c t o r s . In order to n e u t r a l i z e the m i c r o b i o s t a t i c a c t i o n of serum, enough i r o n must be provided to saturate at l e a s t 60-80% - 28 -of the serum t r a n s f e r r i n (Weinberg, 1974). Iron dextran i s a p a r t i c u l a r l y safe i r o n supplement as no more than 1-3% of the i r o n can be d i r e c t l y t r a n s f e r r e d to t r a n s f e r r i n ( S z i l a g y i and E r s l e v , 1970). I r o n administra-t i o n during experimental i n f e c t i o n s of r a t s has been shown to d r a m a t i c a l l y lower the LD 5 0 dose for a v a r i e t y of b a c t e r i a , provided the i r o n i s i n a form able to d i f f u s e to the s i t e of b a c t e r i a l r e p l i c a t i o n , and i s adminis-tered by i v , i p or im routes. Consequently, i r o n dextran and i r o n d e x t r i n are i n a c t i v e i n enhancing m i c r o b i a l growth, whereas f e r r i c ammonium c i t r a t e , i r o n s o r b i t a l c i t r a t e and hemoglobin are hi g h l y a c t i v e (Weinberg, 1971). A c c o r d i n g l y , i r o n supplementation of i r o n - d e f i c i e n t animals appears to confer l i t t l e r i s k , e s p e c i a l l y when i r o n i s provided as i r o n dextran, w h i l e the improvement i n i r o n s t a t u s allows optimal f u n c t i o n i n g of many aspects of the immune system. - 29 -SELENIUM FUNCTIONS OF SELENIUM B i o l o g i c a l f u n c t i o n s of selenium includ e the maintenance of muscle and membrane ( e r y t h r o c y t e , vascular endothelium, and c e l l o r g a n e l l e ) i n t e g r i t y ; s t i m u l a t i o n of antibody and ubiquinone synthesis; maintenance of e s s e n t i a l enzyme systems, pancreatic f u n c t i o n , and vigor and m o b i l i t y of sperm ( C a l v i n _et _ a l . , 1981; Combs and Bunk, 1981; Ganther jet a±., 1976; Hi d i r o g l o u et a_l., 1968; S p a l l h o l z et a l . , 1975). Many of these f u n c t i o n s i n v o l v e g l u t a t h i o n e peroxidase. The selenoenzyme, g l u t a t h i o n e peroxidase (GSH-Px, EC 1.11.1.9) i s pa r t of a complex mechanism, i n c l u d i n g the superoxide dismutases, c a t a l a s e , and vitamin E, which defends the c e l l against c y t o t o x i c oxygen d e r i v a -t i v e s . Due to the i m p o s s i b i l i t y of i s o l a t i n g these compounds i n v i v o , the models proposed for the a c t i o n of GSH-Px are s t i l l t e n t a t i v e (Flohe jet al., 1979). GSH-Px protects the c e l l from damage caused by peroxides and f r e e r a d i c a l s . GSH-Px probably c a t a l y z e s the reduction of many hydroperoxides to t h e i r corresponding a l c o h o l s , or H 20, i n the case of H 20 2 . H 20 2 i s a na t u r a l by-product of many enzyme r e a c t i o n s , i n c l u d i n g those of xanthine oxidase and d-amino ac i d oxidase (Rotruck, 1981). Free r a d i c a l s may be produced by mitochondrial r e s p i r a t i o n , a u t o o x i d a t i o n , i r r a d i a t i o n damage or environmental p o l l u t a n t s (Csallany et a_l., 1981) and by i n t e r a c t i o n between H 20 2 and metal ions or 0 2 (Rotruck, 1981). 0 2 i s generated i n l a r g e amounts by many e l e c t r o n transport steps i n mitochondrial enzyme systems, and c a t a l y z e s the peroxidation of membrane polyunsaturated f a t t y a c i d s ( D i p l o c k , 1981). - 30 -The inti m a t e r e l a t i o n s h i p between vitamin E and selenium can p a r t l y be explained by t h e i r r e l a t e d f u n c t i o n s . GSH-Px, located both i n the c y t o s o l and mitochondria i n a 70:30 r a t i o , reduces l i p i d peroxides to nontoxic hydroxy f a t t y a c i d s . Vitamin E, located w i t h i n membranes, prevents or decreases the formation of l i p i d peroxides. Hoekstra (1974) has discussed the n u t r i t i o n a l i m p l i c a t i o n s of t h i s scheme, but emphasized th a t GSH-Px should not be considered the only biochemical f u n c t i o n of Se. While more than 9 d i f f e r e n t selenium-containing proteins have been found i n lamb t i s s u e s one of these was present only i n heart and muscle of normal lambs and not i n lambs a f f e c t e d with White Muscle Disease. S i g n i f i -c a n t l y , heart and muscle t i s s u e are the target s i t e s during Se/vitamin E d e f i c i e n c y i n sheep. Furthermore, a l i n k between oxygen generation or energy u t i l i z a t i o n and WMD i s i n d i c a t e d by the nature of t h i s p r o t e i n . I t i s a cytochrome containing a heme group i d e n t i c a l to cytochrome c;, but has an amino a c i d composition and weight s i m i l a r to cytochrome (Whanger et^ a l . , 1974). I t has been hypothesized that a primary r o l e of selenium i s as an o x i d a n t - l a b i l e selenide i n a c l a s s of non-heme i r o n proteins present i n the mitochondria and smooth endoplasmic r e t i c u l u m , and protected from o x i d a t i o n by vitamin E (Diplock and Lucy, 1973; Diplock, 1974; C a y g i l l and Di p l o c k , 1973; Giasuddin j^t _ a l . , 1975). D e f i c i e n c y of vitamin E would lead to a replacement of the selenide with a more s t a b l e , but l e s s c a t a l y t i c a l l y a c t i v e , sulphur group, as the se l e n i d e i n modified non-heme Fe p r o t e i n s i s much more vulnerable to o x i d a t i o n than the sulphur group (D i p l o c k , 1970). Increasing evidence suggests a major f u n c t i o n of selenium may be i n e l e c t r o n t r a n s p o r t , p o s s i b l y i n v o l v i n g the same p r o t e i n i n v e s t i g a t e d by - 31 -D i p l o c k (1970) and Whanger et a l . (1974). Levander et _ a l . (1974) were the f i r s t to present evidence of a r o l e f o r Se i n c a t a l y z i n g e l e c t r o n t r a n s -f e r . They point out that with adequate d i e t a r y Se, the Se could "short-c i r c u i t " the r e s p i r a t o r y chain, avoiding the H 20 2 generating step, by d i r e c t l y t r a n s f e r r i n g e l e c t r o n s from GSH to cytochrome _c. SELENIUM METABOLISM The d i e t a r y selenium sources of ruminants are mainly organic com-pounds of Se i n f e e d s t u f f s and s e l e n i t e or selenate s a l t supplements. Ruminants absorb s i g n i f i c a n t l y l e s s of both i n o r g a n i c and organic sources of selenium than do monogastrics. Based on the i n s o l u b l e nature of f e c a l Se and the known a b i l i t y of the anaerobic, highly reducing rumen environ-ment to reduce l e s s s u s c e p t i b l e sulphur compounds, i t i s l i k e l y that rumen microbes reduce Se compounds to u n a v a i l a b l e forms such as se l e n i d e or elemental selenium. At l e a s t 50% of f e c a l selenium i s i n such i n s o l u b l e forms (Cousins and Cairney, 1961). Conversely, rumen microbes can increase the a v a i l a b i l i t y of d i e t a r y Se. They concentrate Se at 2 to 78 times the d i e t a r y concentration. These organic forms, i n c l u d i n g selenoamino aci d s incorporated i n t o m i c r o b i a l p r o t e i n , are more e a s i l y absorbed (Whanger et a l . , 1978a) . The spontaneous recovery of WMD-affected lambs which s u r v i v e to 6 weeks of age may be explained by the increased a v a i l a b i l i t y of Se incorporated i n t o rumen microorganisms (Whanger _et aL., 1970). The proportion of selenium absorbed increases with decreased d i e t a r y selenium and/or d e f i c i e n t Se s t a t u s , and i s not a f f e c t e d by vitamin E l e v e l . S i m i l a r l y , Se r e t e n t i o n of i n j e c t e d Se i s also i n v e r s e l y propor-t i o n a l to d i e t a r y selenium (Ku _et a l . , 1972; K i n c a i d _et a l _ . , 1977; Van - 32 -F l e e t , 1975). Sodium s e l e n i t e i s more e f f e c t i v e than selenomethionine i n in c r e a s i n g t i s s u e and blood l e v e l s of Se and GSH-Px, when supplemented at 0.1 ppm Se. However, at a d i e t a r y l e v e l of 1.0 ppm, selenomethionine i s more e f f e c t i v e than sodium s e l e n i t e (Moknes and Norheim, 1983). The main s i t e of Se absorption i s the duodenum. Selenium i s not absorbed from the rumen or abomasum i n sheep. S e l e n i t e and s e l e n o c y s t i n e are absorbed by passive t r a n s p o r t , while selenomethionine i s transported against a concentration g r a d i e n t . As methionine and selenomethionine share the same tr a n s p o r t system, methionine can i n h i b i t selenomethionine uptake. This may p a r t l y e x p l a i n the p r o t e c t i v e e f f e c t of high p r o t e i n d i e t s a gainst s e l e n o s i s . Methionine may be e f f e c t i v e only i n combination with adequate l e v e l s of vitamin E (Levander, 1976; White and Somers, 1977). Selenium i s transported by plasma p r o t e i n s (Porter ^ t a l . , 1979). I t i s g radually taken up from the blood by the l i v e r and kidney cortex, and l e s s r e a d i l y by the spleen, muscle, heart and lungs. Organic forms of selenium tend to be retained more te n a c i o u s l y by t i s s u e s compared to i n o r -ganic forms (Martin and Gerlach, 1972). Wright and B e l l (1964) suggest that the t i s s u e s with highest Se content a f t e r dosing have the greatest a b i l i t y to synthesize organoselenium compounds from inorganic selenium. Subsequently, these compounds are r e d i s t r i b u t e d , e s p e c i a l l y to the muscle which i s vulnerable to Se d e f i -ciency i n sheep. S u r p r i s i n g l y , S e 7 5 s t u d i e s by Wright and B e l l (1964) and others, i n d i c a t e that 48 hours a f t e r dosing, S e 7 5 uptake i n muscle from S e - d e f i c i e n t sheep i s lower than i n muscle from Se-supplemented sheep. - 33 -Selenium i s excreted by f e c a l , u r i n a r y and r e s p i r a t o r y routes. Fecal e x c r e t i o n i s more important to ruminants than monogastrics, due to lower r a t e s of absorption. At high l e v e l s of d i e t a r y Se, selenium d e t o x i f i c a t i o n by GSH-dependent methylation becomes important. Dimethyl selenide and trimethylselenonium ion are the major pulmonary and u r i n a r y Se metabolites, r e s p e c t i v e l y (Levander, 1976). Selenium i s found i n many p r o t e i n s , i n c l u d i n g heme pr o t e i n s (hemoglo-b i n , cytochrome c;, myoglobin), enzymes (myosin, a l d o l a s e , urokinase, f i b r i n a s e ) and nucleoproteins. P r e v i o u s l y , Se was thought to be a contam-inant mistaken f o r S due to i t s cl o s e chemical s i m i l a r i t y . While seleno-methionine i s " a c c i d e n t a l l y " incorporated i n t o p r o t e i n s i n place of methionine (McConnell et a^., 1970), selenocysteine i s now known to form the a c t i v e center of various selenoproteins which have been i n v e s t i g a t e d (Wilhelmsen et a l . , 1981). Selenium metabolism, while sharing some pathways with sulphur, i s othewise unique (Levander, 1976). Animals r e a d i l y reduce such forms of i n o r g a n i c Se as selenate or s e l e n i t e , but cannot reduce even s u l f a t e or s u l f i t e w i t h i n body c e l l s . Reduction i s c r u c i a l to s e l e n i t e metabolism, as selenium must be reduced from the +4 o x i d a t i o n s t a t e to the -2 o x i d a t i o n s t a t e . Ganther and Hsieh (1974) have described a probable biochemical mechanism. SELENIUM INTERACTIONS Selenium i n t e r a c t i o n s with a v a r i e t y of n u t r i e n t s have been docu-mented, most commonly with a r s e n i c , cadmium, coper, lead, mercury, s i l v e r , t e l l u r i u m , z i n c and sulphate. Selenium metabolism i s i n f l u e n c e d by - 34 -selenium's tendency to complex with heavy metals. These minerals reduce the t o x i c i t y of high l e v e l s of d i e t a r y selenium, and also high l e v e l s can induce a d e f i c i e n c y when d i e t a r y selenium i s marginal (Lee and Jones, 1976). Selenium a l s o i n t e r a c t s , d i r e c t l y and i n d i r e c t l y , with vitamin E, p r o t e i n , c e r t a i n carbohydrate f r a c t i o n s , and other f a c t o r s which remain to be i d e n t i f i e d . D i e t a r y sulphur at l e v e l s found n a t u r a l l y i n feeds a f f e c t s selenium absorption and r e t e n t i o n s i g n i f i c a n t l y . High l e v e l s of sulphate increase the Se requirement i n sheep (Ekermans and Schneider, 1982). Major changes i n blood Se occur between 0.05 and 0.10% d i e t a r y S, but l i t t l e change occurs at higher l e v e l s . I n creasing d i e t a r y sulphur increases u r i n a r y Se s e c r e t i o n . On a high sulphur d i e t , the increased population of Desulpho- v i b r i o b a c t e r i a i n the rumen may reduce more selenium to H 2Se through sulphate reduction pathways. H 2Se i s thought to d i s s o c i a t e to more s t a b l e but l e s s s o l u b l e forms, e i t h e r elemental selenium or hig h l y i n s o l u b l e metal selenides (Pope et a_l., 1979). The selenium content of rumen microorganisms, and p o t e n t i a l l y the a v a i l a b i l i t y of Se, i s reduced by sulphur d e f i c i e n c y (Whanger et a_l. , 1978a). Sulphur d e f i c i e n c y a l s o increases s e l e n o s i s at high Se l e v e l s , as sulphur i n gl u t a t h i o n e and gl u t a t h i o n e reductase i s needed for the forma-t i o n of excretory products such as dimethyl s e l e n i d e and t r i m e t h y l selenon-ium ion (Pope et a l . , 1979). C o n f l i c t i n g evidence of the e f f e c t of sulphur on i n c r e a s i n g the i n c i -dence of white muscle disease may be resolved by the observation that sulphur competes with selenium only when present as the selenium analogue. Thus, "inorganic sulphur may a l t e r the metabolism of inorganic selenium more than organic selenium" (Whanger et a l _ . , 1969a). - 35 -P r o t e i n source may be a major i n f l u e n c e on the absorption of selen-ium. Studies with beef c a t t l e i n d i c a t e d that d i e t a r y selenium l e v e l s may be more c r i t i c a l on d i e t s marginal or d e f i c i e n t i n p r o t e i n . However, a response i n weight gain to a d d i t i o n a l inorganic selenium was apparent i n growing, but not i n f i n i s h i n g , c a t t l e . The degree of p r o t e i n d e p o s i t i o n may be a f a c t o r ( T r e v i s , 1979). Feeding a high p r o t e i n d i e t , on the other hand, has been recommended i n cases of Se t o x i c i t y (Ekermans and Schneider, 1982). Linseed o i l meal has long been known to have a p r o t e c t i v e e f f e c t against chronic s e l e n o s i s . Two cyanogenic glycosides i n l i n s e e d o i l meal re l e a s e CN- which i n t e r a c t s with metabolic selenium to form SeCN-. This i n t e r a c t i o n could ,be detrimental i n animals marginally d e f i c i e n t i n se l e n -ium (Palmer, 1981). Dystrophogenic f a c t o r s i n feeds may reduce the a v a i l a b i l i t y of sele n -ium or vitamin E. The vitamin E i n a l f a l f a may not be completely a v a i l a b l e for calves or c h i c k s . A compound extracted i n ethanol from a l f a l f a i n -creased e x c r e t i o n of a - t o c o p h e r o l from both a l f a l f a and added sources (Pudelkiewicz and Matterson, 1960). A succinoxidase i n h i b i t o r antagonized by a - t o c o p h e r o l may occur i n high l e v e l s i n dystrophogenic feeds (Diplock, 1970). DIETARY SELENIUM AND TOCOPHEROL Selenium d e f i c i e n c y occurs n a t u r a l l y i n large parts of the world, i n c l u d i n g the P a c i f i c Northwest; Midwest, Southeast and Northeast North America. In Canada, low selenium s o i l s are prevalent i n c e n t r a l B.C., west-central A l b e r t a , Northern Ontario, the A t l a n t i c provinces and parts of - 36 -Quebec ( U l l r e y , 1981). Forages sampled i n the Fraser V a l l e y of B.C. were a l s o c o n s i s t e n t l y inadequate i n selenium (Cathcart et a K , 1980). The a v a i l a b i l i t y of Se to plants i s increased i n we l l - a e r a t e d , a l k a l i n e s o i l s and decreased i n a c i d , water-logged s o i l s . Studies i n the Kootenays i n B.C. demonstrated s t r i k i n g d i f f e r e n c e s between Se a v a i l a b i l i t y i n adjacent s o i l types, which were more highly c o r r e l a t e d with s o i l pH and moisture regime than s o i l Se (Van Ryswyk _et a l _ . , 1976). Selenium concentration i n forage can be markedly and suddenly decreased by changing c u l t u r a l p r a c t i s e s , as shown by a dramatic increase i n WMD i n New Zealand a f t e r 1957. Increasing y i e l d s by f e r t i l i z a t i o n , seeding of more productive species, i r r i g a t i o n (and associated leaching) seems to d i l u t e the amount of selenium i n the feed, and heavy cropping may remove more selenium than i s r e c y c l e d . I n t e n s i v e s t o c k i n g may a l s o decrease the a v a i l a b i l i t y of Se to p l a n t s , as much f e c a l selenium i s t i e d up i n elemental and other i n s o l u b l e forms of selenium (Counsins and Cairney, 1961). Tocopherol content of forages does not seem to be a f f e c t e d by selen-ium l e v e l . However, i t i s s e n s i t i v e to stage of maturity, drying losses i n hay, r a i n , processing and storage l o s s e s (Kivimae and Carpena, 1973). Tocopherol i n concentrates i s destroyed by g r i n d i n g , mixing with minerals or f a t , and p e l l e t i n g (MacDonald et , 1976). SELENIUM REQUIREMENT The minimum selenium requirement of sheep i s approximately 0.1 ppm (NRC, 1975, p. 47). The form of d i e t a r y selenium, the c r i t e r i a used to assess adequacy, and the d i e t composition i n c l u d i n g v i tamin E, w i l l - 37 -i n f l u e n c e the requirement. Levels of 0.11 to 0.12 ppm Se were required i n order to maintain t i s s u e PSH-Px l e v e l s i n sheep fed a p r a c t i c a l - t y p e r a t i o n (Oh ^ t al., 1974). Selenium-responsive d i s o r d e r s i n ruminants become i n c r e a s i n g l y prevalent as d i e t a r y Se f a l l s below 0.08 mg /kg D.M. Di e t a r y selenium l e v e l s between 0.03 and 0.05 ppm are marginal (ARC, 1980, p. 243). Under B.C. c o n d i t i o n s , 0.2 ppm i s considered adequate, and <0.2 ppm may be marginal ( P u i s , 1981, pp. 76-87). The d i e t a r y vitamin E may be important only at "marginal" intakes o f Se (ARC, 1980, p. 244-251). I t was suggested that the d i e t a r y Se req u i r e -ment for a l l species i s 0.1 - 0.15 ppm when vitamin E i s s u f f i c i e n t , but may be as high as 0.5 - 1.0 ppm when vitamin E i s low (Hoffman and La Roche, 1971). The selenium requirement may be markedly increased when dystropho-genic feeds such as c u l l kidney beans are included i n the r a t i o n . The a d d i t i o n of 0.17 ppm Se to a kidney bean and hay r a t i o n during l a c t a t i o n did not s i g n i f i c a n t l y reduce c l i n i c a l NMD i n lambs; the authors suggested that a l e v e l of 1.0 ppm added Se may not be completely e f f e c t i v e under these circumstances (Hintz and Hogue, 1964). SELENIUM DEFICIENCY AND WHITE MUSCLE DISEASE Numerous diseases have been i d e n t i f i e d as responsive to selenium and vitamin E. Some respond to only vitamin E or only selenium, while others such as white muscle disease (WMD) may respond to e i t h e r depending on the d e f i c i e n t n u t r i e n t and other n u t r i t i o n a l s t r e s s e s . By f a r the most common worldwide Se/Vitamin E d e f i c i e n c y i n ruminants i s white muscle disease, a l s o known as n u t r i t i o n a l muscular dystrophy or s t i f f lamb di s e a s e . - 38 -Recently, retained placenta i n d a i r y cows, lameness i n breeding stock, "sawdust l i v e r " i n f e e d l o t s t e e r s , and i l l t h r i f t , p e r i d o n t a l disease, reduced f e r t i l i t y and poor wool production i n sheep have a l s o been a s s o c i -ated with selenium d e f i c i e n c y ( J u l i e n et a l . , 1976; MacDonald et a_l., 1976; S c a l e s , 1976; U l l r e y et a l _ . , 1977; Ammerman and M i l l e r , 1975). WMD most commonly a f f e c t s young calves and lambs between b i r t h and weaning, or sometimes j u s t a f t e r weaning or other s t r e s s e s . M o r b i d i t y may be 65% or greater, although immediate Se/Vitamin E treatment u s u a l l y helps prevent severe l o s s e s . Seasonally, WMD i s most common i n s p r i n g , and f r e q u e n t l y , lambs and ewes are on lush pasture, presumably r i c h i n a - t o c o p h e r o l . Symptoms of WMD are s i m i l a r for lambs and c a l v e s . M o r t a l i t y from uncomplicated WMD i n v a r i a b l y r e s u l t s from c a r d i a c f a i l u r e , and may not be properly diagnosed when c l i n i c a l signs do not precede death. In pigs the disease i s c a l l e d mulberry heart disease because extensive c a r d i a c hemor-rhage r e s u l t s i n a reddish-purple appearance. WMD a f f e c t s heart f u n c t i o n even before h i s t o p a t h o l o g i c a l l e s i o n s develop. D e f i c i e n t calves d i s p l a y a marked decrease i n heart r a t e at the same time as i n i t i a l signs of muscular dystrophy. D i f f e r e n c e s i n ECG t r a c i n g s were observed between calves on normal and dystrophogenic d i e t s ( Safford ejt a_l., 1954). S i m i l a r abnormali-t i e s have been seen i n vitamin E d e f i c i e n t lambs as w e l l (Bacigalupo ejt a l . , 1953). More information on the r o l e of Se i n heart f u n c t i o n may come from current s t u d i e s on Keshan disease, a cardiomyopathy a f f e c t i n g thou-sands of people i n China but now c o n t r o l l e d by large s c a l e Se supplementa-t i o n (Chen e t ^ a l . , 1981; Shamberger, 1981). - 39 -The f i r s t s i g n of s t i f f lamb disease i s reluctance to walk, and then a d e f i n i t e s t i f f n e s s , e s p e c i a l l y of the back legs, and the c h a r a c t e r i s t i c arched back stance. The muscles of the f r o n t and hind legs are a f f e c t e d f i r s t and most severely, then those of the shoulder, rump, l o i n and neck may be i n v o l v e d . In extreme cases l e s i o n s may a f f e c t the diaphragm, i n t e r c o s t a l muscles and tongue ( C u l i k et a l _ . , 1951). However, l o s s of a p p e t i t e i s r a r e . P a t h o l o g i c a l l y , WMD i s c h a r a c t e r i z e d by degeneration of s t r i a t e d muscle, e i t h e r s k e l e t a l or c a r d i a c muscle, or both. Degeneration seems c l o s e l y r e l a t e d to the degree of muscle s t r e s s (Young and Keeler, 1962). White or grey spots and streaks i n muscle t i s s u e i n d i c a t e l o c a l i z e d damage and calcium p r e c i p i t a t i o n . Two types of l e s i o n s - f i b e r and vascular - are found i n WMD, and one or both may occur i n other Se/vitamin E responsive diseases, such as exudative d i a t h e s i s i n c h i c k s . F i b e r l e s i o n s are very s i m i l a r i n swine and i n ruminants. Van F l e e t et a l . (1977a) studied u l t r a s t r u c t u r a l changes i n f i b e r s of Se-vitamin E d e f i c i e n t swine, and found evidence of concurrent m y o f i b r i l l a r and mito-c h o n d r i a l changes, both probably i n i t i a t e d by c e l l u l a r p e r o x i d a t i o n . S i m i l a r l y , Godwin et a l . (1974) observed an increase i n membrane l a b i l i t y i n o r g a n e l l e s i n Se-vitamin E d e f i c i e n t t i s s u e s . The membrane damage appears to a f f e c t i n t r a c e l l u l a r f l u i d balance and energy production. Damaged f i b e r s e v e n t u a l l y become m i n e r a l i z e d , and the healed l e s i o n s p e r s i s t as patches of stromal condensation and f i b r o s i s . Regeneration may occur i n sheep s k e l e t a l muscle, but not i n c a r d i a c muscle, at l e a s t i n swine (Van F l e e t et a l . , 1977a). - 40 -Vascul a r damage i s fr e q u e n t l y found i n the hearts and other t i s s u e s of selenium or vitamin E d e f i c i e n t animals. Microvascular l e s i o n s and hemorrhages occur i n the kidney, i n t e s t i n e , l i v e r , s k e l e t a l muscle, stomach and s k i n as w e l l as the heart i n swine (Van F l e e t et a_l., 1977b), developing independently of f i b e r l e s i o n s . The heart of lambs and calves with WMD i s t y p i c a l l y edematous. The vascular l e s i o n s apparently a r i s e from l i p o p e r o x i d a t i o n damage to the e n d o t h e l i a l c e l l s l i n i n g a r t e r i o l e s and c a p i l l a r i e s . The t h i n , t i g h t l y j o i n e d e n d o t h e l i a l c e l l s l i n i n g the lumen of normal ves s e l s give way to loo s e l y attached, thickened but i n t a c t endothelium. The increased perme-a b i l i t y allows leakage of blood p r o t e i n s i n t o the vesse l w a l l , causing accumulation of f i b r i n o i d , and p e r i v a s c u l a r edema. Sudden massive hemor-rhaging may develop i n cases of spontaneous WMD. SELENIUM AND DISEASE RESISTANCE S i n c e 1972 numerous reports have been published on the e f f e c t s of selenium and vitamin E on both humoral and c e l l u l a r immunity. Most s t u d i e s have been conducted using sodium s e l e n i t e , however, organic Se compounds appear l e s s e f f e c t i v e than equivalent amounts of Se as s e l e n i t e or s e l e n i d e ( S p a l l h o l z , 1981). D e f i c i e n c i e s of selenium or vitamin E are associated with impaired immunity and increased s u s c e p t i b i l i t y to experimental bacter-i a l and fungal i n f e c t i o n s , but supplementation of "normal d i e t s " leads to a fu r t h e r increase i n antibody production. Antibody production i n response to various vaccines or SRBC-antigen i s enhanced i n c a t t l e , dogs, mice, r a b b i t s , and chicks simultaneously i n j e c t e d with Se ( S p a l l h o l z , 1981). G e n e r a l l y , d i e t a r y Se l e v e l s above 0.1 - 41 -ppm are als o e f f e c t i v e . The number of plaque-forming c e l l s i n spleen of mice were increased p r o p o r t i o n a t e l y as d i e t a r y s e l e n i t e was increased from 0 to 1.25 ppm ( S p a l l h o l z et a l . , 1973). (Plaque-forming c e l l s produce antibody.) Diets containing 1 to 3 ppm s e l e n i t e , l e v e l s g r e a t l y i n excess of the normal requirement, p o t e n t i a t e d the synth e s i s of IgM and IgG immunoglobulins i n mice ( S p a l l h o l z jst a_l., 1974). However, when given i n t r a p e r i t o n e a l l y , the amount of Se required to enhance the primary immune response i n mice was not much greater than the estimated d a i l y Se requirement ( S p a l l h o l z jet a l _ . , 1975). Some e f f e c t s of selenium and vitamin E on the immune system may be interchangeable. The 2-week-old chick r e q u i r e s both Se and vitamin E f o r optimal imune f u n c t i o n , but by 3 weeks, Se alone can f a c i l i t a t e optimal immune f u n c t i o n (Baumgartner, 1979). Selenium apparently can d u p l i c a t e some of the e f f e c t s of vitamin E on the immune system, which may i n v o l v e a r o l e i n ubiquinone and prostaglandin s y n t h e s i s ( H e i n z e r l i n g jet ad., 1974; Tengerdy and Nockels, 1975; Tengerdy et a l . , 1978). A l t e r n a t i v e l y , some of the immunostimulatory e f f e c t s of Se and vitamin E may in v o l v e p r o v i s i o n of the proper biochemical environment f or c e l l u l a r i n t e r a c t i o n s , and may be re p l a c e a b l e by s y n t h e t i c a n t i o x i d a n t s . Some of the n o n s p e c i f i c mitogenic f a c t o r s of macrophages are a n t i o x i d a n t s , and i t i s suggested that a n t i o x i -dants such as Se and vitamin E have a s i m i l a r adjuvant e f f e c t , i e . they a t t r a c t macrophages (Baumgartner, 1979). D e f e c t i v e m i c r o b i c i d a l a c t i v i t y i s t y p i c a l l y observed i n n e u t r o p h i l s of selenium d e f i c i e n t animals. The v i a b i l i t y of ne u t r o p h i l s and i n g e s t i o n of b a c t e r i a l or fungal c e l l s i s not impaired, but the a b i l i t y to k i l l these c e l l s i s g r e a t l y impaired f or both r a t s and c a t t l e , among others (Serfass - kl -and Ganther, 1975; Boyne and Arthur, 1978). The selenium d e f i c i e n c y reduces n e u t r o p h i l GSH-Px, and consequently the a b i l i t y to metabolize H 20 2. The H 20 2 accumulation r e s u l t s i n d e s t r u c t i o n of the -generating system, which i s not a l t e r e d by vitamin E s t a t u s , at l e a s t i n r a t s (Baker and Cohen, 1983). In c o n t r a s t , Bass _et a l . (1977) compared b a c t e r i c i d a l a c t i v i t y of ne u t r o p h i l s from species varying g r e a t l y i n normal GSH-Px content, and concluded that post-phagocytic o x i d a t i v e responces and bacter-i a l k i l l i n g "were not compromised by complete absence of GSH-Px, even i n species with the highest n a t u r a l l e v e l s of t h i s enzyme". Sex d i f f e r e n c e s i n antibody response to Se dosage have been observed by s e v e r a l groups working with c h i c k s , but have not been i n v e s t i g a t e d i n other species. At d i e t a r y l e v e l s i n s l i g h t excess of that required to prevent d e f i c i e n c y diseases, antibody t i t e r i s s i g n i f i c a n t l y depressed i n male but not female chicks (Marsh et a l . , 1981; Baumgartner, 1979). - 43 -IRON AND SELENIUM INTERACTIONS Three p o s s i b l e areas of metabolic i n t e r a c t i o n between selenium and i r o n have been documented. Selenium d e f i c i e n c y may reduce red c e l l l i f e -span, i n d i r e c t l y i n c r e a s i n g i r o n turnover, and i n severe cases causing hemolytic anemia. Selenium d e f i c i e n c y may be d i r e c t l y involved i n the u t i l i z a t i o n of i r o n f o r heme s y n t h e s i s . F i n a l l y , i r o n s t a t u s i n f l u e n c e s l e v e l s of the selenoenzyme, GSH-Px i n red blood c e l l s . SELENIUM DEFICIENCY AND ANEMIA Selenium and/or vitamin E d e f i c i e n c i e s are associated with hemolytic anemia i n swine, monkeys and other animals. Both vitamin E and selenium are important i n maintaining RBC membrane i n t e g r i t y , but only selenium, as GSH-Px, i s e f f e c t i v e against Hb o x i d a t i o n , due to the membrane l o c a l i z a t i o n of vitamin E (Hoekstra, 1974). C e l l s c o n taining o x i d i z e d Hb, or Heinz bodies, break down prematurely. S i m i l a r l y , an i n h e r i t e d d e f i c i e n c y of reduced g l u t a t h i o n e (GSH), the substrate f o r GSH-Px, a l s o reduces the p o t e n t i a l l i f e s p a n of RBC's i n sheep and i n man (Tucker, 1974). SELENIUM AND HEME SYNTHESIS Tissue Enzymes Recent research on Se i n heme synthesis and catabolism suggests a r o l e f or selenium, d i s t i n c t from GSH-Px, i n the u t i l i z a t i o n of i r o n . A marked increase i n both heme synthesis and catabolism was found i n l i v e r but not i n spleen of S e - d e f i c i e n t r a t s (Correia and Burk, 1976). Further i n v e s t i g a t i o n s revealed that selenium i s e s s e n t i a l f o r normal u t i l i z a t i o n of heme i n rat l i v e r , and selenium d e f i c i e n c y leads to "wasted" heme; that - kk -the e f f e c t of selenium i s not mediated by GSH-Px, nor i s an a n t i o x i d a n t e f f e c t of the element involved; and the n u t r i t i o n a l selenium requirement of the r a t i s lower f o r maintenance of hepatic heme u t i l i z a t i o n than f o r main-tenance of hepatic GSH-Px l e v e l s (Burk and C o r r e i a , 1981). Whanger et al_. (1977) reported the f i r s t data on the e f f e c t s of selenium on ovine hepatic microsomal heme p r o t e i n s . Hepatic microsomal cytochrome P ^ Q and t o t a l heme content were s i g n i f i c a n t l y lower i n WMD lambs, but cytochrome b 5 content was not a f f e c t e d . Hepatic m i t o c h o n d r i a l heme p r o t e i n content and l e v e l s of cytochromes a, b, and c + c 1 d i d not d i f f e r between normal and WMD lambs. Thus microsomal but not mi t o c h o n d r i a l heme pro t e i n s were a f f e c t e d i n t h i s case. Whanger et a l . (1977) f e l t that a d e f i c i e n c y of both vitamin E and Se was necessary to a l t e r microsomal heme compounds i n the ovine l i v e r . The r o l e of vitamin E i n the r e g u l a t i o n of heme synthesis has been i n v e s t i g a t e d by Nair et a l . (1972), while a d e t a i l e d study on the e f f e c t of vitamin E d e f i c i e n c y on c e l l u l a r membranes and membrane-bound enzymes has a l s o been reported ( H u l s t a e r t jet ^1_., 1975). The r e l a t i o n s h i p between selenium and vitamin E and heme metabolism i s not yet f u l l y understood. SELENIUM AND HEME SYNTHESIS Erythropoiesis Selenium and/or v i t a m i n E d e f i c i e n c y may a l s o i n t e r f e r e with e r y t h r o p o i e s i s . An i n d i r e c t e f f e c t of selenium i n s t a b i l i z i n g heme enzyme systems may occur, as was observed i n l i v e r (Whanger jet a l . , 1977). Bone marrow abnormalities - erythrocyte hyperplasia and m u l t i n u c l e -ated precursor c e l l s - were found i n Se-E d e f i c i e n t swine (Niyo jst a l . , - 45 -1980). M u l t i n u c l e a t e d erythrocyte precursor c e l l s are a s s o c i a t e d with delayed erythrocyte maturation, which could eventually be manifested by low blood hemoglobin l e v e l s . Hemoglobin g r a d u a l l y decreased i n vitamin E d e f i -c i e n t lambs ( C u l i k et jrL., 1951). Baustad and Nafstad (1972) observed changes i n swine hematology con-s i s t e n t with impairment of hematopoiesis during vitamin E d e f i c i e n c y . The r e t i c u l o c y t e count i n vitamin E-treated p i g l e t s was s i g n i f i c a n t l y higher than i n untreated l i t t e r m a t e s at 2 weeks of age (6.95 vs. 3.26% of erythro-c y t e s ) . T o t a l Hb, PCV, and RBC count were a l s o higher i n vitamin E-treated p i g l e t s . A d d i t i o n a l l y , bone marrow abn o r m a l i t i e s t y p i c a l of those des-c r i b e d by Nafstad (1973) were found i n vitamin E d e f i c i e n t p i g l e t s of any age from newborn to 5 weeks. Fontaine et _ a l . (1977a, 1977b) demonstrated that selenium, but not vitamin E, may have a s p e c i f i c r o l e i n e r y t h r o p o i e s i s . Much work remains to be done i n t h i s area. So f a r , the l i m i t e d information on Se and ery-t h r o p o i e s i s i n sheep i s confusing, as two papers reported that selenium treatment depressed Hb l e v e l s . Buchanan-Smith et c Q . (1969) observed a depression i n PCV, and more sl o w l y i n Hb, i n 4 month old lambs supplemented with selenium compared to s e l e n i u m - d e f i c i e n t c o n t r o l s . Horton et a l . (1978) compared four methods of Se/vitamin E supplementation, f i n d i n g the greatest depression i n red c e l l count and Hb with the most e f f e c t i v e methods of Se supplementation. IRON AND SELENIUM UTILIZATION I r o n status a f f e c t s the concentration of GSH-Px i n red blood c e l l s , i n d i c a t i n g an unanticipated r o l e for i r o n i n selenium metabolism. Studies - 46 -i n humans demonstrate that red blood c e l l GSH-Px i s s i g n i f i c a n t l y decreased i n i r o n d e f i c i e n c y anemia ( C e l l e r i n o ejt a i l . , 1976; Macdougall, 1972). The e f f e c t was not dependent on anemia per se, as GSH-Px was e i t h e r unaffected or increased i n other types of anemia ( C e l l e r i n o et a l . , 1976). Furthermore, GSH-Px was s i g n i f i c a n t l y c o r r e l a t e d with serum i r o n l e v e l s . Red c e l l GSH-Px was als o reduced i n i r o n d e f i c i e n t calves (Horber et a l . , 1980), and i r o n - d e f i c i e n t r a b b i t s (Rodvien j3t a l . , 1974). The decrease i n GSH-Px observed with i r o n d e f i c i e n c y may r e s u l t from an i n a b i l i t y to u t i l i z e d i e t a r y Se f o r GSH-Px synthesis (Ganther jst _ a l . , 1976). However, t h i s theory would not e x p l a i n why GSH-Px a c t i v i t y i n S e - d e f i c i e n t animals might be inf l u e n c e d by high l e v e l s of i r o n . Red blood c e l l GSH-Px l e v e l s may be a f f e c t e d by i r o n when selenium i s d e f i c i e n t . A s i n g l e study was done using r a t s (Lee et a l . , 1981). High l e v e l s of d i e t a r y i r o n f a i l e d to i n f l u e n c e RBC GSH-Px when Se was ade-quate. However, GSH-Px was higher i n Se- and E - d e f i c i e n t r a t s fed 1255 ppm i r o n than those fed 305 ppm i r o n . Unfortunately, the e f f e c t of d e f i c i e n t l e v e l s of i r o n was not considered. - 47 -MATERIALS AND METHODS EXPERIMENTAL DESIGN I n a l l t r i a l s , lambs were born over a 3-6 week lambing period, and randomly a l l o c a t e d at b i r t h to one of the i n j e c t i o n treatments. The number of treatments v a r i e d according to the t r i a l . Iron Supplementation (Trial 1) T r i a l 1 i n v e s t i g a t e d the e f f e c t s of i r o n supplementation using 17 c o n t r o l and 18 i r o n treated lambs. The i r o n dosages were 0 and 500 mg of i r o n , administered w i t h i n 3 days of b i r t h . As breed (Finn or Dorset s i r e d ) , sex and rearing (as s i n g l e or tw i n ) , may markedly i n f l u e n c e weight gains and p o s s i b l y other parameters, these f a c t o r s were included i n the experimental model. Consequently, the t r i a l was set up as a completely randomized 2X2X2X2 design. Weight, hemoglobin and PCV were measured weekly from b i r t h to 11 weeks of age. Plasma samples taken at 24-25 days of age were analyzed f o r a blood p r o f i l e i n c l u d i n g calcium, in o r g a n i c phosphate, glucose, blood urea n i t r o g e n , t o t a l p r o t e i n , albumin, a l k a l i n e phosphatase, l a c t a t e dehydro-genase, aspartate transaminase and plasma i r o n . This p r o f i l e was se l e c t e d to assess the e f f e c t of i r o n treatment and/or anemia on other p h y s i o l o g i c a l parameters. For example, glucose and c h o l e s t e r o l are a f f e c t e d by i r o n d e f i c i e n c y anemia i n humans. The f o l l o w i n g l e a s t squares model was used to analyze a l l the T r i a l 1 data: - 48 -Y i j k l = u + Tj^  + Bj + + Ri + Ti Bj + TjS^ + T^Ri + BjS^ + BjR^ + S kRi + W i j k i + E i j k i . where Yj.jkl = the dependent v a r i a b l e hemoglobin, PCV, e t c . u = the o v e r a l l mean common to a l l samples T^ = the e f f e c t of the i ' t h treatment B j = the e f f e c t of the j ' t h breed = the e f f e c t of the k'th sex R l = the e f f e c t of the l ' t h r e a r i n g T^B-j = the i n t e r a c t i o n of the i ' t h treatment with the j ' t h breed T^S^ = the i n t e r a c t i o n of the i ' t h treatment with the k'th sex T^R| = the i n t e r a c t i o n of the i ' t h treatment with the l ' t h r e a r i n g B-JSJ,. = the i n t e r a c t i o n of the j ' t h breed with the k'th sex BjR i = the i n t e r a c t i o n of the j ' t h breed with the l ' t h r e a r i n g S^Ri = the i n t e r a c t i o n of the k'th sex with the l ' t h r e a r i n g W i j k l = the c o v a r i a b l e b i r t h weight E i j k l = the unexplained r e s i d u a l e r r o r a s s o ciated with each sample The above model was a l t e r e d f o r a n a l y s i s of the plasma p r o f i l e data by the a d d i t i o n of a hemolysis c o v a r i a b l e f o r T r i a l s 1 and 2. Hemolysis was ranked on the scal e of 0 (no hemolysis) to 4 (severe hemolysis) as some hemolysis was unavoidable. - 49 -Level of Iron Supplementation (Trial 3) T r i a l 2 compared the e f f e c t s of 3 l e v e l s of i r o n treatment: 0, 250 mg, and 500 mg i r o n . As before, breed, sex and r e a r i n g were other sources o f v a r i a t i o n , r e s u l t i n g i n a 3X2X2X2 design. S i x t y - s i x lambs were used. The same l e a s t squares model was used as i n T r i a l 1. Iron and/or Selenium Supplementation (Trial 3) I n T r i a l 3, i r o n and selenium treatments were combined i n t o 4 t r e a t -ment combinations. These were c o n t r o l , 1.5 mg Se only, 500 mg Fe only, and 1.5 mg Se + 500 mg Fe. The experiment was r e p l i c a t e d 3 times, with a t o t a l of 121 lambs d i v i d e d between the 3 lambing periods. Hb, PCV, weight and plasma p r o f i l e data were c o l l e c t e d as before. However, plasma selenium and plasma p r o t e i n data were obtained from r e p l i c a t e s 2 and 3 only, and hemag-g l u t i n a t i o n and soremouth data from r e p l i c a t e 3 only. The f o l l o w i n g l i n e a r model was used to measure the e f f e c t s of r e p l i -c a t e , treatment, breed, sex and r e a r i n g on T r i a l 3 Hb, PCV, weight and plasma p r o f i l e data: Y i j k l m = u + Pi + Tj + ^ + S X + + P i T j + PiBk + TjBk + + T j ^ n + + E i j k l m where Y i j k l m = t n e dependent v a r i a b l e Hb, PCV, e t c . u = the o v e r a l l mean common to a l l samples P i = the e f f e c t of the i ' t h r e p l i c a t e T j = the e f f e c t of the j ' t h treatment B k = the e f f e c t of the k'th breed S i = the e f f e c t o the l ' t h sex R m = the e f f e c t of the m'th r e a r i n g - 5 0 -P j j j = the i n t e r a c t i o n of the i ' t h r e p l i c a t e with the j ' t h treatment Pj^B^ = the i n t e r a c t i o n of the i ' t h r e p l i c a t e with the k'th breed T j B ^ = the i n t e r a c t i o n of the j ' t h treatment with the k'th breed T j S | = the i n t e r a c t i o n of the j ' t h treatment with the I ' t h sex T j R m = the i n t e r a c t i o n of the j ' t h treatment with the m'th r e a r i n g E^ k^ m = the i n t e r a c t i o n of the j ' t h breed with the m'th r e a r i n g ^ i j k l m = t n e b i r t h w e i g h t c o v a r i a b l e F-ijklm = the unexplained r e s i d u a l e r r o r a s s o c i a t e d with each sample Plasma pr o t e i n s were analyzed using a s i m p l i f i e d l i n e a r model, which in c l u d e d t o t a l p r o t e i n as a c o v a r i a b l e : Y i j k l m = u + T i + B j + S k + Ri + P 1^1 + E i ; j k l where ^ i j k l = t n e dependent v a r i a b l e albumin, b e t a - g l o b u l i n , e t c . u = the o v e r a l l mean common to a l l samples l " i = the e f f e c t of the i ' t h treatment Bj - the e f f e c t of the j ' t h breed S|< = the e f f e c t of the k'th sex Ri = the e f f e c t of the I'th r e a r i n g F>ijkl = the t o t a l p r o t e i n c o v a r i a b l e F - i j k l = the unexplained r e s i d u a l e r r o r a s s o c i a t e d with each sample Plasma selenium data were analyzed using t h i s model: - 51 -Y i j k l m = u + ^ + Xj + Bk + S x + Rn, + ^ X j + X ^ + E i ( } k l m where v i j k l m = the dependent v a r i a b l e selenium u = the o v e r a l l mean common to a l l samples 1^ = the e f f e c t of the i ' t h i r o n treatment X j = the e f f e c t of the j ' t h selenium treatment B k = the e f f e c t of the k'th breed S i = the e f f e c t of the I'th sex R m = the e f f e c t of the m'th r e a r i n g I^X-j = the i n t e r a c t i o n of the i ' t h i r o n treatment with the j ' t h selenium treatment X j S i = the i n t e r a c t i o n of the j ' t h selenium treatment w i t h the I'th sex F-ijklm = the unexplained r e s i d u a l e r r o r a s s ociated with each sample The model f o r a n a l y s i s of the hemagglutination data was the same, w i t h the a d d i t i o n of the i n t e r a c t i o n s of selenium with r e a r i n g , i r o n with sex, and i r o n with r e a r i n g . STATISTICAL ANALYSIS A n a l y s i s of variance was done using UBC BMD:10V (1975), a General Linear Hypothesis packaged program. A major advantage of t h i s program was i t s a b i l i t y to manipulate unbalanced c e l l s and missing dtaa, although not missing c e l l s . A n a l y s i s of covariance was used instead of ANOVA when a concomitant v a r i a b l e , which could be measured but not c o n t r o l l e d , a f f e c t e d a dependent v a r i a b l e . For example, despite random a l l o c a t i o n of lambs to treatments, mean b i r t h weight tended to be higher f o r some treatments. As - 52 -b i r t h weight i s r e l a t e d to weight gain, i t was very u s e f u l to be able to adjust means f o r the e f f e c t of b i r t h weight on weight. ANOVA with BMD:10V a l s o enabled the t e s t i n g of s i n g l e degrees of freedom c o n t r a s t s . A_ p r i o r i , orthogonal hypotheses were: f o r a l l i r o n l e v e l data, T r i a l 2: 1. Control lambs do not d i f f e r from i r o n treated lambs. 2. High l e v e l of i r o n i n j e c t i o n does not d i f f e r from low l e v e l . s i m i l a r l y , f o r data i n T r i a l 3: 1. Iron t r e a t e d lambs do not d i f f e r from non-iron t r e a t e d lambs. 2. Selenium treated lambs do not d i f f e r from non-selenium t r e a t e d lambs. 3. Iron and selenium do not i n t e r a c t . The BMD:10V program was run f o r a l l sets of data c o l l e c t e d , changing the model as warranted. When i n s i g n i f i c a n t i n t e r a c t i o n s were obtained, the SS 1 and d.f.'s were added i n t o the experimental e r r o r to increase the pre-c i s i o n , then the F's were r e c a l c u l a t e d by the program. Consequently, three-way and four-way i n t e r a c t i o n s were normally e l i m i n a t e d , and many b i o l o g i c a l l y meaningless two-way i n t e r a c t i o n s . The f i r s t model given was adjusted i n t h i s manner, while the remaining models represent the f i n a l versions of the complete i n i t i a l models. This frequently r e s u l t e d i n h i g h l y s i g n i f i c a n t main e f f e c t s . C o r r e l a t i o n s between T r i a l 2 plasma metabolites and plasma i r o n , Hb, PCV, weight gain, and b i r t h weight were i n v e s t i g a t e d using UBC TRP (1978), a t r i a n g u l a r regression package. They were of i n t e r e s t as covariance a n a l y s i s was not appropriate f o r lo o k i n g at c o r r e l a t i o n s , yet the existence - 53 -o f c e r t a i n c o r r e l a t i o n s had a major impact on the data. TRP used a forward stepwise regression technique to derive regression equations. UBC TRP (1978) was a l s o used to do p l o t s . Throughout the t r i a l s , i t was tedious to measure both Hb and PCV, when they might be of equal value i n assessing i r o n d e f i c i e n c y . Regression a n a l y s i s derived p r e d i c t i v e equa-t i o n s f o r Hb from PCV at d i f f e r e n t ages, and was used to assess the c l o s e -ness of the r e l a t i o n s h i p between the two v a r i a b l e s . TRP a l s o p l o t t e d scat-tergrams of Hb versus PCV, with and without a severe l e v e l of o u t l i e r r e j e c t i o n of 5% of the data. Simple regression equations were c a l c u l a t e d for a l l p l o t s . A l l means are given with v a r i a t i o n expressed as the standard e r r o r , and not as the standard d e v i a t i o n . ANIMAL MANAGEMENT Lambs were housed with t h e i r dams i n sawdust-bedded group pens w i t h i n an open-eaved unheated b u i l d i n g on the U n i v e r s i t y of B r i t i s h Columbia research farm. Dams were Dorset and FinnXDorset breeding. Dorset, Finn and S u f f o l k rams were used. Lambs were docked at 3-7 days of age. Males were not c a s t r a t e d . Lambs were weaned at an average age of eight weeks. Water and c o b a l t - i o d i z e d s a l t were a v a i l a b l e ^d l i b . Ewes were fed a l f a l f a cubes and a barley-based g r a i n mixture twice d a i l y . S t a r t i n g at ten days of age, the lambs began eating small amounts of a creep-feed r a t i o n having a c a l c u l a t e d i r o n content of 95 ppm (Appendix 1). By s i x weeks of age, lambs were consuming about 0.5 kg of creepfeed per head per day. - 54 -Health problems were never severe. Contagious p u s t u l a r d e r m a t i t i s (soremouth) was endemic, appearing i n each lamb crop s e v e r a l weeks a f t e r the s t a r t of lambing. I s o l a t e d cases of both J E . c o l i scours and Corynebac- terium o v i s j o i n t abscesses occurred; the l a s t r a r e l y a f f e c t e d growth r a t e . White muscle disease a f f e c t e d some lambs at various ages between b i r t h and 4 weeks of age i n T r i a l s 1 and 2 only. The a f f e c t e d animals u s u a l l y responded to a combined selenium/vitamin E i n j e c t i o n . O v e r a l l m o r t a l i t y was low and var i e d from 0 to 7%; main causes were premature/ d i f f i c u l t b i r t h s , trampling and pneumonia. Lambs were treated with i r o n and/or selenium depending on the e x p e r i -ment. Both i r o n and selenium were administered by intramuscular i n j e c t i o n between 0 and 3 days of age. The products used were Haemalift and Dysto-s e l , both from the Rogar/STB d i v i s i o n of BTI Products, Inc., London, Ontario. Haemalift provided 100 mg a c t u a l i r o n per ml as a f e r r i c hydrox-id e complex with dextran. Dystosel contained 3 mg selenium per ml as sodium s e l e n i t e , and 163 IU d-alpha tocopheryl acetate. ANALYTICAL PROCEDURES Weight Lambs under 15 kg were weighed i n a p a i l with a spring balance (accuracy ± 0.1 kg). When lambs reached 15 kg, a large beam balance with an accuracy of ±0.5 kg was used. Blood Samples A l l samples were obtained by j u g u l a r venipuncture using 20 gauge needles. Five ml tubes containing a small amount of sodium heparin were - 55 -used to c o l l e c t small (2 ml) i n i t i a l samples f o r hemoglobin and packed c e l l volume determinations. Subsequently, 10 ml heparinized vacutainer tubes were used to c o l l e c t blood for Hb, PCV, plasma p r o f i l e , and mineral analy-ses. Vacutainer tubes containing potassium oxalate instead of heparin were used to c o l l e c t samples for plasma p r o t e i n e l e c t r o p h o r e s i s . P l a i n or s i l i c o n - c o a t e d vacutainers were used when serum was required. Hemoglobin Blood hemoglobin l e v e l s were measured by the cyanmethemoglobin tech-nique (Schoen and Solomon, 1962; E i l e r s , 1967). I t i s a c o l o r i m e t r i c tech-nique which measures a l l hemoglobin d e r i v a t i v e s using cyanide reagents. Duplicate analyses were done f o r each sample w i t h i n 2k hours of c o l l e c t i o n ; however, hemoglobin i s s t a b l e f o r over 7 days at 4°C, or a month and more at -20°C. The equipment involved consisted of Vanlab 20 mm3(±1%) dispos-able m i c r o p i p e t t e s , Hycel No. 117 Cyanmethemoglobin C e r t i f i e d Standard, and a G i l f o r d Stasar I I spectrophotometer. Packed Cell Volume D u p l i c a t e microhematocrits were done on each blood sample, g e n e r a l l y on the day of c o l l e c t i o n . Dade and Canlab heparinized microhematocrit c a p i l l a r y tubes were two-thirds f i l l e d with blood, capped with C r i t o s e a l , and c e n t r i f u g e d f o r 15 minutes i n a Canlab I n t e r n a t i o n a l M i c r o c a p i l l a r y C e n t r i f u g e with Reader (Models MB and CR). Plasma Profile Plasma was extracted from 10 ml whole blood samples, frozen and l a t e r analyzed by a commercial laboratory (B.C. Biomedical Laboratories L t d . , - 56 -7845 Edmonds, Burnaby, B.C.). Eleven plasma c o n s t i t u e n t s were measured -calcium (Ca), in o r g a n i c phosphate ( P ^ ) , glucose, blood urea nitrogen (BUN), c h o l e s t e r o l , t o t a l p r o t e i n , albumin, a l k a l i n e phosphatase (AP), l a c t a t e dehydrogenase (LDH), a s p a r t a t e transaminase (AT), and i r o n . References to a n a l y t i c a l procedures f o r each metabolite are given i n Appendix 2. <. Plasma Protein Electrophoresis and Total Protein Plasma p r e p a r a t i o n . Plasma was separated from whole blood c o l l e c t e d i n vacutainer tubes containing potassium o x a l a t e . Heparin i s a un s u i t a b l e a n t i c o a g u l a n t as i t may i n t e r f e r e with various p r o t e i n bands i n e l e c t r o -phoresis. A f t e r thawing, samples were c e n t r i f u g e d and decanted as necessary to remove t u r b i d i t y caused by l i p i d s and denatured p r o t e i n s . Hemolysis was evident i n some samples but was not judged severe enough to n e c e s s i t a t e a n a l y s i s of Hb and use of a c o r r e c t i o n f a c t o r . (The cyanmethemoglobin technique i s not appropriate f o r Hb concentrations l e s s that 4 g/d£.) However, hemolysis could w e l l be a source of err o r i n both t o t a l p r o t e i n (TP) and plasma p r o t e i n e l e c t r o p h o r e s i s . T o t a l p r o t e i n . The b i u r e t method (Go r n a l l ejt a l _ . , 1949; Cannon ejt a 1., 1974) was chosen for t o t a l p r o t e i n determination, as i t produces a stable colour that obeys Beer's Law and i s unaffected by the r a t i o of albumin to g l o b u l i n . P r o t e i n f r a c t i o n s . Plasma p r o t e i n f r a c t i o n s were separated by e l e c t r o p h o r e s i s on prepared agarose gel i n the Corning Cassette E l e c t o r -phoresis C e l l System. The Corning procedure was followed ("Determination - 57 -of serum p r o t e i n s (Amido Black 10B)", Corning Medical, Corning Glass Works, Me d f i e l d , Massachusetts). In order to maximize the r e s o l u t i o n of the pro-t e i n f r a c t i o n s , various b u f f e r s were tested at varying pH's and i o n i c strengths (Cannon et _ a l . , 1974). The optimum combination seemed to be sodium b a r b i t a l b u f f e r , i o n i c strength u=0.05, pH=8.6, i n preference to Corning U n i v e r s a l B a r b i t a l Buffer containing EDTA. The electrophoretograms were scanned i n the Transidyne General 2980 Scanning Densitometer. P r o t e i n f r a c t i o n s were c a l c u l a t e d as percentages of t o t a l p r o t e i n by c a l c u l a t i n g the r e l a t i v e surface area under each peak. A c t u a l values of the 5 p r o t e i n f r a c t i o n s were then c a l c u l a t e d from the percentages using t o t a l p r o t e i n values obtained by the b i u r e t technique. Production of Anti-erythrocyte Serum A s e p t i c c o l l e c t i o n of chicken blood. Laying hens from the U n i v e r s i t y of B r i t i s h Columbia Department of P o u l t r y Science supplied the e r y t h r o c y t e s f o r both antiserum production and t e s t i n g . Using d i f f e r e n t b i r d s each time, ten or more b i r d s were bled three times weekly. Ten mis of blood were withdrawn from a medial vein on the underside of the wing (Garvey et a l . , 1977, p. 31). The equipment included ethanol, cotton gauze, 21 guage needles rinsed with concentrated sodium heparin s o l u t i o n , 12 ml syringes containing 2-3 mis of Alsever's s o l u t i o n plus sodium heparin, and c o l l e c -t i o n f l a s k s c o n taining Alsever's s o l u t i o n . Heparin was necessary to pre-vent c o a g u l a t i o n , e s p e c i a l l y i n the needle and s y r i n g e . A l l equipment and s o l u t i o n s were s t e r i l e . S t a n d a r d i z a t i o n of chicken e r y t h r o c y t e s . Blood samples were combined i n a l a r g e volume of Alsever's s o l u t i o n . C e l l s were washed by mixing with - 58 -s t e r i l e c i t r a t e / s a l i n e s o l u t i o n (Garvey £t a K , 1977, p. 524), c e n t r i f u g e d i n s t e r i l e 40 ml tubes i n a r e f r i g e r a t e d c e n t r i f u g e , then the supernatant was decanted. These steps were repeated 4-6 times to remove plasma pr o t e i n s and l i p i d s . The e r y t h r o c y t e concentration was standardized by adapting a method for the photometric s t a n d a r d i z a t i o n of sheep erythrocytes (Garvey et a l . , 1977, pp. 140-143). Two mis of a 2.5-3.0% c e l l suspension were added to 10 mis of d i s t i l l e d water. The l y s a t e was read against a d i s t i l l e d water blank at a wavelength of 520 nm, and adjusted to give an o p t i c a l d ensity of 0.500. The o r i g i n a l suspension was then adjusted accordingly by the addi-t i o n of c e l l s or b u f f e r s o l u t i o n , r e s u l t i n g i n a 2.5% c e l l suspension co n t a i n i n g approximately 4 X 10 8 c e l l s per ml. Antiserum production. Sheep on a l l four i r o n and selenium treatments i n R e p l i c a t e 3 of T r i a l 3 were i n j e c t e d i n t r a p e r i t o n e a l l y with 1 ml of the fre s h l y - p r e p a r e d c e l l suspension at weekly i n t e r v a l s from 4 to 9 weeks of age. This was done three times weekly at the usual sampling times to minimize age v a r i a b i l i t y w i t h i n the sampling periods. Simultaneously, blood samples were taken f o r serum. As every e f f o r t was made to ensure a s e p t i c c o n d i t i o n s , no d e l e t e r i o u s side e f f e c t s r e s u l t e d from the erythro-cyte i n j e c t i o n s . The sheep blood samples were allowed to c l o t at room temperature, and c e n t r i f u g e d . Serum was decanted and i n i t i a l l y , r e f r i g e r a t e d at 4°C f o r immediate a n t i s e r a testng using a passive hemagglutination method. Because of time r e s t r a i n t s , most samples were frozen and tested 4-5 months l a t e r . The i n i t i a l serum samples were a l s o frozen and r e t e s t e d . - 59 -Hemagglutination Test The passive hemagglutination t e s t was performed on the thawed, heat-treated antiserum samples f o l l o w i n g the method described by Garvey et a l . (1977, pp. 356-360). As the technique s p e c i f i e d t a n n i c a c i d t r e a t e d sheep rbc's coated with bovine serum albumin (BSA) as antigen, and anti-BSA from r a b b i t s , some m o d i f i c a t i o n s were necessary: 1) the antigen was chicken rbc's, the antiserum was sheep a n t i - c h i c k e n - r b c ; 2) the antiserum required at most 1:2 and 1:10 d i l u t i o n , rather than 1:10,000 d i l u t i o n ; 3) erythro-cytes rather than BSA were the antigen, t h e r e f o r e tannic a c i d coating onto rbc's was unnecessary; and 4) c o n t r o l e r y t h r o c y t e s could not be used f o r the same reason as 3 ) . T i t r e s were obtained as d i l u t i o n u n i t s , and con-verted to n a t u r a l log u n i t s f or s t a t i s t i c a l a n a l y s i s . - 60 -RESULTS AND DISCUSSION TRIAL 1 IRON SUPPLEMENTATION Effect of Iron on Hemoglobin, PCV, and Plasma Iron I r o n supplementation of lambs s u b s t a n t i a l l y a f f e c t e d the patter n of hemoglobin and hematocrit changes during the f i r s t 11 weeks of l i f e . The ra p i d d e c l i n e i n hemoglobin and PCV between b i r t h and three weeks of age i n c o n t r o l lambs was prevented by i r o n dextran i n j e c t i o n . The s i n g l e i n j e c -t i o n of 500 mg i r o n at b i r t h s i g n i f i c a n t l y increased Hb, PCV and plasma i r o n from 2 to 11 weeks of age (P<0.05). Although few c o n t r o l lambs became anemic, Hb, PCV, and plasma i r o n values i n d i c a t e d that e r y t h r o p o i e s i s was r e s t r i c t e d by i r o n d e f i c i e n c y i n c o n t r o l lambs. The i n i t i a l blood samples were taken at various times between b i r t h and 3 days of age. Mean Hb was 14.12±0.30 g/d£, which i s s i m i l a r to the mean b i r t h hemoglobin value of 14.17 g/d& reported by Holz et a l . (1961). Mean i n i t i a l values of both Hb and PCV were s l i g h t l y higher f o r the i r o n -t r e a t e d group than the c o n t r o l group, with means of 14.5±0.4 vs. 13.7±0.6 g/d£ Hb, and 40.2±1.2 vs. 37.8±1.5% PCV. This d i f f e r e n c e was not s i g n i f i -cant (P<0.05) due to the high v a r i a b i l i t y of Hb and PCV at t h i s age. Co n t r i b u t i n g f a c t o r s i n c l u d e polycythemia i n some lambs at b i r t h , and a ra p i d drop i n t o t a l red c e l l s during the f i r s t 12 hours of l i f e , followed by a slower decrease i n Hb and PCV (B l u n t , 1975). Consequently, Hb and PCV values change r a p i d l y during the f i r s t three days of l i f e . The Hb l e v e l s of both c o n t r o l and i r o n - t r e a t e d lambs continued t o f a l l u n t i l 1 week of age (12.3±0.5 vs. 12.9±0.2 g/d&), but were not s i g n i -f i c a n t l y d i f f e r e n t (P>0.5). However, at 2 weeks of age the Hb was f u r t h e r - 61 -depressed to 10.9±0.4 g/d£ i n the c o n t r o l group, but had increased to 13.3±0.2 g/d£ i n the i r o n t r e a t e d group (P<0.001). The Hb l e v e l i n the c o n t r o l group f e l l to 10.7±0.4 g/d£ at 3 weeks, then increased slowly to 12.0±0.2 g/d£ at 7 weeks and s t a b i l i z e d at that l e v e l u n t i l the end of the t r i a l at 11 weeks (Appendix 3). In comparison, the Hb l e v e l i n the i r o n - t r e a t e d group increased s t e a d i l y from a low of 12.9±0.2 g/djj, at 1 week to 13.7±0.3 g/djj, at 4 weeks, and remained near that l e v e l u n t i l 7 weeks. As shown i n Figure 1, the Hb l e v e l d eclined s l i g h t l y from 8 to 11 weeks but remained higher than i n the c o n t r o l s . The treatment d i f f e r e n c e was s t i l l s i g n i f i c a n t (P<0.001) at 7 weeks, and remained s i g n i -f i c a n t at 11 weeks (P<0.05). Changes i n hematocrit values i n the two treatment groups p a r a l l e l e d changes i n hemoglobin almost e x a c t l y , as shown i n Figure 2. Treatment e f f e c t was s i g n i f i c a n t at 2 weeks (31.2+1.156 vs. 38.8±0.8% PCV, P<0.001). Subsequently, PCV d e c l i n e d i n the c o n t r o l group to a low of 30.9±1.3% at 3 weeks of age, and increased s t e a d i l y from 3 to 11 weeks, reaching a mean value of 36.0+0.7%. PCV l e v e l s i n the i r o n - t r e a t e d group increased from a low of 35.8±0.8% at 1 week to a high of 39.5±0.7% at 5 weeks. The t r e a t -ment e f f e c t was s i g n i f i c a n t (P<0.001) from 2 to 7 weeks of age, and re-mained s i g n i f i c a n t (P<0.05) at 11 weeks. Data i s given i n Appendix 3. While a c t u a l Hb and PCV values may vary from study to study, the same pattern of both minimum l e v e l s i n c o n t r o l s and maximum response to i r o n a t 3 weeks has been c o n s i s t e n t l y observed (Holz et a l , , 1961; R i c k e t t s et a l . , 1965; U l l r e y et a l . , 1965; T a i t and Dubeski, 1979). U l l r e y et a l . (1965) measured average Hb and PCV values of 6.2 g/d£ Hb and 20.5% PCV i n 3 week ol d c o n t r o l lambs, compared to an i r o n - t r e a t e d group with 10.8 g/djj, Hb and 15.0 A Weeks of Age F i g u r e 1 . E f f e c t o f i r o n s u p p l e m e n t a t i o n on h e m o g l o b i n ( T r i a l 1 ) . 30 I 1 1 ] 2 3 4 5 6 7 8 9 10 11 Weeks of Age Figure 2. E f f e c t of i r o n supplementation on packed c e l l volume ( T r i a l 1). - 64 -33.6% PCV. In another study, the minimum Hb l e v e l averaged 8.37 g/d& i n 3 week old lambs (Holz et a l . , 1961). More r e c e n t l y , Wohlt (1982) found l e v e l s of 23% PCV and 9.4 g/d£ Hb i n 42 14-day o l d lambs that had not been docked. In the present study, the minimum Hb and PCV l e v e l s i n c o n t r o l s were much higher, at 10.7 g/d£ Hb and 30.9% PCV, yet i r o n treatment s t i l l r e s u l t e d i n a dramatic hematological response. Rearing c o n s i s t e n t l y a f f e c t e d Hb and PCV, but the e f f e c t was s i g n i f i -cant only at 2 and 3 weeks of age (P<0.05). Both parameters were s l i g h t l y higher i n s i n g l e s compared to twins at b i r t h (14.25±vs. 13.99 g/d£ Hb, 39.24 vs. 38.86% PCV). By 2 and 3 weeks of age s i n g l e t o n s had s i g n i f i -c a n t l y higher PCV and Hb l e v e l s than twins, w i t h i n each treatment group. This i n d i c a t e s that higher body reserves of i r o n present i n the s i n g l e lamb at b i r t h may s i g n i f i c a n t l y a f f e c t hematology i n e a r l y l i f e . Work with other species provides evidence that twinning leads to reduced storage of i r o n i n each f e t u s . A study of newborn d a i r y calves revealed a s i g n i f i c a n t incidence of severe neonatal anemia with 15.8% of s i n g l e c a l v e s , and 37.5% of twin calves having <20% PCV at b i r t h (Tennant et al^., 1975b). P o s s i b l y blood t r a n s f u s i o n between fetuses r e s u l t e d from unequal f u n c t i o n i n g of the two c i r c u l a t o r y systems, as occurs i n human twins. Twinning, low birthweight and prematurity predispose i r o n - l a c k anemia i n human i n f a n t s (Betke, 1970). The type and degree of transplacen-t a l i r o n t r a n s f e r would determine whether twinning or low birthweight pre-dispose anemia i n sheep. So f a r the l i m i t e d information on the subject i s i n c o n c l u s i v e ( H i d i r o g l o u , 1980; Hoskins and Hansard, 1964). Mean plasma i r o n at 4 weeks was 185 ug/d£. Iron treatment s i g n i f i -c a n t l y (P<0.005) elevated plasma i r o n from a mean of 141±17 yg/d£ i n the - 65 -c o n t r o l s to 223±15 pg/d£ i n the i r o n - t r e a t e d lambs. Breed, sex and r e a r i n g did not a f f e c t plasma i r o n . However, c o n t r o l s i n g l e males had the highest growth r a t e , and tended to have extremely low i r o n l e v e l s . Thus, even though s i n g l e lambs are presumed to have greater t i s s u e i r o n stores at b i r t h , they may be most b e n e f i t t e d by supplementary i r o n due to t h e i r high growth ra t e . Normal l e v e l s of plasma i r o n were reported to be 159 yg/d£ i n 2 week old lambs, and 228 yg/dj;, i n 6 week old lambs ( H i d i r o g l o u & 3enkins, 1971). In older lambs normal values were between 200 and 300 yg/d£, which i s much higher than the normal l e v e l of 115 yg/d£ i n humans (Bothwell et a_l., 1979, p. 297). I t i s not known i f the plasma i r o n threshold f o r i r o n - d e f i c i e n t e r y t h r o p o i e s i s i s the same f o r a l l species. Plasma Profile V a r i a b i l i t y was high for most metabolites. In s p i t e of minimizing age v a r i a t i o n , and accounting f o r breed, sex and r e a r i n g e f f e c t s , the high o v e r a l l v a r i a b i l i t y tended to obscure s u b t l e treatment d i f f e r e n c e s , i f any. A l k a l i n e phosphatase was the only metabolite s i g n i f i c a n t l y a f f e c t e d by i r o n treatment (Table I ) . The a p p l i c a b i l i t y of the plasma p r o f i l e t e s t to n u t r i t i o n a l s tudies has been questioned (Rowlands, 1980). Large numbers of animals per treatment may be required to compensate for high innate v a r i a b i l i t y i n the data. Calcium The mean plasma calcium value at 24-25 days of age was 11.56±0.12 mg/d£, w i t h i n the normal range of 9-12 mg/d£ (Simesen, 1980). S i m i l a r l y , Mitruka and Rawnsley (1977) gave a range of 10.4-14.0 mg/d£ from the - 66 -TABLE I. Effect of iron treatment on plasma profile at 24-25 days (Trial 1) CONTROL IRON S.E.M. n 17 18 Calcium (mg/d£) 11.7 11.4 0.1 Inorganic Phosphate (mg/d£) 10.3 10.0 0.3 Glucose (mg/d£) 95.3 93.9 2.8 Blood Urea Nitrogen (mg/d£) 16.4 17.4 0.8 C h o l e s t e r o l (mg/dji) 142 132 7 Total P r o t e i n (g/djt) 6.04 5.92 0.09 Albumin (g/djj.) 3.28 3.20 0.06 A l k a l i n e Phosphatase (IU/i) 755 a 627 b 30 Lactate Dehydrogenase (IU/£ ) 569 587 31 Aspartate Transaminase (IU/& ) 104.6 113.1 2.8 Plasma Iron (yg/£) 141 a 223 b 13 a-b Denotes s t a t i s t i c a l d i f f e r e n c e s between treatment means i n the same row, P<0.05. - 67 -l i t e r a t u r e , with a mean of 11.4 mg/d£. Plasma calcium and phosphorus have been observed to vary with age i n lambs (Long et a l _ . , 1965; Moodie, 1975) as w e l l as i n calves (Simesen, 1970, p. 320). A comprehensive study of lamb hematology reported 2 week serum calcium l e v e l s of 12.2+0.19 mg/d£, f a l l i n g to 11.5+0.14 mg/d£ at 4 weeks (Long jet a l . , 1965). Iron treatment d i d not s i g n i f i c a n t l y a f f e c t plasma calcium (P>0.05). Calcium values were 11.73±0.15 mg/d£ and 11.41+0.17 mg/dz f o r the c o n t r o l and i r o n - t r e a t e d groups, r e s p e c t i v e l y . Phosphorus The mean plasma i n o r g a n i c phosphate (P^) was 10.14+0.29 mg/d£. Values i n the l i t e r a t u r e show great v a r i a b i l i t y because of age and d i e t a r y e f f e c t s . Long et a l . (1965) obtained very s i m i l a r r e s u l t s to the present study. The mean from 2-4 weeks of age was 11.0 mg/d£ . Weight gain and feed intake are c o r r e l a t e d with P^, r e s u l t i n g i n high v a r i a b i l i t y even among animals fo the same age and d i e t (Moodie, 1975; L i t t l e et a l . , 1977). Plasma P^ i s more s e n s i t i v e than plasma Ca to d i e t a r y f a c t o r s . P i v a r i e s markedly with d i e t a r y phosphorus, and al s o i n t i m a t e l y l i n k e d w i t h carbohydrate metabolism (Simesen, 1970). Iron treatment d i d not s i g -n i f i c a n t l y (P>0.05) a f f e c t P i # Plasma contained 10.33±0.46 mg/d£ P j i n the c o n t r o l group compared to 9.97+0.37 mg/d£ Pj_ i n the i r o n - t r e a t e d group. Glucose Blood glucose l e v e l s i n young ruminants and monogastrics are comparable, but are s i g n i f i c a n t l y lower i n the adult ruminant. Blood - 68 -glucose has been p o s i t i v e l y c o r r e l a t e d with feed intake and weight gain i n calves ( L i t t l e et a l . , 1977) and i n one year o ld sheep (Bensadoun et a l . , 1962). L i t e r a t u r e values of ovine plasma glucose range from 55.0 to 131 mg/djj, (Mitruka and Rawnsley, 1977). Hackett _et al_. (1957) reported a mean of 53.5±2.3 mg/dJL, with no d i f f e r e n c e between ewes and lambs under range c o n d i t i o n s , but lamb age was not s p e c i f i e d . Lindsay and Leat (1975) l i s t e d mean glucose l e v e l s of 103.4 mg/d£ and 96.9 mg/d£ for 17-24 day old and 25-32 day old lambs, r e s p e c t i v e l y . These means are s i m i l a r to the mean i n T r i a l 1 of 94.6+2.8 mg/d£. The blood glucose f a l l s s t e a d i l y with advancing age i n the growing lamb, reaching adult l e v e l s at 6-9 weeks (Reid, 1953). In s p i t e of the metabolic s h i f t i n emphasis from glucose to v o l a t i l e f a t t y a c i d s , the change i n blood glucose does not have a c l o s e r e l a t i o n s h i p with rumen development. Various workers have a t t r i b u t e d the blood glucose changes to a s h i f t i n erythrocyte metabolism, associated with the replacement of f e t a l e r y t h r o c y t e s with adult-type c e l l s (Reid, 1953; Kappy, 1982). Adult erythrocytes d i f f e r i n hemoglobin type, i o n i c composition, glucose meta-bolism and enzyme a c t i v i t y ( Blunt, 1975). Plasma glucose may not then be appropriate f o r assessing energy s t a -t u s i n the young lamb. The response of plasma FFA but not blood glucose to ovine growth hormone supports t h i s viewpoint (Lindsay and Leat, 1975). Rowlands (1980) concluded that i n sheep, u n l i k e i n c a t t l e , "FFA concentra-t i o n s c o r r e l a t e better with energy intake than do glucose concentrations." As expected, plasma glucose was not a f f e c t e d (P>0.05) by i r o n t r e a t -ment (95.3±3.0 mg/d£ vs. 94.9±4.7 mg/d£ i n c o n t r o l and i r o n - t r e a t e d groups). - 69 -T o t a l P r o t e i n , Albumin and BUN Plasma t o t a l p r o t e i n , albumin, and BUN (blood urea nitrogen) are t y p i c a l l y included as i n d i c e s of p r o t e i n metabolism i n the metabolic pro-f i l e . Results are frequently ambiguous as these parameters are not s e n s i -t i v e to small q u a n t i t a t i v e or q u a l i t a t i v e d i e t a r y changes. For example, decreased serum p r o t e i n turnover has been observed to maintain serum l e v e l s f o r animals on a low or z e r o - p r o t e i n d i e t . Serum albumin, t o t a l p r o t e i n , Hb and PCV are found to v a r i a b l y and slowly respond to p r o t e i n d e f i c i e n -c i e s . P r o t e i n intake seems to a f f e c t serum albumin but not g l o b u l i n i n sheep (Rowlands, 1980). In t h i s study, t o t a l p r o t e i n , albumin and BUN were normal. There was no reason to a n t i c i p a t e s i g n i f i c a n t treatment responses. To t a l p r o t e i n values averaged 5.98±0.09 g/d£, with no s i g n i f i c a n t treatment e f f e c t at P>0.05. T y p i c a l mean values i n the l i t e r a t u r e average 5.81±0.54 g/d£ ( K u t t l e r and Marble, 1960) and 5.46 g/d£ ( I r f a n , 1967). T o t a l p r o t e i n increases with age, mainly because of i n c r e a s i n g gamma globu-l i n s , whereas albumin decreases p r o p o r t i o n a t e l y l e s s (Dimopoullos, 1970). Consequently, serum p r o t e i n ranges from 5.70-9.10 g/dj, i n sheep (Mitruka and Rawnsley, 1977), depending on the age of the animal. Mean plasma albumin was 3.24±0.06 g/d£. L i t e r a t u r e values of albumin vary from 2.70-4.55 g/d£ (Mitruka and Rawnsley, 1977). The mean BUN was 16.0±0.8 mg/d£. Controls and i r o n - t r e a t e d lambs d i d not d i f f e r s i g n i f i c a n t l y (P>0.05), with means of 16.4±1.2 vs. 17.4±1.1 mg/d£ . BUN has been reported to range from 15.0 to 36.0 mg/djj, i n normal sheep (Mitruka and Rawnsley, 1977). - 70 -C h o l e s t e r o l Sheep have a low plasma l i p i d c oncentration compared to other rumin-ants, u s u a l l y l e s s than 200 ug/d£. C h o l e s t e r o l e s t e r s are a major compon-ent, and with a s i m i l a r amount of phospholipid, t o t a l from 70-80% of the plasma l i p i d (Nelson, 1969; Lindsay and Leat, 1975). Workers i n Germany have measured serum c h o l e s t e r o l to p r e d i c t several metabolic diseases i n e a r l y l a c t a t i o n p o s s i b l y associated with l i v e r malfunction (Manston and A l l e n , 1981), however i t s use i n ovine n u t r i t i o n s tudies remains to be c l a r i f i e d . Mean plasma c h o l e s t e r o l was 137±7 mg/d£, which i s higher than the mean of 57.8±8 mg/d£ reported by Smith jet al_. (1978) and 64.6±3.3 reported by Hackett et a_l. (1957), but w i t h i n the range of 50.0-140 (Mitruka and Rawnsley, 1977). The high l e v e l of c h o l e s t e r o l i s probably an age e f f e c t , as plasma c h o l e s t e r o l decreases when adult rumen fu n c t i o n develops. A l k a l i n e Phosphatase Iron treatment s i g n i f i c a n t l y (P<0.05) a f f e c t e d plasma a l k a l i n e phos-phatase. Plasma AP a c t i v i t y was much greater i n the c o n t r o l lambs, with a mean of 755±40 IU/jl compared to 627±40 IU/£ i n the i r o n - t r e a t e d lambs. Healy (1975c) mesured mean AP a c t i v i t y of 741±66 IU/a i n 28 lambs at 4 weeks of age. Age, isoenzyme source, blood group, n u t r i t i o n , feed i n t a k e , and r a t e of growth are among the major sources of v a r i a t i o n known to a f f e c t a l k a l i n e phosphatase a c t i v i t y . In the current study, breed, sex, and r e a r i n g were not s i g n i f i c a n t , while blood group was not i s o l a t e d as a source of v a r i a t i o n . - 71 -The range i n AP a c t i v i t y may be maximum at b i r t h . Serum AP a c t i v i t y ranged from 220-8500 Will i n newborn lambs, at which time v i r t u a l l y a l l a c t i v i t y was of s k e l e t a l o r i g i n (Healy, 1975b). AP a c t i v i t y peaks at 2k hours as i n t e s t i n a l AP enters the c i r c u l a t i o n . Serum a l k a l i n e phosphatase a c t i v i t y tends to decrease with i n c r e a s i n g age i n sheep. The isoenzymes a l s o vary with age. At 2 weeks of age, isoenzyme analyses showed that 79% of serum AP a c t i v i t y was s k e l e t a l type, the remainder i n t e s t i n a l . The high AP a c t i v i t y i n lambs and the gradual f a l l with age are considered to r e f l e c t the changing ra t e of o s t e o b l a s t i c a c t i v i t y a s s o ciated with s k e l e t a l development. By maturity, l i v e r and/or i n t e s t i n a l isoenzymes predominate (Healy, 1975a). Many n u t r i t i o n a l f a c t o r s a f f e c t plasma a l k a l i n e phosphatase. When d i e t s varying i n the r a t i o of wheat to a l f a l f a were fed at maintenance l e v e l s , both t o t a l AP a c t i v i t y and the proportion of serum heat r e s i s t a n t AP ( i n t e s t i n a l isoenzyme) were a f f e c t e d by d i e t and blood group (Healy and Davis, 1975). Healy and Mclnnes (1975) a l s o observed that d i e t a r y i n t a k e s i n f l u e n c e d serum AP a c t i v i t y i n lambs fed to gain at d i f f e r e n t r a t e s on the same d i e t , and i n s p i t e of the absence of isoenzyme s t u d i e s , concluded that the AP response r e f l e c t e d the i n f l u e n c e of the d i e t a r y i n t a k e on s k e l e t a l development. Serum AP i s reduced i n z i n c d e f i c i e n c y i n pigs (Furugouri, 1972) and calves ( M i l l e r _et a l . , 1965) and could be u s e f u l i n the diagnosis of z i n c d e f i c i e n c y i n ruminants (Blackmon et a l . , 1967). A l k a l i n e phospha-tase a l s o responds q u i c k l y to changes i n d i e t a r y phosphorus i n c a l v e s , with a c t i v i t y varying i n v e r s e l y to serum P i (Wise jet ^1_., 1958). The r e l a -t i o n s h i p between i r o n d e f i c i e n c y and plasma AP has not been i n v e s t i g a t e d . - 72 -L a c t a t e Dehydrogenase and Aspartate Transaminase Plasma l a c t a t e dehydrogenase (LDH) and aspartate transaminase (AT .or GOT) are used to diagnose selenium and vitamin E d e f i c i e n c y diseases. Mean plasma l e v e l s of l a c t a t e dehydrogenase (LDH) and aspartate transaminase (AT or SGOT) were 578±31 IU/SL and 108.9±2.8 IU/£, respec-t i v e l y . Smith et a l . (1978) reported a mean of 311±55 IU/£, of LDH i n ad u l t ewes, and 71±26 IU/n of AT. Data from Horton _et a l . (1978) were s i m i l a r , with lamb serum containing higher amounts of LDH and AT. Mean LDH and AT values were 643 IU/£ and 35 IU/£ i n the lambs i n j e c t e d with vitamin E and selenium, and 869 IU/jj, LDH and 176 IU/x, AT i n the c o n t r o l lambs (Horton et a l . , 1978). The lambs i n the current study d i d not have AT and LDH l e v e l s i n d i c a t i v e of selenium and/or vitamin E d e f i c i e n c y . Iron treatment did not a l t e r a c t i v i t y of e i t h e r enzyme. WEIGHT Weight data are given i n Appendix 3. At the time of i n i t i a l t r e a t -ment, i r o n - t r e a t e d lambs averaged 4.2±0.3 kg compared to 3.9±0.2 kg i n the c o n t r o l group, but the d i f f e r e n c e was not s i g n i f i c a n t . Between 2 days and 1 week of age the i r o n - t r e a t e d group gained almost twice as f a s t as the c o n t r o l group (1.1 kg vs. 0.6 kg). The weight d i f f e r e n c e continued to increase between the i r o n - t r e a t e d and c o n t r o l groups u n t i l 7 weeks, r e s u l t -ing i n a s i g n i f i c a n t (P>0.05) i r o n treatment e f f e c t from 6 weeks to 9 weeks of age. Rearing s i g n i f i c a n t l y (P<0.01) a f f e c t e d lamb weight throughout the study. At 2 days, s i n g l e lambs weighed 4.5 kg and twins, 3.6 kg. At 11 weeks, s i n g l e s weighed 56.5 kg and twins, 47.3 kg. Dorsets and Finns - 73 -weighed 4.0 kg and 4.1 kg at b i r t h , and 23.6 and 23.4 kg at 11 weeks. A s i g n i f i c a n t sex X re a r i n g i n t e r a c t i o n was observed from 2 days to 11 weeks (P<0.05). Male s i n g l e s were on average heavier than female s i n g l e s , but male twins were l i g h t e r than female twins. TRIAL 2 LEVEL OF IRON SUPPLEMENTATION Effect of Iron Level on Hemoglobin, PCV, and Plasma Iron Mean Hb and PCV values did not d i f f e r i n the three groups of lambs at 2 days of age, j u s t p r i o r to treatment. At 16, 30 and 44 days of age d i f f e r e n c e s between the c o n t r o l and i r o n - t r e a t e d groups were s i g n i f i c a n t (P<0.001) for both Hb and PCV. Hb and PCV were higher (P<0.05) for those lambs which received 500 mg of i r o n compared to those which received 250 mg, at 30 and 44 days of age. In the c o n t r o l group, Hb f e l l from 13.6 g/d£ at 2 days to a low of 10.3 g/d£ at 30 days, and then increased to 11.8 g/d£ at 44 days (Figure 3). S i m i l a r l y , PCV decreased from 38.0% at 2 days to 29.2% at 30 days, and reached 34.0% at 44 days (Figure 4 ) . In co n t r a s t , mean Hb and PCV values f o r both i r o n - i n j e c t e d groups increased from 2 days to 30 days, then decreased s l i g h t l y . (Data are given i n Appendix 4.) The maximum d i f f e r e n c e s between the c o n t r o l and high i r o n (500 mg) groups occurred at 30 days i n t h i s study. At t h i s time, mean Hb and PCV values were 40.7% and 40.1% higher r e s p e c t i v e l y i n the high i r o n group. Results i n T r i a l 1 and those reported i n the l i t e r a t u r e i n d i c a t e that minimum Hb and PCV values are reached at 3 weeks i n s u c k l i n g lambs. PCV and Hb probably continued to decrease to 3 weeks, and then increased, i n s t e a d of plateauing from 16 to 30 days of age as shown i n Figures 3 and 15.0i 14.01 13.01 12.0J 1 1 .04 10.0-1 I , , , 1 r r 1 2 3 4 5 6 Weeks of Age Figure 3. E f f e c t of i r o n l e v e l on hemoglobin ( T r i a l 2). Weeks of Age Figure 4. E f f e c t of i r o n l e v e l on packed c e l l volume ( T r i a l 3 ) . - 76 -4. Minimum l e v e l s of Hb and PCV could w e l l have been even lower at 3 weeks than observed at the 2 and 4 week sampling periods. B i r t h weight was used as a c o v a r i a b l e i n the s t a t i s t i c a l a n a l y s i s . The s i g n i f i c a n t e f f e c t of b i r t h weight on Hb at 16 and 44 days, and on PCV at 16 and 30 days, i n d i c a t e s that b i r t h weight i s r e l a t e d to i r o n s t o r e s . This appears to be confirmed by the r e l a t i o n s h i p between b i r t h weight and plasma i r o n . Plasma i r o n increased with i r o n dosage. Treatment means at 30 days of age were 132+22, 204±18, and 230±14 yg/d£ f o r the c o n t r o l , low and high i r o n groups. Orthogonal c o n t r a s t s comparing the c o n t r o l vs. i r o n groups, and the low vs. high i r o n l e v e l s , were both s i g n i f i c a n t at P<0.05. Dorsets had s i g n i f i c a n t l y lower plasma i r o n l e v e l s than Finn c r o s s lambs (P<0.05, 171±12 vs. 224±18 yg/d£), however more of the Finn cross lambs were e i t h e r twins or t r i p l e t s and thus t h e i r slower growth rate could have co n t r i b u t e d to t h i s e f f e c t . The s i g n i f i c a n c e of b i r t h weight as a c o v a r i a b l e (P<0.05) suggests th a t lamb b i r t h weight a f f e c t s i r o n s t o r e s , as occurs i n humans (Betke, 1970). Even though plasma i r o n was measured at 30-31 days of age, b i r t h weight s t i l l had a major i n f l u e n c e on plasma i r o n , according to the regres-sion a n a l y s i s . S i g n i f i c a n t p a r t i a l c o r r e l a t i o n c o e f f i c i e n t s with plasma i r o n (P<0.05) were 0.3289 f o r b i r t h weight, 0.4839 for hemoglobin, and 0.4772 f o r PCV (Appendix 5). Thirteen out of 65 lambs had plasma i r o n l e v e l s lower than 100 yg/d£ at 30-31 days of age, i n s p i t e of the f a c t that i r o n status i s expected to improve from 21 days as lambs s t a r t to eat creep feed. The 13 lambs included 10 out of 20 c o n t r o l lambs, and 3 out of 23 lambs i n j e c t e d w i t h - 77 -250 mg of i r o n at b i r t h . Thus i f the purpose of i r o n i n j e c t i o n i s to main-t a i n plasma i r o n l e v e l s above 100 ug/d£ from b i r t h to 4 weeks, a dosage of between 250 and 500 mg i r o n i s warranted. I t was assumed that 100 pg/d£ plasma i r o n i s at or above the threshold f o r u n r e s t r i c t e d e r y t h r o p o i e s i s i n lambs, as i n humans. However, the optimum plasma i r o n l e v e l i n sheep i s unknown. As p r e v i o u s l y mentioned, normal l e v e l s of 8 week old lambs are higher than 200 ug/d£ ( H i d i r o g l o u and Jenkins, 1971) and that l e v e l may be optimum. Hemoglobin and PCV are more commonly used to assess anemia i n farm animals than i s plasma i r o n . Schalm et j a l . (1975) have defined anemia as being c h a r a c t e r i z e d by a 20% reduction i n e i t h e r PCV or hemoglobin. The data from t h i s study were assessed on t h i s basis assuming normal values of 35.0% f o r PCV and 11.5 g/d£ f o r hemoglobin (Schalm et _ a l . , 1975). At 4 weeks of age 42% of the c o n t r o l lambs could be considered anemic w i t h PCV values below 28%. On the basis of hemoglobin, 21% of the c o n t r o l lambs could be considered anemic with l e v e l s below 9.2 g/d£. The d i f f e r e n c e between Hb and PCV for assessing anemia was caused by the a r b i t r a r y l e v e l s set for "normal" Hb and PCV values, and not by the a c t u a l techniques. Apparently, the normal l e v e l of 11.5 g/d£ Hb was too low f o r the UBC f l o c k , based on average values for 8 week old lambs (12.4 g/d£ Hb and 36.4% PCV). Plasma Profile Plasma p r o f i l e data were analyzed by r e g r e s s i o n as w e l l as covariance techniques. The e f f e c t s of weight gain and birthweight on the metabolites were of major i n t e r e s t , but both v a r i a b l e s could not be included i n the covariance model, due to confounding. Consequently, regression a n a l y s i s was used separately to i n v e s t i g a t e c o r r e l a t i o n s between metabolites and - 78 -b i r t h weight, plasma i r o n , hemoglobin, PCV and most impo r t a n t l y , average d a i l y gain to 30 days. Many plasma c o n s t i t u e n t s may be a f f e c t e d by growth r a t e . Glucose, BUN, P^, g l o b u l i n , albumin and serum i r o n have been c o r r e l a t e d with plane o f n u t r i t i o n and growth rat e i n c a t t l e (Kitchenham et^ , 1975; L i t t l e et a l . , 1977; Kitchenham et a_l., 1977). A l s o , plasma AP i s p o s i t i v e l y corre-l a t e d with growth rate i n lambs (Healy and Mclnnes, 1975). In the current study, glucose was p o s i t i v e l y c o r r e l a t e d with weight gain, whereas choles-t e r o l and AT were neg a t i v e l y c o r r e l a t e d with weight gain (P<0.05). Results of regression a n a l y s i s are given i n Appendix 5 and 6. Covariance a n a l y s i s of the plasma p r o f i l e data i n d i c a t e d that i r o n treatment had a s i g n i f i c a n t e f f e c t (P<0.001) on ten out of eleven plasma c o n s t i t u e n t s measured (Table I I ) . The s i g n i f i c a n c e of T r i a l 2 compared to T r i a l 1 r e s u l t s may be r e l a t e d to the higher o v e r a l l growth r a t e , espe-c i a l l y c o n s i d e r i n g the higher incidence of twins; sampling at 30 ins t e a d of 24 days of age; much l a r g e r number of experimental u n i t s per treatment; use of orthogonal c o n t r a s t s f or means separation; and v i r t u a l absence of hemo-l y s i s i n blood samples. Calcium Plasma calcium data were not a v a i l a b l e . The vacutainer tubes were apparently contaminated with disodium EDTA, or m i s l a b e l l e d as co n t a i n i n g sodium heparin as ant i c o a g u l a n t . Phosphorus The response of plasma P^ to i r o n i n j e c t i o n was small but s i g n i f i -cant (P<0.001). Means were 8.9±0.2, 8.7±0.3, and 8.4+0.1 mg/da f o r the TABLE II. Effect of 3 levels of iron treatment on plasma profile 1 at 30-31 days of age (Trial 2) TREATMENTS 0 mg Fe 250 mg Fe 500 mg Fe X" ± SE 1 n 20 22 24 Inorganic Phosphate (mg/d£) 8.9 + 0.2 8.7 + 0.3 8.4 + 0.1 C1***, c2* Glucose (mg/dx.) 104.9 + 2.5 101.3 + 2.6 92.9 + 2.0 p *** p *** c l ' ^2 BUN (mg/di) 17.0 + 0.8 15.6 + 0.8 18.4 + 0.7 p *** L 2 C h o l e s t e r o l (mg/d£) 96.1 + 4.5 108.5 + 3.1 123.5 + 5.8 p *•** p * n ' L 2 T o t a l P r o t e i n (g/d£ ) 5.57 + 0.06 5.49 + 0.15 5.20 + 0.06 p •***• p *** Albumin (g/d£) 2.91 + 0.06 2.86 + 0.08 2.90 + 0.06 P *#* p * > ^2 A l k a l i n e Phosphatase (lU/l) 1098 + 116 881 + 84 725 + 43 p •**# p * L l » L 2 L a c t a t e Dehydrogenase (IU/z) 570 + 34 608 + 39 563 + 30 p *•** Aspartate Transaminase (IU/£ ) 146 + 38 116 + 6 111 + 6 p * p * Plasma Fe ( u g/d£ ) 132 + 22 , 204 + 18 230 + 14 p p * n » *-2 Calcium data not a v a i l a b l e due to manufacturer 's contamination of vacutainer tubes with disodium EDTA. Predetermined orthogonal c o n t r a s t s were as fo l l o w s : C1 = Control vs. both l e v e l s of i r o n treatment; setter ^ o ? 8 i r e ^ o M ( 5 o ° m g F e ) i e v e l s ; Significance of Contrasts 2 - 80 -c o n t r o l , low, and high i r o n groups. tends to decrease during i n -creased carbohydrate u t i l i z a t o n (Simesen, 1970, p. 319). This would i n d i -cate more e f f i c i e n t carbohydrate u t i l i z a t i o n i n the ir o n - t e a t e d groups, probably through increased synthesis of iron-dependent enzymes. While plasma inorganic phosphate l e v e l s are " i n t i m a t e l y r e l a t e d to the i n t e r -mediary metabolism of glucose", these changes are s a i d to occur indepen-dently of blood glucose (Latner, 1975, p. 46). Regression a n a l y s i s con-firmed that plasma was not s i g n i f i c a n t l y c o r r e l a t e d with blood g l u -cose, nor with weight gain, Hb, PCV or plasma i r o n , i n s p i t e of the s i g n i -f i c a n t response to i r o n treatment. Glucose Plasma glucose means were 104.9±2.5, 101.3±2.6, and 92.9+2.0 mg/d£ for the c o n t r o l , low and high i r o n treatments. The d i f f e r e n c e between the c o n t r o l and both i r o n treatments, and the low and high l e v e l s of i r o n treatment, were both s i g n i f i c a n t (P<0.001). There may be two aspects to the r e l a t i o n s h i p between i r o n and glucose. As p r e v i o u s l y discussed i n T r i a l 1, plasma glucose changes appear to be r e l a t e d to the s h i f t i n erythrocyte metabolism i n young lambs. Iron supplementation would enhance the s h i f t by a c c e l e r a t i n g the production of ad u l t types of red c e l l s . This hypothesis i s supported by the s i g n i f i c a n t c o r r e l a t i o n (P<0.05) of Hb, PCV, and plasma i r o n with plasma glucose at 4 weeks of age, with p a r t i a l c o r r e l a t i o n c o e f f i c i e n t s of -0.3055, -0.2533, and -0.3981 (Appendix 5 ) . A l t e r n a t i v e l y , i r o n supplementation may prevent a s l i g h t e l e v a t i o n of blood glucose associated with anemia. In anemia, the - 81 -t r a n s f e r of glucose from blood to t i s s u e s i s retarded. However, no d i s t u r -bance of t i s s u e glucose u t i l i z a t i o n i s involved (Latner, 1975, p. 65). Knight jet _ a l . (1983) observed a s i g n i f i c a n t depression i n f a s t i n g serum glucose f or i r o n - i n j e c t e d p i g l e t s compared to c o n t r o l s . Breed and re a r i n g were a l s o s i g n i f i c a n t sources of v a r i a t i o n (P<0.05) for plasma glucose. The p o s i t i v e r e l a t i o n s h i p between intake and growth ra t e with plasma glucose e x p l a i n s the higher glucose l e v e l s i n s i n g l e com-pared to twin lambs (104.7 vs. 98.1 mg/d£). Means were 103.2 mg/d£ f o r the Dorsets and 91.6 mg/dj, f o r the Finn cross lambs. T o t a l P r o t e i n T o t a l plasma p r o t e i n was s i g n i f i c a n t l y depressed with i n c r e a s i n g i r o n dosage. Mean t o t a l p r o t e i n values were 5.57±0.06, 5.49±0.15, and 5.20±0.06 g/d£ for dosages of 0, 250 and 500 mg or i r o n . The d i f f e r e n c e between c o n t r o l and i r o n - t r e a t e d groups, and between the low and high i r o n groups, were both s i g n i f i c a n t (P<0.001). Regression a n a l y s i s confirmed a strong negative c o r r e l a t i o n of TP with plasma i r o n , and l e s s so with hemoglobin. P a r t i a l c o r r e l a t i o n c o e f f i c i e n t s were -0.4243 and -0.3276 (P<0.001 and P<0.01). Iron may i n d i r e c t l y a f f e c t plasma t o t a l p r o t e i n s by enhancing the synthesis of hemoglobin. Albumin Albumin was s i g n i f i c a n t l y depressed (P<0.001) by i r o n treatment. Mean values were 2.91±0.06, 2.86±0.08, and 2.90±0.06 g/d£ for the dosage l e v e l s of 0, 250 and 500 mg i r o n . As there was no l i n e a r trend with t r e a t -ment, these r e s u l t s may not be meaningful, although one would expect a - 82 -depression i n albumin s i m i l a r to that i n t o t a l p r o t e i n . However, albumin was not s i g n i f i c a n t l y c o r r e l a t e d with plasma i r o n , nor with t o t a l p r o t e i n . BUN BUN was s i g n i f i c a n t l y a f f e c t e d by i r o n treatment (P<0.001), but be-cause of the major i n f l u e n c e of b i r t h weight, the trend was not l i n e a r . Means were 17.0±0.08, 15.6±0.8, and 18.4±0.7 mg/d£. BUN was hi g h l y corre-l a t e d with b i r t h weight, with a p a r t i a l c o r r e l a t i o n c o e f f i c i e n t of 0.4595 (P<0.001); BUN was a l s o p o s i t i v e l y c o r r e l a t e d with plasma i r o n , with a p a r t i a l c o r r e l a t o n c o e f f i c i e n t of 0.2651 (P<0.05). C h o l e s t e r o l C h o l e s t e r o l was s i g n i f i c a n t l y increased by i r o n treatment (P<0.001). Means were 96.1±4.5, 108.5±3.1, and 123.5±5.8 mg/d£. The response to i r o n may be explained by the p o s i t i v e c o r r e l a t i o n of plasma c h o l e s t e r o l with packed c e l l volume. P r a c t i c a l l y a l l human p a t i e n t s with severe hypochromic anemia demonstrate hypocholesterolemia. The r i s e i n plasma c h o l e s t e r o l upon i r o n treatment i s proportionate to the r i s e i n r e t i c u l o c y t e concentra-t i o n . The b a s i s of the response i s not c l e a r . The reduced c h o l e s t e r o l i n anemia i s associated with other changes i n plasma l i p i d s , i n c l u d i n g reduced phospholipids (Latner, 1975, p. 142). C h o l e s t e r o l was lower i n s i n g l e than twin lambs (P<0.01; 99.0 v s . 113.4 mg/djj,). Regression a n a l y s i s confirmed the negative c o r r e l a t i o n of c h o l e s t e r o l with weight gain (Appendix 5 ) . C h o l e s t e r o l was al s o lower i n Dorset lambs than i n Finn cross lambs (P<0.05, 105.9 vs. 119.5 mg/d£). The breed d i f f e r e n c e may be gen e t i c , r e f l e c t i n g the d i f f e r e n c e s i n l i p i d meta-bolism between the Finn breed and the various B r i t i s h breeds (Boylan e_t a l . , 1976). - 83 -A l k a l i n e Phosphatase A l k a l i n e phosphatase was s e n s i t i v e to se v e r a l of the f a c t o r s t e s t e d . Breed a f f e c t e d AP (P<0.05). The 45 Dorset lambs had a mean plasma AP a c t i v i t y of 987 IU/£, compared to a mean of 680 IU/£ i n the 20 Finn cross lambs. I r o n treatment and b i r t h weight were a l s o major sources of v a r i a -t i o n . Regression a n a l y s i s demonstrated a s i g n i f i c a n t negative c o r r e l a t i o n (P<0.0005) of AP with b i r t h weight, plasma i r o n , Hb, and PCV. B i r t h weight and hemoglobin were the most important regression c o e f f i c i e n t s (Appendix 6 ) . The response of plasma AP to i r o n supplementation cannot be r e a d i l y explained. Means were 1098, 881, and 725 IU/£ f o r c o n t r o l , low and high i r o n lambs. C l i n i c a l l y , an increase i n plasma AP r e f l e c t s o s t e o b l a s t i c p r o l i f e r a t i o n or increased a c t i v i t y , u s u a l l y i m p l i c a t i n g inadequate miner-a l i z a t i o n . This occurs i n r i c k e t s and osteomalacia, for example (Latner, 1975, p. 562). P o s s i b l y i r o n d e f i c i e n c y a l s o a f f e c t s plasma AP through a disturbance i n bone metabolism. Iron d e f i c i e n c y i s known to cause s k e l e t a l a b n o r m a l i t i e s i n both man and experimental animals, proportionate to the s e v e r i t y of the d e f i c i e n c y (Beutler and Fairbanks, 1980). More commonly, i r o n d e f i c i e n c y i n c h i l d r e n i s f r e q u e n t l y associated with temporary episodes of high blood a l k a l i n e phosphatase, presumably caused by a tempor-ary disturbance of bone metabolism. Isoenzyme analyses of plasma AP are necessary to determine the source of the increased AP a c t i v i t y of i r o n d e f i c i e n c y - whether he p a t i c , skele-t a l , or i n t e s t i n a l . I f s k e l e t a l , i t i s important to d i s t i n g u i s h between o s t e o b l a s t and marrow sources. AP i s known to be p r i m a r i l y l o c a l i z e d i n the o s t e o b l a s t , p a r t i c u l a r l y i n growng bone and c a r t i l a g e (McComb et. a l . , - 84 -1979, p. 579), but vascular and r e t i c u l o e n d o t h e l i a l sources, i n c l u d i n g marrow, are thought to make a s u b s t a n t i a l c o n t r i b u t i o n to plasma AP l e v e l s (Wolfe, 1970). One can speculate that increased AP a c t i v i t y i n marrow may accompany the e r y t h r o i d hyperplasia of anemia. Lactate Dehydrogenase and Aspartate Transaminase Lactate dehydrogenase and aspartate transminase values were w i t h i n the normal range as i n T r i a l 1. Grand means were 580±2.1 IU/z f o r LDH, and 123+12 IU/£ f o r AT. For both enzymes, the orthogonal c o n t r a s t comparing the c o n t r o l treatment with the combined i r o n - i n j e c t e d groups, was s i g n i f i -cant (P<0.00001), yet there was no d i f f e r e n c e between low and high i r o n treatments (P>0.05). LDH means by treatment were 570±34, 608+39, and 563±30 IU/£ f o r the c o n t r o l , 250 mg, and 500 mg i r o n groups. The data were extremely v a r i a b l e , ranging from a low of 339 IU/z i n a c o n t r o l lamb with 48 pg/d£ plasma i r o n , to highs of 1110 and 1020 IU/jj, i n two lambs from the low i r o n group, which had plasma i r o n l e v e l s of 196 and 221 yg/d£. LDH was not s i g n i f i c a n t l y c o r r e l a t e d with weight gain, Hb or plasma i r o n using l i n e a r regression a n a l y s i s . A l l i n a l l , a true response of LDH to i r o n treatment was not evident. Aspartate transaminase a c t i v i t i e s v aried markedly w i t h i n treatments, apparently because of a s i g n i f i c a n t c o r r e l a t i o n with weight gain. Means f o r the c o n t r o l , low and high i r o n treatment groups were 146+38, 116±6, and 111±6 IU/il AT. In s p i t e of the apparent trend, i t could be erroneous to conclude that i r o n reduced plasma AT. For instance, the maximum AT a c t i v -i t y was 226 , i n a high i r o n lamb with 245 yg/d£ plasma i r o n . The minimum a c t i v i t y was 78 IU/£, i n a c o n t r o l lamb with 31 pg/d£ plasma i r o n . - 85 -M u l t i p l e l i n e a r r egression i d e n t i f i e d weight gain and plasma i r o n as the major regression c o e f f i c i e n t s . Plasma i r o n was p o s i t i v e l y r e l a t e d to AT, whereas weight gain was ne g a t i v e l y r e l a t e d to AT a c t i v i t y (Appendices 5 and 6 ) . These r e s u l t s demonstrate the importance of accounting f o r unfixed f a c t o r s such as i n d i v i d u a l growth rate which a f f e c t plasma parameters. WEIGHT Age i n days, and a c t u a l b i r t h weight, were c o v a r i a b l e s used i n analyzing weekly weight data from 2 days to 51 days. Exact age and b i r t h weight were s i g n i f i c a n t c o v a r i a b l e s (P<0.0001) at time of treatment between 0-3 days of age. B i r t h weight s t i l l had a s i g n i f i c a n t e f f e c t (P<0.001) at the f i n a l sampling at 7 weeks of age. I n i t i a l weight and growth rate to 4 weeks were both higher i n T r i a l 2, than i n T r i a l 1, i n s p i t e of the higher percentage of twin lambs (79% and 54% twins, r e s p e c t i v e l y ) . Rearing was the only s i g n i f i c a n t main e f f e c t . S i n g l e lambs were heavier (P<0.05) than twin lambs at 1 week, (8.0 kg compared to 6.1 kg). The d i f f e r e n c e was s i g n i f i c a n t (P<0.0005) at 30 days, at which time s i n g l e s weighed 15.3 kg compared to 11.2 kg f o r twins. The increased milk i n t a k e of s i n g l e lambs during the f i r s t month of age accounts for t h i s e f f e c t ; a f t e r 30 days, s o l i d food consumption increases markedly and twin weight gain begins to catch up. Iron treatment had no e f f e c t on lamb weight (Appendix 7 ) . Average weights for the c o n t r o l and high i r o n groups of lambs were i d e n t i c a l from i n i t i a l to f i n a l samplings. The low-iron group was only 1 kg heavier than the c o n t r o l group at 51 days. At no time were d i f f e r e n c e s s i g n i f i c a n t . - 86 -A weight response to i r o n supplementation was observed i n T r i a l 1 and, as w i l l be shown l a t e r , a l s o i n T r i a l 3. The reason for the lack of response i n growth ra t e i n t h i s t r i a l cannot be r e a d i l y explained. TRIAL 3 IRON AND/OR SELENIUM SUPPLEMENTATION Effect of Treatment on Hemoglobin and PCV Combined data from the three r e p l i c a t e d experiments i n T r i a l 3 were analyzed by a n a l y s i s of variance (Appendices 8 and 9 ) . The four treatments were c o n t r o l , selenium, i r o n and i r o n plus selenium. M u l t i p l e range t e s t s and orthogonal c o n t r a s t s were used f o r means separation. The c o n t r a s t s tes t e d were i r o n e f f e c t , selenium e f f e c t , and i n t e r a c t i o n of i r o n and selenium. They were necessary as i r o n and selenium were not considered as separate f a c t o r s i n the design, but were combined together under treatment. Figures 5 and 6 i l l u s t r a t e the e f f e c t s of treatments on Hb and PCV f o r lambs from 2 days to 8 weeks of age. Hemoglobin and PCV decreased from 2 days to 3 weeks of age i n c o n t r o l and selenium treated lambs. The values then increased slowly from 3 to 8 weeks of age. In lambs tr e a t e d with i r o n or i r o n plus selenium, the Hb and PCV values declined from 2 days to 1 week, then increased reaching a plateau at 3 weeks of age. As i n the previous t r i a l s , the maximum d i f f e r e n c e between the i r o n t r e a t e d lambs and those not trea t e d with i r o n occurred at 3 weeks of age. Hb was 11.3% higher at 1 week, 24.5% higher at 2 weeks, 25.6% higher at 3 weeks, and 20.5% higher at 9 weeks i n i r o n - t r e a t e d lambs. Mean Hb l e v e l s at 3 weeks were 10.9±0.2, 10.6±0.3, 13.6±0.2 and 13.4±0.2 g/d£ f o r c o n t r o l , selenium, i r o n and i r o n plus selenium t r e a t e d lambs r e s p e c t i v e l y . Corres-ponding PCV values were 31.2+0.4, 31.9±0.4, 41.4±0.3, and 39.2±0.4%. 1 I 1 I f 1 I I • 1 2 3 4 5 6 7 8 Weeks of Age F i g u r e 5. E f f e c t o f i r o n a n d s e l e n i u m s u p p l e m e n t a t i o n o n h e m o g l o b i n ( T r i a l 3 ) . Figure 6. E f f e c t of i r o n and selenium supplementation on blood packed c e l l volume ( T r i a l 3 ). - 89 -On the basis of Hb and PCV response to i r o n treatment, v i r t u a l l y a l l c o n t r o l and selenium lambs were d e f i c i e n t i n i r o n , and a s i g n i f i c a n t number could be classed as anemic. As p r e v i o u s l y , the c r i t e r i a of <9.2 g/d& Hb or <28% PCV were used to assess the proportion of c o n t r o l lambs anemic at any time. Using t h i s a r b i t r a r y Hb l e v e l , 22% of c o n t r o l s were anemic at 3 weeks, and 1196 at 4 weeks. S i m i l a r l y , using the l e v e l of <28% PCV as the d e f i n i t i o n of anemia, 25% of c o n t r o l lambs were anemic at 2 weeks, 23% at 3 weeks, and 17% at 4 weeks. I r o n - i n j e c t e d lambs were never anemic. Selenium d i d not a f f e c t Hb or PCV at any time between 2 days and 8 weeks, nor was an i n t e r a c t i o n with i r o n evident (P<0.05). The data there-fore did not support the r e s u l t s of Horton et al_. (1978) and Buchanan-Smith et a l . (1969), who found selenium supplementation depressed Hb l e v e l s . The use of reg r e s s i o n a n a l y s i s of t h e i r data would have elimin a t e d a growth response to Se as the reason f or the Hb depression. The current study a l s o f a i l e d to Confirm the e r y t h r o p o i e t i c response to selenium observed by Niyo et a l . (1980) and by Fontaine et aJL (1977a, 1977b). The absence of a hematological response to selenium i s not conclu-s i v e . I t only suggests that the selenium status of the c o n t r o l lambs i n T r i a l 3 was adequate f o r hemoglobin production and/or red c e l l maintenance, p o s s i b l y because vitamin E l e v e l s were good. On the other hand, blood hemoglobin was probably a crude i f not in a p p r o p r i a t e technique to assess the importance of Se i n heme metabolism. The e f f e c t of selenium status on heme synthesis and catabolism i n bone marrow has not yet been thoroughly i n v e s t i g a t e d , although l i m i t e d information i s a v a i l a b l e f or l i v e r , spleen and muscle (Burk jet _ a l . , 1974; Burk and C o r r e i a , 1978; Whanger ejt a l . , 1977). - 90 -Hemoglobin versus Hematocrit Much d i s c u s s i o n i n the l i t e r a t u r e on i r o n d e f i c i e n c y concerns the comparative e f f i c i e n c y of hemoglobin and hematocrit f o r d e t e c t i o n of anemia. D i f f e r e n t r e s u l t s are f r e q u e n t l y obtained, depending on which parameter i s used. The hematocrit t e s t i s cheaper and e a s i e r than the hemoglobin t e s t , but can be l e s s e f f e c t i v e than hemoglobin i n i d e n t i f y i n g anemia. For example, the hematocrit f a i l s to detect 20-50% of c h i l d r e n who would be considered anemic based on hemoglobin l e v e l s ( G r a i t c e r ejt a l _ . , 1981). The large amount of data c o l l e c t e d on lamb hematology i n t h i s t r i a l provided an opportunity to compare the Hb and PCV t e s t s . Hemoglobin was p l o t t e d against PCV, and r e g r e s s i o n equations were developed with and without o u t l i e r r e j e c t i o n . A l l equations given were derived using 5% o u t l i e r r e j e c t i o n . The c o r r e l a t i o n between hemoglobin and hematocrit was very high from b i r t h to 6 weeks of age. The c o e f f i c i e n t of l i n e a r c o r r e l a t i o n was 0.93 at 2 days of age, 0:92 at 1 week, 0.96 at 2 weeks, 0.94 at 3 weeks, 0.89 at 4 weeks, 0.93 at 5 weeks, 0.86 at 6 weeks, 0.75 at 7 weeks, and 0.74 at 8 weeks. The regression equations f o r p r e d i c t i o n of Hb from PCV according to these age groups are given i n Appendix 10. The regression analyses showed that the r e l a t i o n s h i p between Hb and PCV was c l o s e at b i r t h (Figure 7) and even c l o s e r at 4 weeks (Figure 8). As Figure 9 i l l u s t r a t e s , the r e l a t i o n s h i p between PCV and Hb was not as t i g h t by 8 weeks. When data from a l l sampling periods of T r i a l 3 were analyzed together, the c o e f f i c i e n t of l i n e a r c o r r e l a t i o n was 0.9084. Excluding the THE ... ARE USED 10 PLOT THE REGRESSION LINE; THE — IS USED XHEN * PLOT POINT COVERS DATA P O I N T S _ I cr 13.30 10.70 17.(0 / 1 1 / I . • is.so -i i ?..«. « a . at i" i i. a i i « iii i i • i «3 9 0 i • : : » cn '. i i a i / i i.. i i i • i i i i. ii i 1 i • i i a i i cn '. . i i o f i •' • « " ' . 1 1 1 11 i . 1 . 1 .• 1 7 i ') '.' i a n i i 7 ' / / / • » / 9. 100 I " MA MM 44 39.OO . v % * ]4 00 89.00 3« 00 3 » 0 0 DISTANCE BETWEEN SLASHES ON THE X-AXIS IS O.2SO0 % Packed CelI VoIume VO Figure 7. Hemoglobin versus PCV at 2 days ( T r i a l 3 ) . Y = 0.4851 + 0.3419X R 2 = 0.8624 AND ••• ARE USED TO PLOT THE REGRESSION LINE; THE ••• IS USED WHEN A PLOT POINT COVERS DATA POINTS I 1 • 1 I i . • i i i » i i i i • . i i i i i i • . i i ' i 1 31*. 11 1 1 1 «11 1 1 . . . . . . . . . . . . 1 1 121 '•. 1 • 1 11 . 1 2 1 • •.1 1 1 • 1 1 11 • I I 1 • i . • i i . . . a i . 2 i i i i i i !• . I 11 1 i ' )\)uni)n\nuiuu\unnui\nuunl\inu)n)\l)nnin\)))ll)))nn)nn)WnU)))nuHn))n^ 14 00 21 00 2».O0 35.OO ' w DISTANCE BETWEEN SLASHES ON THE X-AXIS IS 0.3500 % P a c k e d C e l I Vo Iume Figure 8. Hemoglobin versus PCV at k weeks ( T r i a l 3 ) . Y - 0.2120 + 0.3346X R 2 = 0.9228 THE V AND ARE USED TO PLOT THE REGRESSION L I N E ; THE • • • IS USED WHEN A PLOT POINT COVERS DATA P O I N T S 1 1 1 1. 1 3 . 8 0 _o CD O e 1 1 . 8 0 1 0 . 8 0 / I a 1 i i t i i i n i i • . . 1 • . ."" i i 1 2 1 1 i • i i i i 7 / 1 / / 2 9 . 0 0 3 1 . 8 0 3 4 . S O DISTANCE BETWEEN SLASHES ON THE X - A X I S IS 0 . 1 4 0 0 8 . B O O % Packed CelI Volume 4 3 . 0 0 Figure 9. Hemoglobin versus PCV at 8 weeks ( T r i a l 3 ) . Y = 4.084 + 0.2334X R 2 = 0.5409 •a .o O CT O E 0) THC " . • » N O • • • ARE USEO TO PLOT THE REGRESSION L I N E : THE • • • IS USED WHEN A PLOT POINT COVERS DATA P O I N T S A N INTEGER " I " . B E T W E E N 1 AMD 9, REPRESENTS APPROXIMATELY 2*1 OATA P O I N T S ; ' O - REPRESENTS 1 OR FEWER DATA P O I N T S 17.20 - O 1 17.20 0 0 O 16.99 O 0 0 16.72 0 0 0 0 1 0 0 O 00 I 6 10 O 00 OO 16.49 16.24 16.00 19.76 19.92 19.28 14.90 / 1 o 6 6i oii 0 11 021102 ••« 0 0 1 02001•••0 10 0 0 0 0 14.80 14.96 1 6b obiti30iii'^'ooo 0 1001121*.• 1 O001101121t2«««11 101 00 00 14.32 14.08 13.84 / / 6 OlCOli 13 02 il2«« 124 201 01 010 00311 2042*.12 020 0 13.36 12.40 / 00001112202««»202 10 00 0 0 01 01 O20334»»«1441430011 00 1 12.64 12.40 / / 101 01301••002020 OO 6 011115O23**0201l 30 10 0 0 120121*.0111 21 01 1 0 12. 16 11.92 11.68 1 1 6b 2ti«« i 200 i 6 0141»««2 11111 OOOO 01««*011 120100 11.44 11.20 10.96 / 1 b i i66«.»2i 63 6b bo obbb 0 0.** 010 2 0 0 0 0 10*. 11 10 0 10.48 10.24 10.00 1 1 6o2««ib 2  6 6 0 1 21 1 01 0 000*. 0 1 00001 9.760 9.920 1 . • 6 1 6 b •.01 0100 0 .• 00 1 9.280 9.040 8.800 1 1 1 • • 6 .. 0 01 0 2 0 0 8.320 8.080 7.9O0 / .6 0 ib 6 6 0 7.600 7.360 / 1 6.880 6.640 f / 0 6. 160 9.920 9.200 / : I 9.440 9.200 ~ ixtim, 14.00 DISTANCE mwrmnmh 21 BETWEEN SLASHES '77/ oo ON / / / / l / / / / / / / / / l7 / / / / / / / / l / / / / / /7 / ' / i / ; / ; / / / / / i / / / / /7 / / / i / / / / 28.00 39.00 42.OO THE X-AXIS IS 0.3900 rnmm rrrn 49.00 VO % Packed CelI Volume F i g u r e 10. Hemoglobin versus PCV, a l l T r i a l 3 data (5% o u t l i e r e x c l u s i o n l e v e l ) 9 = 0.8015 + 0.3221X R 2 = 0.8251 • AND — ARE USEO TO PLOT THE REGRESSION L I N E ; THE — IS USED WHEN A PLOT POINT COVERS DATA P O I N T S EGER I " . B E T W E E N 1 AND 9 . REPRESENTS APPROXIMATELY 2 * I DATA P O I N T S ; ' O ' REPRESENTS 1 OR FEWER DATA P O I N T * THE AN INTEGER " I " , B E T W E E N 1 AND 9 , REPRESENTS A P P H U X l l M I t L i J-i u . . . r u i » , , , „ . . . . . . . . . . . . . JO.16 - o 26.ib / 1 9 . 8 0 / 1 9 . 5 0 / 1 8 . 9 0 / 1 8 . 6 0 / 0 0 1 8 . 0 0 / 1 7 . 7 0 1 7 . 1 0 - O 1 1 7 . 1 0 / O OOOO O 1 6 . 8 0 / OO 1 • • 1 6 . 2 0 / O O 0 0 1 . • • 1 5 . 9 0 / 0 0 O 0 O 0 0 O 0 O . • • 1 5 . 3 0 / 0 0 0 0 0 110011 . . . » O O 1 5 . 0 0 / O 0 0 1013 1 1 2 1 1 » « « 1 O O O 1 4 . 4 0 1 4 . 1 0 - 01 1 2 1 1 3 2 1 2 . • . 2 0 0 0 0 1 4 . 1 0 / 01 1 0 0 3 1 1 2 2 0 4 1 l » « « 1 1 1 1 2 0 1 OO 0 1 3 . 5 0 / 0 0 2 1 0 1 0 2 3 3 2 2 1 » « » I 2 1 I I 1 0 3 1 0 O 1 3 . 2 0 / 0 0 0 1 0 1 1 1 2 3 4 0 * * " 2 1 4 1 1 0 0 O 0 0 0 0 0 0 0 1 2 . 6 0 / I 10 1403 I • • • 3 2 2 4 0 2 2 0 0 0 1 0 0 O 1 2 . 3 0 / 0 0 1 2 1 2 4 « » » 0 4 1 2 21 11 2 0 1 0 1 1 . 7 0 / .0 0 0 0 1 2 2 « " 2 2 3 1 0 1 0 O 1 1 . 4 0 / 1 O . • • • 2 1 13 2011O 0 0 0 0 0 1 0 . 8 0 / 1 0 . • • • 2 221 2100 1 0 0 0 1 0 . 5 0 / O . « « 3 21 20 10 O 0 0 9 . 9 O 0 - " 9.60O 9 . 6 0 0 ) •• 1 i 1 6 6 6 6 6 o n 9.366 1 1 10010 O 0 . O O 9 . O 0 0 8 . 7 0 0 6 6 8 . 4 0 0 8 . 100 7 . 8 0 0 / o 7. soo / o 7.200 t . 6 . 9 0 0 / O 6 . 3 0 0 6 . 0 0 O / ... 01 010  o/ 1 0 0 0 0 / ;: 0 6 6 0 6 8.100 - . 0 2 1 0 / o 10 0 o / 5 . 7 0 0 / • 0 5 . 4 0 0 / 0 5 . 1 0 0 1 4 . O 0 2 1 . 0 O 2 8 . O O D I S T A N C E BETWEEN SLASHES ON THE X - A X I S IS 0 . 3 5 O 0 4 9 . 0 0 % Packed Ce l I Volume Figure 11. Hemoglobin versus PCV f o r a l l T r i a l 3 data. Y = 1.613 + 0.2996X R 2 = 0.6571 - 96 -o u t l i e r s , 1081 complete p a i r s of observations were used i n the a n a l y s i s . The simple l i n e a r r e gression equation derived from t h i s data was Y = 0.8015 + 0.3221X. where Y = the dependent v a r i a b l e hemoglobin X - the independent v a r i a b l e PCV. The p l o t of Y vs. X and of the regression l i n e i s shown i n F i g u r e 10. Figure 11 shows the same data p l o t t e d without e x c l u s i o n of the o u t l i e r s . The hematocrit t e s t appeared to be a good estimator of lamb i r o n status between b i r t h and 8 weeks. However, the hemoglobin technique was p r e f e r a b l e f o r lambs over 6 weeks of age, depending on the accuracy d e s i r e d . A f t e r 6 weeks of age, the r e l a t i o n s h i p between the two v a r i a b l e s d e t e r i o r a t e d , and i t was not known i f the c o r r e l a t i o n would become even lower a f t e r 8 weeks. Since the concentration of Hb i n the erythrocyte i s r e l a t i v e l y constant except during anemia, t h i s may not occur. PLASMA SELENIUM Plasma selenium data were obtained f o r 39 lambs i n R e p l i c a t e 2, and 36 lambs i n R e p l i c a t e 3 of T r i a l 3. Mean selenium values were 0.092±0.004 ppm i n R e p l i c a t e 2, and 0.091±0.004 ppm i n R e p l i c a t e 3. As r e p l i c a t e s d i d not a f f e c t plasma selenium (P<0.05), data from the r e p l i c a t e s was combined. Selenium treatment s i g n i f i c a n t l y increased plasma Se l e v e l s at 4 weeks (P<0.05). Means were 0.086 ppm Se for the 38 c o n t r o l lambs, and 0.098 ppm Se f o r the 37 selenium-injected lambs. - 97 -The range i n plasma selenium values was l a r g e , extending from d e f i -c i e n t to normal l e v e l s w i t h i n both treatments (Figure 12). The assumption that the f l o c k had a marginal selenium status was j u s t i f i e d by plasma s e l -enium values i n the c o n t r o l lambs. Control lambs had l e v e l s ranging from 0.016-0.118 ppm Se. Between 0.080 and 0.500 ppm Se i s considered adequate fo r serum selenium l e v e l s i n sheep i n B.C. ( P u i s , 1981). Based on t h i s c r i t e r i o n , 25 out of 38 c o n t r o l s , or 66%, had adequate plasma selenium l e v e l s at 4 weeks. The l e v e l of .08 ppm i s much higher than that c o n s i -dered normal i n some other s t u d i e s ( i e . 0.02 ppm Se may be adequate), but was chosen as l e v e l s of < 0.07 ppm Se were c u r r e n t l y associated with Se-responsive WMD i n the UBC f l o c k . However, no WMD occurred i n any lamb used i n T r i a l 3. Corresponding ranges for selenium-injected lambs were 0.029-0.170 ppm Se. A l a r g e r percentage of selenium-injected lambs had plasma l e v e l s w i t h i n the nromal range. Out of 37 i n j e c t e d lambs, 30 animals or 81% could be considered to have adequate plasma selenium values. Due to the very low selenium l e v e l s i n some i n d i v i d u a l s , the recommended Se i n j e c t i o n dosage of 1.5 mg selenium as sodium s e l e n i t e may not ensure adequate selenium l e v e l s i n a l l t r eated lambs. According to Thompson jst ja_l. (1976), sheep appear to form two d i s t i n c t groups, one having high blood selenium l e v e l s ranging from 133-249 ng/mjj, , and the other having low l e v e l s ranging from 21-67 ng/m£. Blood selenium data from the present study appeared to have a normal d i s t r i b u t i o n ( F i g u r e 12). Breed, sex and r e a r i n g had no e f f e c t on plasma selenium l e v e l s (P>0.05). - 98 -50' C o n t r o l Lambs 4 0 ' 30" .o 1 2 0 ' 10-— 1 — .02 .04 .06 .08 .10 .12 .14 .16 .18 P lasma S e l e n i u m (ppm) 50 40 30 _a E fD " 2 0 10 .02 .04 .06 .08 .10 .12 .14 .16 .18 P lasma S e l e n i u m (ppm) Figure 12. E f f e c t of selenium treatment at b i r t h on plasma selenium at 4 weeks. SeI e n i u m - i n j e c t e d Lambs J=L - 99 -The i n t e r a c t i o n of i r o n with selenium was s i g n i f i c a n t (P<0.05). Selenium l e v e l s were s i g n i f i c a n t l y higher i n those lambs i n j e c t e d with selenium and not i n j e c t e d with i r o n , than i n the other three treatment com-bin a t i o n s (P<0.05, Newman-Keuls m u l t i p l e range t e s t ) . Means were 0.085 ppm (-Fe-Se); 0.086 ppm (+Fe-Se); 0.107 ppm (-Fe+Se); and 0.088 ppm Se (+Fe+Se), with 18 to 20 lambs per treatment combination. These d i f f e r e n c e s may r e f l e c t a d i f f e r e n c e i n the p a r t i t i o n i n g of blood selenium but not n e c e s s a r i l y i n t o t a l selenium l e v e l s . Selenium i s incorporated i n t o the er y t h r o c y t e only during i t s formation, mainly as GSH-Px (Ganther et a l _ . , 1976). As selenium was i n j e c t e d simultaneously with i r o n , which g r e a t l y stimulated e r y t h r o p o i e s i s , p o s s i b l y more selenium was incorporated i n t o the erythrocyte f r a c t i o n of +Se+Fe lambs than i n +Se-Fe lambs. Analyses of Se and GSH-Px i n both plasma and red c e l l s would provide valuable information. Iron d e f i c i e n c y , but not anemia per se, may r e s t r i c t the sy n t h e s i s of GSH-Px (Rodvien et a l . , 1974). Iron d e f i c i e n c y anemia i s associated with decreased erythrocyte GSH-PX a c t i v i t y , and i r o n supplementation induces a rapid increase i n GSH-Px. A study with humans i n d i c a t e d that the decrease i n GSH-Px was p r o p o r t i o n a l to the decrease i n Hb (Macdougall, 1972). How-ever, a study with r a b b i t s showed that erythrocyte GSH-Px a c t i v i t y was markedly depressed by i r o n d e f i c i e n c y , even when expressed per u n i t of hemoglobin (Rodvien et a l . , 1974). The s i g n i f i c a n t i n t e r a c t i o n i n the current study between i r o n and selenium treatments on plasma selenium l e v e l s suggests a r o l e f o r i r o n i n selenium metabolism. As GSH-Px does not contai n i r o n (Ganther jet a l . , 1976), i t i s l i k e l y that i r o n - c o n t a i n i n g enzymes may be involved i n the - 100 -s y n t h e s i s or r e g u l a t i o n of GSH-Px. A l t e r n a t e l y , plasma selenium could be decreased i n +Se+Fe lambs merely because of the increased red c e l l mass c o n t a i n i n g GSH-Px. Plasma P r o f i l e The plasma p r o f i l e included the same parameters as i n the previous two t r i a l s . The s t a t i s t i c a l a n a l y s i s tested r e p l i c a t e , treatment, breed, sex, r e a r i n g and i n t e r a c t i o n s , using orthogonal c o n t r a s t s to i s o l a t e the e f f e c t s of i r o n and selenium. R e p l i c a t e s i g n i f i c a n t l y a f f e c t e d every parameter, as can be seen i n Table I I I . However, mean values of each parameter were w i t h i n normal ranges. R e p l i c a t e means were highest f or Pj_, BUN, LDH and AT i n R e p l i -c a t e 2, and lowest i n R e p l i c a t e 1. Means were highest f or calcium, g l u -cose, c h o l e s t e r o l and AP i n R e p l i c a t e 1, and lowest i n R e p l i c a t e 2. A l l R e p l i c a t e 3 means were intermediate. The data i n d i c a t e d that R e p l i c a t e 2 samples suffered some d e t e r i o r a -t i o n i n storage, and R e p l i c a t e 3 samples a l e s s e r amount of d e t e r i o r a t i o n . Plasma samples were stored i n a freezer at -10°C, but due to temperature f l u c t u a t i o n s the samples were often found to be i n a semi-frozen s t a t e upon removal f o r a n a l y s i s . T r i a l 2 samples were stored f o r the longest time and th e r e f o r e were the most a f f e c t e d . While twenty of the most commonly measured plasma c o n s t i t u e n t s , i n c l u d i n g glucose, are s t a b l e when frozen f or three or more years, re-peated f r e e z i n g and thawng must be avoided (Caraway, 1962). A l k a l i n e phosphatase was the only parameter s i g n i f i c a n t l y a f f e c t e d by i r o n i n a l l r e p l i c a t e s of T r i a l 3 (Table I V ) . As i n T r i a l s 1 and 2, the a c t i v i t y of plasma a l k a l i n e phosphatase was c o n s i s t e n t l y lower i n plasma of - 101 -TABLE III. Effect of replicate on plasma profile at 4 weeks (Trial 3) METABOLITE X ± SEM R1 R2 R3 Significant Main E f f e c t 1 . 2 ——————— = = = = = = = — — = = -Calcium (mq/dl) 10.82 ± .12 11.56 10.41 10.60 Rep**, Rear* PL (mg/d£) 11.27 ± .15 10.14 12.10 11.44 Rep** Glucose (mg/dji) 49.8 ± 3.7 94.6 18.1 42.4 Rep*** BUN (rng/di) 19.3 ± 0.5 16.9 22.6 18.1 Rep*** C h o l e s t e r o l (mg/d£) 123.1 ± 3.6 132.6 106.5 126.8 Rep** TP (g/d£) 6.20 ± 0.05 6.98 6.45 6.17 Rep* Albumin (g/d£) 3.72 ± 0.04 3.24 4.00 3.87 Rep*** AP (lU/i) 605 ± 18 691 527 604 Rep*, Fe* LDH (IU/£) 696 ±21 578 756 775 Rep***, Sex* AT (IU/£) 161 ± 10 109 188 180 Rep** n (111) (33) (37) (41) ^ a i n e f f e c t s t e s t e d were r e p l i c a t e , treatment, breed, sex and r e a r i n g . P redetermined orthogonal c o n t r a s t s were as f o l l o w s : FE. C o n t r o l + Se treatments vs. Fe + Fe/Se treatments SE. Co n t r o l + Fe treatments vs. Se + Fe/Se treatments FEXSE C o n t r o l + Fe/Se treatments vs. Se + Fe treatments 3*P<0.05; **P<0.01; ***P<0.001 TABLE IV. Effect of iron and selenium supplementation on plasma profile (Trial 3) PLASMA METABOLITE REPLICATE 1 REPLICATE 2 REPLICATE 3 Control Se Fe Se/Fe Control Se Fe Se/Fe Control Se Fe Se/Fe C a l c i u m (mg/d£) 1 1 . 7 6 1 1 . 7 0 1 1 . 7 1 1 1 . 0 6 1 0 . 8 2 1 0 . 5 0 1 0 . 1 3 1 0 . 1 3 1 0 . 0 6 1 1 . 0 2 1 0 . 8 5 1 0 . 4 0 P i ( m g / d i ) 1 0 . 4 4 1 0 . 2 2 9 . 9 7 9 . 9 6 1 1 . 9 4 1 2 . 5 7 1 1 . 6 7 1 2 . 2 3 1 1 . 8 5 1 1 . 2 6 1 1 . 2 8 1 1 . 3 9 G l u c o s e (mg/d£) 9 7 . 0 9 3 . 5 9 4 . 8 9 3 . 0 1 9 . 5 1 2 . 0 2 5 . 8 1 5 . 1 4 0 . 2 4 3 . 4 41 .5 4 5 . 0 BUN (mg/d£) 1 7 . 2 1 5 . 6 1 8 . 4 1 6 . 2 2 2 . 2 2 1 . 2 2 3 . 6 2 3 . 7 1 8 . 3 1 7 . 0 2 0 . 1 1 6 . 6 C h o l e s t e r o l (mg/d£) 1 4 8 . 0 1 3 5 . 7 1 2 9 . 4 1 3 4 . 5 111 . 3 9 4 . 0 1 1 4 . 2 1 0 5 . 9 1 2 0 . 8 1 2 3 . 8 1 3 3 . 0 1 2 8 . 4 TP ( g / d j i ) 6 . 1 2 5 . 9 7 6 . 0 2 5 . 8 0 6 . 4 2 6 . 4 1 6 . 3 9 6 . 5 8 6 . 0 1 6 . 5 6 . 1 3 6 . 0 2 A l b u m i n ( g / d £ ) 3 . 2 6 3 . 2 9 3 . 2 3 3 . 1 8 3 . 9 5 3 . 9 7 4 . 0 8 4 . 0 1 3 . 8 4 3 . 9 4 3 . 7 8 3 . 9 4 AP ( l U / i ) 799 717 6 1 2 6 4 3 5 5 3 5 3 8 5 0 7 5 0 8 6 4 5 6 4 6 5 7 0 5 5 6 LDH ( I U / £ ) 552 585 582 593 7 9 4 7 3 0 7 3 3 7 6 2 7 7 5 7 3 5 7 2 6 7 2 5 AT ( I U / £ ) 110 100 112 115 262 150 172 159 176 172 2 0 4 1 6 0 n 8 8 9 8 10 9 9 9 10 10 12 9 - 103 -i r o n - t r e a t e d lambs. Mean AP i n 56 c o n t r o l lambs was 645±28 compared to 565±28 IU/1 i n 56 i r o n - t r e a t e d lambs. Selenium treatments were not associated with any changes i n plasma parameters i n d i c a t i v e of white muscle disease. The d e t e r i o r a t i o n of muscle t i s s u e that occurs i n WMD induces a host of biochemical changes i n plasma as we l l as muscle t i s s u e . For example, inorganic phosphate and a l k a l i n e phosphatase are increased ( K o v a l ' s k i i and Ermakov, 1970), while serum calcium and magnesium are unaffected (Godwin ejt a_l_., 1974). Plasma l e v e l s of t i s s u e enzymes are s e n s i t i v e to muscle damage, i n c r e a s i n g even during s u b c l i n i c a l WMD. Consequently, plasma malate dehydrogenase, a l a n i n e amino-t r a n s f e r a s e , l a c t a t e dehydrogenase, and aspartate aminotransferase are commonly used to diagnose WMD i n ruminants (Whanger et ai ^ . , 1969b; Boyd, 1973). Except for plasma selenium and GSH-Px, few plasma parameters respond t o v a r i a t i o n s i n selenium s t a t u s i n the absence of WMD. Glucose and f a t t y acid metabolism may be a f f e c t e d at various l e v e l s of selenium a v a i l a b i l -i t y . Selenium s t a t u s i n f l u e n c e d r a t e of glucose metabolism, r a t e of f a t t y acid metabolism, and t i s s u e f a t t y a c i d content i n one study (Fischer and Whanger, 1977). Supplementing d i e t a r y Se to an adequate Se d i e t decreased blood sugar, pyruvic a c i d , and P^, and increased t i s s u e glycogen and muscle ATP ( K o v a l ' s k i i and Ermakov, 1970, p. 68). On the other hand, Whanger et aJL (1969b) were unable to measure a response i n blood glucose, l a c t a t e or c h o l e s t e r o l to WMD, although t i s s u e c h o l e s t e r o l i s presumed to increase. The high v a r i a b i l i t y c o n t r i b u t e d by r e p l i c a t e and other sources of v a r i a t i o n may have obscured i r o n and selenium treatment e f f e c t s . Contrary - 104 -t o e x pectation, Se d i d not s i g n i f i c a n t l y (P>0.05) a f f e c t LDH and AT l e v e l s . The d i f f e r e n c e i n selenium status at 4 weeks between the c o n t r o l and selenium treatments may have been too narrow to be r e f l e c t e d i n plasma metabolites. The scanty l i t e r a t u r e on the subject i n d i c a t e s that a sub-c l i n i c a l Se d e f i c i e n c y may be expected to have only a s u b t l e e f f e c t on plasma c o n s t i t u e n t s . Hence plasma p r o f i l e a n a l y s i s , except f o r c e r t a i n muscle enzymes, may be of doubtful value for i n v e s t i g a t i n g selenium even under c a r e f u l l y c o n t r o l l e d experimental c o n d i t i o n s . We igh t The weight data i n T r i a l 3 was taken from a t o t a l of 121 lambs. There were 35, 43, and 43 lambs r e s p e c t i v e l y i n r e p l i c a t e s 1, 2 and 3. R e p l i c a t e was a s i g n i f i c a n t main e f f e c t from 1 week to 8 weeks of age. However, d i f f e r e n c e s between r e p l i c a t e s were s l i g h t . I n i t i a l weights were 4.05±0.99, 3.84±0.88, and 3.90±0.98 kg i n R e p l i c a t e s 1, 2 and 3. F i n a l weights at 8 weeks were 18.6±4.4, 19.0±4.0, and 19.4±3.9 kg. Rearing s i g n i f i c a n t l y a f f e c t e d weight throughout the t r i a l (P<0.001). The 46 s i n g l e lambs were heavier than the 75 twin lambs from b i r t h (4.47±0.97 vs. 3.59±0.77 kg) to 8 weeks (21.8±3.9 vs. 17.3±3.1 kg). I n i t i a l weights were 3.79±0.75 kg f o r 63 females, compared to 4.06±1.10 kg for 58 males. F i n a l weights at 8 weeks averaged 17.9±3.2 kg for females, and 20.2±4.5 kg for males, however the d i f f e r e n c e was not s i g n i f i c a n t (P>0.05). Iron treatment s i g n i f i c a n t l y a f f e c t e d lamb weight gain from 2 weeks t o the end of the t r i a l (Appendix 11). As shown i n Figure 13, the d i f f e r -ence between i r o n - t r e a t e d lambs and other c a t e g o r i e s increased slowly throughout the t r i a l . - 106 -The treatment i n t e r a c t i o n s with sex and with r e a r i n g were not s i g n i -f i c a n t (P>0.05), undoubtedly because i r o n and selenium combinations were considered together as treatments. However, i f the e f f e c t s of selenium are ignored (selenium having no apparent i n f l u e n c e on weight gain at P>0.05), some very i n t e r e s t i n g r e s u l t s emerge. The f a s t e s t gaining c a t e g o r i e s of lambs - s i n g l e lambs rather than twins, and males rather than females -appear to respond most favourably to i r o n treatment. The 29 i r o n i n j e c t e d male lambs gained 17.2 kg., while the 29 male lambs not i n j e c t e d with i r o n gained 15.1 kg. This was a d i f f e r e n c e of 2.1 kg due to i r o n treatment of male lambs. In comparison, 31 female i r o n -t r e a t e d lambs gained 14.3 kg compared to 13.9 kg f o r 32 female c o n t r o l s , a d i f f e r e n c e of only 0.4 kg. S i m i l a r l y , weight gain was improved by i r o n more markedly i n s i n g l e than i n twin lambs. The 25 i r o n - i n j e c t e d s i n g l e lambs gained 18.2 kg, w h i l e the 21 c o n t r o l s i n g l e s gained 16.2 kg, a d i f f e r e n c e of 2 kg. The 35 i r o n - i n j e c t e d twins gained 13.9 kg, which was only 0.3 kg more than the 40 c o n t r o l twins gained. These r e s u l t s i n d i c a t e that fast-growing lambs may b e n e f i t most by i r o n treatment, p a r t i c u l a r l y s i n g l e and/or male lambs. Under c o n d i t i o n s where intake i s r e s t r i c t e d , i r o n may not become l i m i t i n g for growth. The lambs i n t h i s study were Dorset and Dorset/Finn crosses. S u f f o l k or Hamp-s h i r e lambs might demonstrate a greater response to i r o n due to t h e i r high growth r a t e . Iron supplementation may enhance growth rate i n s e v e r a l ways. L o g i -c a l l y , the a v a i l a b i l i t y of i r o n for hemoglobin, myoglobin and enzyme syn-t h e s i s prevents a r e s t r i c t i o n i n growth. A d d i t i o n a l l y , s e v e r a l d e l e t e r i o u s s i d e - e f f e c t s of i r o n d e f i c i e n c y are avoided. - 107 -The g a s t r o i n t e s t i n a l mucosa i s e s p e c i a l l y s u s c e p t i b l e to i r o n d e f i -ciency i n young animals of most species so f a r stu d i e d , i n c l u d i n g human, dog, and p i g . Atrophy of the g a s t r o i n t e s t i n a l mucosa and d e f i c i e n c i e s i n t i s s u e enzymes are common i n i r o n d e f i c i e n t animals and may impair absorp-t i o n of i r o n , vitamin A, and other n u t r i e n t s (Beutler and Fairbanks, 1980; Guha et a l _ . , 1968). This problem has not been studied i n the lamb. Another s i d e - e f f e c t of i r o n d e f i c i e n c y i s increased s u s c e p t i b i l i t y to scours and e n t e r i t i s , which could w e l l c o n t r i b u t e to growth depresson ( L a r k i n and Hanran, 1983). Plasma Protein Electrophoresis The e l e c t r o p h o r e s i s of plasma p r o t e i n s y i e l d e d some i n t e r e s t i n g r e s u l t s , although the technique i s not commonly used i n n u t r i t i o n s t u d i e s . While serum p r o t e i n s are s e n s i t i v e to n u t r i t i o n a l i n f l u e n c e s , i n most cases the changes are sub t l e and d i f f i c u l t to detect and i n t e r p r e t (Kaneko, 1975). Over one hundred plasma p r o t e i n s have been described. However, only f i v e plasma p r o t e i n bands are obtained from most species by agarose g e l e l e c t r o p h o r e s i s . Thus a change i n a s p e c i f i c p r o t e i n i s r a r e l y of s u f f i c i e n t magnitude to produce a c l i n i c a l change i n the associated p r o t e i n band (Latner, 1975, p. 200). As i n the plasma p r o f i l e samples, some storage d e t e r i o r a t i o n was e v i -dent, which may have caused some p r o t e i n denaturation. The t o t a l p r o t e i n values were higher than those obtained i n the plasma p r o f i l e . D i f f i c u l t y i n d i s t i n g u i s h i n g peaks may have c o n t r i b u t e d to the exper-imental e r r o r . Fibrinogen t r a i l s the b e t a - g l o b u l i n f r a c t i o n (Kaneko, - 108 -1975), which tends to obscure the boundary between the beta- and gamma-globulin bands when plasma i s used instead of serum. In a u s e f u l d i s c u s s i o n of agarose g e l e l e c t r o p h o r e s i s , Johansson (1972) suggested adding heparin to the plasma sample to improve separation of beta l i p o p r o t e i n s . Otherwise, use of serum instead of plasma might have improved the r e s o l u t i o n of the beta and gamma bands. The densitometric t r a c i n g s were very s i m i l a r to those obtained by Keay and Doxey (1982). The alpha-1 and alpha-2 g l o b u l i n zones could be e a s i l y subdivided, and the alpha-2 g l o b u l i n zone was much greater q u a n t i t a -t i v e l y (Figure 14). In the present study, the alpha-1 zone was even sm a l l -e r , and i n some cases non-existent. The major d i f f e r e n c e was a no t i c e a b l y smaller gamma g l o b u l i n peak i n the current study. As Keay and Doxey (1982) did not q u a n t i t a t e t h e i r r e s u l t s , the data couldn't be compared. Compari-son of r e s u l t s with other l i t e r a t u r e values are summarized i n Table V. Many of the p r o t e i n f r a c t i o n s were h i g h l y c o r r e l a t e d with the t o t a l p r o t e i n c o v a r i a b l e (Table V I ) . T o t a l p r o t e i n was c o n s i s t e n t l y r e l a t e d to albumin (P<0.001) and gamma g l o b u l i n (P<0.05) l e v e l s . Data from R e p l i c a t e 2 were a v a i l a b l e only from 4 week old lambs. R e s o l u t i o n of bands was rather poor, compared to r e p l i c a t e 3, p o s s i b l y because of longer time i n storage. As a r e s u l t , treatment e f f e c t s were obscured. Data from R e p l i c a t e 2 i s shown i n Table V I I , but the d i s c u s s i o n w i l l center around the r e s u l t s from lambs at 2, 4 and 6 weeks of age i n R e p l i c a t e 3. Gamma g l o b u l i n ranged from 0.10-1.21 g/djj. at 2 weeks, and 0.10-0.62 g/d£ at both 4 and 6 weeks of age. O v e r a l l gamma g l o b u l i n l e v e l s d e c l i n e d from 0.69 g/dx, at 2 weeks to 0.37 g/d£, at 6 weeks, r e f l e c t i n g the - 109 -F i g u r e 14. Densitometric t r a c e s of plasma p r o t e i n s . Arrow i n d i c a t e s sample a p p l i c a t i o n s l i t . a. sample from 2-week o l d lamb b. sample from 4-week o l d lamb showing low Y - g l o b u l i n content c. comparative t r a c e from normal sheep, Keay and Doxey (1982) TABLE V. Plasma protein electrophoresis results compared to literature values REPLICATE 3 RESULTS3 REPLICATE 2 a MITRUKA & RAWNSLEY6 IRFANC P R O T E I N ( g / d £ ) 2 w e e k s ( 4 0 ) d 4 w e e k s ( 4 2 ) 6 iveeks ( 3 9 ) 4 iveeks ( 3 9 ) N o r m a l m a l e s h e e p N o r m a l f e m a l e s h e e p N o r m a l r a n g e 3 m o n t h s ( 1 0 ) TOTAL 7 . 8 3 + 0 . 1 6 7 . 7 0 ± 0 . 1 6 7 . 2 8 ± 0 . 1 5 6 . 4 1 ± 0 . 1 5 6 . 8 0 ± 0 . 3 0 7 . 2 0 ± 0 . 3 1 5 . 7 0 - 9 . 1 0 5 . 4 6 A L B U M I N 4 . 3 7 + 0 . 1 2 4 . 5 2 ± 0 . 1 3 4 . 5 2 ± 0 . 1 0 3 . 6 6 ± 0 . 1 3 3 . 7 0 ± 0 . 3 5 3 . 8 1 ± 0 . 3 3 2 . 7 0 - 4 . 5 5 3 . 1 0 «-1 g l o b u l i n 0 . 4 1 + 0 . 0 3 0 . 3 8 ± 0 . 0 2 0 . 3 8 ± 0 . 0 2 0 . 4 3 ± 0 . 0 2 0 . 3 3 ± 0 . 0 8 0 . 3 8 ± 0 . 0 6 0 . 1 5 - 0 . 5 0 0 . 3 5 <*~2 g l o b u l i n 1 . 4 0 + 0 . 0 8 1 . 3 9 ± 0 . 0 7 1 . 2 6 ± 0 . 0 5 0 . 9 9 ± 0 . 0 6 0 . 9 6 ± 0 . 1 3 0 . 7 3 ± 0 . 1 2 0 . 4 5 - 0 . 1 2 0 . 4 8 B - g l o b u l i n 1 . 0 2 + 0 . 0 6 0 . 9 3 ± 0 . 0 6 0 . 7 6 ± 0 . 0 7 0 . 7 7 ± 0 . 0 4 0 . 5 2 ± 0 . 1 0 0 . 9 1 ± 0 . 1 3 0 . 2 5 - 1 . 2 0 0 . 5 0 Y - g l o b u l i n 0 . 6 9 + 0 . 0 4 0 . 4 3 ± 0 . 0 2 0 . 3 7 ± 0 . 0 2 0 . 5 4 ± 0 . 0 3 1 . 3 3 ± 0 . 2 0 i . 3 7 ± 0 . 2 5 0 . 8 2 - 1 . 9 0 1 . 0 3 A L B U M I N / G L O B U L I N 1 . 3 0 + 0 . 0 5 1 . 5 0 ± 0 . 0 6 1 . 7 3 ± 0 . 0 7 1 . 3 6 ± 0 . 0 5 1 . 1 9 ± 0 . 2 0 1 . 1 2 ± 0 . 2 1 0 . 7 0 - 1 . 6 0 1 . 3 1 4 V a r i a t i o n e x p r e s s e d a s S . E . b M i t r u k a , B . M . a n d R a w n s l e y , H . M . 1 9 7 7 . D a t a s u m m a r i z e d f r o m t h e l i t e r a t u r e ; v a r i a t i o n e x p r e s s e d a s S . D . C I r f a n , M. 1 9 6 7 . ^ N u m b e r o f s h e e p s a m p l e s TABLE VI. Effect of age and treatment on plasma proteins (Trial 3, Replicate 3) Protein Fraction (g/dt) Control Se Fe Se/Fe S.E .N. Significance of Contrasts1»2 Significance of Main Effects1»3 2 weeks Gamma globulin Beta Globulin Alpha-2 globulin Alpha-1 globulin Albumin Albumin/Globulin Covar. (TP) 4 weeks Gamma globulin Beta globulin Alpha-2 globulin Alpha-1 globulin Albumin Albumin/Globulin Covar. (TP) 6 weeks Gamma globulin Beta globulin Alpha-2 globulin Alpha-1 globulin Albumin Albumin/Globulin Covar. (TP) 0.73 1.25 1.32 0.40 4.42 1.52 7.83 0.40 1.17 1.29 0.37 3.84 1.48 7.12 0.32 0.78 1.16 0.41 4.43 2.22 7.04 11 0.77 1.03 1.38 0.31 4.29 1.48 7.80 0.50 1.02 1.31 0.46 4.50 1.94 7.76 0.31 0.71 1.34 0.35 4.56 2.11 7.29 11 0.64 1.00 1.45 0.48 4.44 1.74 8.00 0.43 0.86 1.52 0.34 4.93 1.94 8.09 0.41 0.80 1.10 0.34 4.47 2.23 7.15 12 0.57 0.76 1.46 0.45 4.29 1.73 7.61 0.41 0.57 1.45 0.34 4.89 2.20 7.82 0.43 0.71 1.53 0.43 4.67 1.98 7.72 8 NS 0.06 0.08 0.03 0.12 1.07 0.16 0.02 0.06 0.07 0.02 0.13 0.09 0.16 0.02 NS 0.05 0.02 0.10 0.08 0.15 NS Se*, Fe** NS NS NS NS NS Fe*** NS NS Fe*** Fe* Fe* NS NS NS NS NS TP* TP* TP* NS TP*** Sex* TP* Sex* TP***, Sex* NS jp»#» NS TP** Rear* Rear* T/p*#» NS Covariable = Total Protein; Main Effects tested were treatment, breed, sex, rearing. 2FE = Fe + Se/Fe vs. Control + Se treatments. SE = Se + Se/Fe vs. Control + Fe treatments. SE X FE = Control + SE/Fe vs. Fe + Se treatments. 3NS P>0.05, *P<0.05; **P<0.01; ***P<0.001. - 112 -TABLE VII. Effect of treatment on plasma proteins at 4 weeks T r i a l 3 (Trial 3, Replicate 2). Protein (g/di) Control Se Fe Se/Fe S.E.M. Significant Effects 3» b Gamma g l o b u l i n 0.51 0.62 0.50 0.52 0.03 TP** Beta g l o b u l i n 0.73 0.88 0.68 0.78 0.04 TP*** Alpha-2 g l o b u l i n 1.01 1.02 1.08 0.88 0.66 1P*#* Alpha-1 g l o b u l i n 0.40 0.49 0.39 0.42 0.02 TP* Albumin 3.51 3.57 3.96 3.60 0.13 TP*** Albumin/Globulin 1.75 1.62 1.95 1.85 0.16 NS T.P. (Covariable) 6.14 6.59 6.69 6.23 0.15 n 9 9 10 11 a I r o n , selenium, breed, sex, and r e a r i n g were n o n - s i g n i f i c a n t sources of v a r i a t i o n ; TP = t o t a l p r o t e i n ( c o v a r i a b l e ) . D*P<0.05; **P<0.01; ***P<0.001. - 113 -catab o l i s m of maternal a n t i b o d i e s and the apparent immaturity of the lambs' lymphoid system. Sheep serum contains 3 major immunoglobulins: IgA, IgG, and IgM. IgG i s q u a n t i t a t i v e l y the most important, accounting f o r 89% of t o t a l serum immunoglobulin, compared to 1.5% and 9.5% f o r IgA and IgM r e s p e c t i v e l y (Smith et a l . , 1975). The s o - c a l l e d gamma-globulin band i n e l e c t r o p h o r e s i s may contain only IgG, as IgA and IgM may extend i n t o the b e t a - g l o b u l i n band ( L a u r e l l , 1972). The plasma gamma-globulin concentration i n the young lamb may not be re l a t e d to neonatal n u t r i t i o n , except that the consumption, of colostrum determines i n i t i a l l e v e l s . The young lamb i s born with n e g l i g i b l e l e v e l s of serum immunoglobulins, so the i n g e s t i o n of colostrum provides immunoglo-b u l i n s , mainly IgG, which are important f o r the f i r s t weeks of l i f e ( C u r t a i n , 1975). These maternal a n t i b o d i e s probably depress the endogenous antibody production, as occurs i n calves (Husband and L a s c e l l e s , 1975). Selenium d e f i c i e n c y i s known to depress gamma-globulin l e v e l s i n sheep (Keeler and Young, 1961). Given the major c o n t r i b u t i o n of maternal gamma-globulin to lamb plasma l e v e l s , a response to selenium supplementa-t i o n was not expected. A l s o , the dosage of selenium used was not s u f f i -c i e n t to ensure optimum selenium l e v e l s i n a l l t reated lambs. Selenium d i d not s i g n i f i c a n t l y a f f e c t gamma g l o b u l i n s at 2, 4 or 6 weeks, although very low gamma g l o b u l i n l e v e l s were found only i n non-selenium supplemented lambs. S u r p r i s i n g l y , i r o n s i g n i f i c a n t l y increased g l o b u l i n l e v e l s at 6 weeks (P<0.05). Means were 0.43±0.2 g/d£, f o r the nineteen i r o n - t r e a t e d lambs, and 0.32+0.02 g/d£ f o r twenty lambs not i n j e c t e d with i r o n . - 1 1 4 -As expected, the b e t a - g l o b u l i n f r a c t i o n was s i g n i f i c a n t l y lower i n i r o n - t r e a t e d lambs at 2 weeks (P<0.01) and 4 weeks (P<0.001). T r a n s f e r r i n i s a major component of the b e t a - g l o b u l i n band, and increases markedly during i r o n d e f i c i e n c y ( F i e l d i n g , 1980). T r a n s f e r r i n was not a f f e c t e d at 6 weeks because of the recovery of plasma i r o n l e v e l s i n c o n t r o l lambs by t h i s time. With the exception of t r a n s f e r r i n i n i r o n d e f i c i e n c y , increases i n b e t a - g l o b u l i n s are extremely rare (Kaneko, 1975). The e f f e c t of i r o n status on plasma pr o t e i n s with the exception of b e t a - g l o b u l i n i s obscure. In t h i s study, i r o n very s i g n i f i c a n t l y (P<0.001) increased plasma albumin l e v e l s at 4 weeks, but not at 2 or 6 weeks. Hypoalbuminemia i s common i n i r o n d e f i c i e n t i n f a n t s , and may be caused by a malabsorption syndrome (Naiman ejt a l . , 1964). Very l i t t l e i nformation i s a v a i l a b l e on the e f f e c t s of selenium on plasma p r o t e i n s , other than the dramatic increase i n c e r t a i n plasma enzymes during white muscle disease. Keeler and Young (1961) found a marked increase i n a l p h a - g l o b u l i n and a decrease i n b e t a - g l o b u l i n as w e l l as gamma-globulins i n s e l e n i u m - d e f i c i e n t sheep. Mice d e f i c i e n t i n selenium, vitamin E and c y s t i n e had decreased f i b r i n o g e n and t o t a l plasma p r o t e i n l e v e l s (Ganther jet a l . , 1976). The s i g n i f i c a n t depression (P<0.05) of the b e t a - g l o b u l i n f r a c t i o n a t 2 weeks by Se treatment was v i r t u a l l y of the same magnitude as the e f f e c t of i r o n on t r a n s f e r r i n . Means were 1.25 g/d£ f o r c o n t r o l lambs, 1.03 g/d£ f o r +Se lambs, 1.00 g/d£ f o r +Fe lambs, and 0.76 g/d£ f o r +Se+Fe lambs. Selenium treatment a l s o tended to reduce b e t a - g l o b u l i n l e v e l s at 4 weeks, but due to the high v a r i a b i l i t y t h i s e f f e c t was not s i g n i f i c a n t (P<0.05). I t i s p o s s i b l e that a l a r g e r dosage of selenium might give more c o n c l u s i v e - 115 -r e s u l t s . I f selenium treatment does depress b e t a - g l o b u l i n l e v e l s , t h i s would c o n f l i c t with the r e s u l t s of Keeler and Young (1961). The i n f l u e n c e of selenium cannot be explained at t h i s p o i n t . A measureable e f f e c t of selenium on the b e t a - g l o b u l i n band would most l i k e l y i n v o l v e f i b r i n o g e n , t r a n s f e r r i n , hemopexin, or complement f r a c t i o n s (C 3 , C 4, and o t h e r s ) , as these are the major p r o t e i n s i n t h i s band. P o s s i b l y a major increase i n plasma enzymes might increase the beta f r a c t i o n i n selen-ium d e f i c i e n t lambs, but there was no evidence of c l i n i c a l WMD, nor were AT and LDH s i g n i f i c a n t l y increased i n the plasma p r o f i l e at 4 weeks. Selenium a l s o appeared to a f f e c t many of the other p r o t e i n f r a c t i o n s , but r e s u l t s were never s i g n i f i c a n t . For example, alpha-2 g l o b u l i n means at 6 weeks were 1.13±0.05 g/d£ f o r non-Se lambs, and 1.43+0.05 g/d£ f o r +Se lambs, with P<0.06. The l i m i t a t i o n s of the e l e c t r o p h o r e s i s technique, c o n d i t i o n of the samples, number of lambs compared to treatments, and the low selenium dosage were some of the problems faced. The absence of s i g n i f i c a n t Se treatment e f f e c t s f o r most plasma p r o t e i n s was t h e r e f o r e not c o n c l u s i v e . More information i s needed on t h i s s u b j e c t , using a l a r g e r number of lambs to compensate for the high v a r i a b i l i t y i n the data, and using c l e a r l y d i f f e r e n t i a t e d l e v e l s of selenium. The e f f e c t s of i r o n on gamma g l o b u l i n and albumin, and of selenium on beta g l o b u l i n , were unexpected and would bear f u r t h e r i n v e s t i g a t i o n . Effect of Selenium on Epidemiology of Soremouth Throughout R e p l i c a t e 3 of T r i a l 3, lambs were observed weekly during sampling f o r the presence of soremouth l e s i o n s . S e v e r i t y was evaluated - 116 -s u b j e c t i v e l y on the s c a l e of 0-5. Because of the s u b j e c t i v e nature of these observations a s t a t i s t i c a l a n a l y s i s was not c a r r i e d out. However, some i n t e r e s t i n g trends were evident, and f u r t h e r i n v e s t i g a t i o n with a greater number of lambs i s warranted. Soremouth i s an i n f e c t i o u s poxvirus disease of sheep and goats, i n which pustular l e s i o n s develop on the l i p s , o r a l mucous membranes, and udder. Synonyms include contagious p u s t u l a r d e r m a t i t i s and o r f . Trans-mission occurs through minor abrasions or trauma. Pustules develop w i t h i n k days of i n f e c t i o n , scabs b u i l d up, and healing takes about 3 weeks. Soremouth may be d e b i l i t a t i n g i n the case of severe secondary b a c t e r i a l i n f e c t i o n s , or through i n t e r f e r e n c e with eating and d r i n k i n g (Mohanty and Dutta, 1981). Iron d i d not appear to have any e f f e c t on soremouth. The same number of lambs treated with i r o n were i n f e c t e d as those not t r e a t e d . This was contrary to expectation as mouth l e s i o n s are commonly observed i n i r o n d e f i c i e n t people ( F l e t c h e r et a l . , 1975) and a l s o i n i r o n d e f i c i e n t c a l v e s ( B l a x t e r et a l _ . , 1957). Selenium seemed to i n f l u e n c e the incidence, duration and s e v e r i t y of the disease (Table V I I I ) . Selenium-injected lambs were l e s s l i k e l y to develop soremouth, and appeared to contract the disease at a s l i g h t l y o l d e r age, and recover f a s t e r . The mean plasma selenium l e v e l was greatest i n lambs which d i d not develop soremouth, i n both the +Se and -Se groups. The mean Se l e v e l was 0.112 ppm i n healthy +Se lambs, compared to .087 ppm i n soremouth-infected +Se lambs. A d d i t i o n a l l y , i n +Se lambs with soremouth l e s i o n s , selenium l e v e l appeared to be n e g a t i v e l y c o r r e l a t e d with the s e v e r i t y of the disease. - 117 -TABLE VII I . E f f e c t s o f selenium treatment on epidemiology o f soremouth i n f e c t i o n Treatment + Selenium C o n t r o l Number of lambs Incidence -2 weeks of age -3 weeks -4 weeks -5 weeks -6 weeks -7 weeks -8 weeks Severity of disease in infected lambsa -2 weeks -3 weeks -4 weeks -5 weeks -6 weeks -7 weeks -8 weeks Avg. duration of disease in infected lambs Avg. age at infection % lambs infected Avg. 4 week plasma Se in soremouth lambs Avg. 4 week plasma Se in healthy lambs 20 10.0% 15.0% 20.0% 30.0% 15.0% 15.0% 5.0% 2.00 2.66 2.00 1.50 1.30 4.30b 1.00 2.3 weeks 4.4 weeks 50% (10/20) 0.087 ppm 0.112 23 17.4% 34.8% 47.8% 56.5% 21.7% 17.4% 0.0% 2.25 2.75 2.45 2.30 1.60 1.25 2.9 weeks 3.6 weeks 70% (16/23) 0.076 ppm 0.093 aSeverity ranked on sale of 0 (no soremouth lesions) to 5 (severe lesions). bHigh score due to 2 lambs with +5 scores due to late development of soremouth. - 118 -While these resul ts are far from conclusive, they do suggest that selenium has an impact on the epidemiology of soremouth. Possibly a higher dosage of selenium resul t ing in optimum plasma selenium l e ve l s , could s i g -n i f i c an t l y affect the resistance of lambs to certa in diseases. The i n f l u -ence of selenium on diseases of high morbidity and low mortal i ty such as scours or soremouth could explain the benefit of selenium treatment to unthr i f ty f locks and/or growth rate when obvious defic iency does not occur. Hemagglutinaton Results The hemagglutination t i t e r of lambs injected with chicken red blood ce l l s (CRBC) from four to eight weeks of age was influenced by sex, dura-t ion of st imulus, interact ions of selenium with sex and possibly i ron . Data are given in Appendix 12 and Appendix 1 3 . The sheep responded immunologically to repeated antigenic stimulus from CRBC with increasing hemagglutination t i t e r s . At the time of the i n i t i a l in jec t ion at 4 weeks of age, the t i t e r was 0 . With progressive in jec t ions , the mean hemagglutination t i t e r increased to 4 . 8 ± 0 . 4 , 1 5 . 9 ± 0 . 4 , 3 2 . 7 ± 0 . 3 , and 4 2 . 1 ± 0 . 3 at 5 , 6 , 7 , and 8 weeks of age. Because of the very slow i n i t i a l increase in hemagglutination t i t e r , i t may be necessary to continue the experiment for a longer period of time in order to accurately assess treatment ef fects . Iron and selenium did not s i gn i f i c an t l y ( P > 0 . 5 ) a f fect t i t e r at any time, as indicated by f igures 15 and 1 6 . However, selenium-treated lambs had higher mean t i t e r s throughout the t r i a l . Means for controls compared to +Se lambs were 4 . 1 + 0 . 5 vs. 5 .6±0.6 at 4 5 weeks; 1 5 . 6 + 0 . 4 vs. 16 .2±0 .7 at 6 weeks; 2 8 . 5 ± 0 . 4 vs. 38 .2±0 .4 at 7 weeks; and 36 .6±0 .4 vs. 4 8 . 6 ± 0 . 4 at 8 - 119 -5 6 7 8 Weeks o f Age Figure 15. E f f e c t of i r o n on HA t i t e r . 5 6 7 8 Weeks of Age Figure 16. E f f e c t of Se on HA t i t e r . - 120 -weeks. Not s u r p r i s i n g l y , i n view of the la r g e standard e r r o r and a l s o the lack of e f f e c t of selenium on female lambs, the e f f e c t of selenium f a i l e d to be s i g n i f i c a n t (P>0.05). Breed and re a r i n g did not i n f l u e n c e t i t e r s , but sex r e s u l t e d i n s i g -n i f i c a n t e f f e c t s (P<0.05) at 5 and 7 weeks of age (Figure 17). Male lambs had higher hemagglutination t i t e r s at a l l times. Means were 3.4+0.5, and 5.9±0.5, for females and males at 5 weeks. By 8 weeks, mean t i t e r i n females was 38.6±0.4, compared to 45.4±0.4 i n males. An i n t e r a c t i o n between selenium and sex may account f o r t h i s e f f e c t . Selenium seemed to have l i t t l e i f any e f f e c t on t i t e r i n females, but markedly increased t i t e r i n males (Figure 18). Males not tre a t e d with Se had t i t e r s s i m i l a r to those of females. Thus the mean t i t e r was highest f o r selenium-treated males throughout the t r i a l . While i r o n did not a f f e c t t i t e r , the selenium X i r o n i n t e r a c t i o n may have become s i g n i f i c a n t i f the experiment had been continued. I r o n and selenium are both involved i n the immune response, and may p o s s i b l y i n t e r -act a d d i t i v e l y or s y n e r g i s t i c a l l y (Figure 19). Selenium and/or vitamin E s i g n i f i c a n t l y r a i s e d the hemagglutination t i t e r to sheep red blood c e l l s i n a study with weanling swine (Peplowski _et a l . , 1980). Peplowski et a l . (1980) found that an immediate source of these n u t r i e n t s , such as i n j e c t i o n s or a high d i e t a r y concentration pro-vided on a short-term b a s i s , enhanced the immune response i n young pigs m a r g i n a l l y d e f i c i e n t i n Se and/or vitamin E. A higher t i t e r was obtained with high d i e t a r y Se than with the high Se dosage i n j e c t e d . The d i f f e r e n c e i n response to selenium between our r e s u l t s and those of Peplowski et ajl. (1980) can be explained by sev e r a l f a c t o r s , mainly the - 121 -5 6 7 8 Weeks o f Age Figure 18. E f f e c t of selenium x sex i n t e r a c t i o n on hemagglution t i t e r . - 122 -- 123 -dosage and timing of selenium use, i n a d d i t i o n to the presence of Se i n t e r -a c t i o n s . In the present study 1.5 mg Se was i n j e c t e d at b i r t h , and CRBC antigen was i n j e c t e d at 4-4.5 weeks of age and weekly t h e r e a f t e r ; by t h i s time, while plasma selenium was s t i l l s i g n i f i c a n t l y higher i n the +Se group, many lambs i n both groups were marginally d e f i c i e n t i n Se s t a t u s . In c o n t r a s t , Peplowski et a l . (1980) used weaned swine of the same age, the same schedule of antigen i n j e c t i o n s using a s i m i l a r antigen, but the Se treatment simultaneously provided 0.50 ppm Se, which i s f i v e times the d i e t a r y requirement. S i m i l a r l y , Se dosage f o r i n j e c t e d weanling pigs was 6.0 mg, which was four times the l e v e l we used f o r lambs of s i m i l a r s i z e . Work with mice which demonstrated an immunologic response to selenium, a l s o involved high l e v e l s of d i e t a r y selenium ( S p a l l h o l z et a l . , 1973; S p a l l h o l z et a l . , 1974; S p a l l h o l z et a i l . , 1975). The hemagglutination technique i s very s e n s i t i v e but important l i m i t a t i o n s i n c l u d e the "occasional lack of r e p r o d u c i b i l i t y , q u a l i t a t i v e r ather than q u a n t i t a t i v e nature, and o c c a s i o n a l n o n - s p e c i f i c i t y " ( S t a v i t s k y , 1954). According to S t a v i t s k y (1954), i t s s e n s i t i v i t y can be a l i a b i l i t y , i n that small amounts of heterologous antibody or antigen i n a n t i s e r a or t e s t antigen may confuse the estimation of the major a n t i -bodies. The adaptation of the hemagglutination t e s t f o r t h i s experiment, s p e c i f i c a l l y the use of CRBC antigen, probably induced production of heterologous antibody. Because the maximal s i z e of a s p e c i f i c a n t i g e n i c determinant i s equivalent to four to s i x amino aci d s or simple sugars, the p o t e n t i a l number of d i f f e r e n t combining s i t e s on the red c e l l membrane i s extremely large (Garvey et a l _ . , 1977, p. 133). In e f f e c t , then, a - 124 -m u l t i t u d e of p o t e n t i a l antigens, each at extremely low and v a r i a b l e concen-t r a t i o n s , were then i n j e c t e d i n each weekly dose of 1 ml c e l l suspension. The presence of heterologous a n t i b o d i e s f r u s t r a t e d the i n t e r p r e t a t i o n of the s e r i a l d i l u t i o n r e s u l t s . Instead of a r e l a t i v e l y c l e a r cut d i s t i n c -t i o n between p o s i t i v e and negative readings, frequently as many as 4 or 6 d i l u t i o n s i n sequence would appear intermediate between p o s i t i v e and nega-t i v e . This c o n t r i b u t e d to poor r e p r o d u c i b i l i t y of the technique. - 125 -CONCLUSIONS This study has i n v e s t i g a t e d s e v e r a l aspects of i r o n and selenium sup-plementation of newborn lambs. Iron supplementation had a profound i n f l u -ence on lamb metabolism, apparently a f f e c t i n g most blood metabolites measured, as w e l l as b e t a - g l o b u l i n ( t r a n s f e r r i n ) , plasma i r o n , Hb, and PCV. Iron supplementation unexpectedly depressed plasma selenium l e v e l s , and a l s o enhanced gamma g l o b u l i n production at 6 weeks of age, i n d i c a t i n g that lymphoid t i s s u e s may be p a r t i c u l a r l y vulnerable to preweaning i r o n d e f i c i e n c y , as i n r a t s (Baggs and M i l l e r , 1973). However, i r o n had l i t t l e e f f e c t on hemagglutination t i t e r except i n selenium-treated lambs. I n j e c t i o n of 500 mg Fe s i g n i f i c a n t l y (P<0.05) increased hemoglobin from 2 to 11 weeks of age i n T r i a l 1, and from 1 to 8 weeks i n T r i a l 3. While i r o n dosages of e i t h e r 250 or 500 mg prevented the depression of Hb and PCV from b i r t h to 30 days, plasma i r o n was s i g n i f i c a n t l y higher (P<0.05) at 4 weeks i n lambs r e c e i v i n g 500 mg Fe. A s i g n i f i c a n t p roportion of c o n t r o l lambs were anemic at 3-4 weeks of age i n a l l s t u d i e s . The number of lambs defined as anemic v a r i e d according to the t r i a l , and the parameter used. For example, 42%, 21% and 50% of the c o n t r o l lambs were anemic i n T r i a l 2 based on the c r i t e r i a of <28% PCV, <9.2 g/d£ Hb, and <100 ug/d£ plasma i r o n . The assessment of anemia should t h e r e f o r e be based on normal values f o r the f l o c k r a t h e r than l i t e r a t u r e values. PCV i s highly c o r r e l a t e d with Hb i n lambs 0-6 weeks of age; conse-quently, PCV can be measured instead of Hb to assess i r o n s t a t u s . The two selenium treatments d i d not adequately c o n t r a s t l e v e l s of selenium s t a t u s . As plasma selenium l e v e l s i n both the selenium-injected - 126 -l a m b s a n d c o n t r o l l a m b s v a r i e d f r o m m a r g i n a l t o a d e q u a t e l e v e l s , t h e e f f e c t s o f s e l e n i u m t r e a t m e n t c o u l d n o t be c o n s i d e r e d c o n c l u s i v e . P r o b a b l y some s e l e n i u m t r e a t m e n t e f f e c t s w e r e o b s c u r e d . S e l e n i u m t r e a t m e n t d i d n o t a f f e c t Hb a n d PCV v a l u e s s i g n i f i c a n t l y ( P > 0 . 0 5 ) . W h i l e s e l e n i u m may h a v e a r o l e i n heme m e t a b o l i s m i n t h e b o n e m a r r o w , t h e c i r c u l a t i n g h e m o g l o b i n c o n c e n t r a t i o n may n o t be a f f e c t e d e x c e p t a t m a r k e d l y d e f i c i e n t l e v e l s o f S e . A w e i g h t r e s p o n s e t o i r o n t r e a t m e n t , b u t n o t s e l e n i u m t r e a t m e n t , w a s o b s e r v e d i n T r i a l s 1 a n d 3 . I n T r i a l 3 , i r o n - t r e a t e d l a m b s w e r e s l i g h t l y h e a v i e r t h a n c o n t r o l s f r o m 2 w e e k s ( P < 0 . 0 5 ) t o 8 w e e k s ( P < 0 . 0 1 ) o f a g e . T r i a l 1 was c o n t i n u e d t o 11 w e e k s , a n d i n d i c a t e d t h a t c o n t r o l l a m b s b e g a n t o c a t c h up t o i r o n - t r e a t e d l a m b s a f t e r 8 w e e k s o f a g e . A w e i g h t r e s p o n s e t o i r o n t r e a t m e n t was c o n s i s t e n t l y o b s e r v e d i n t h e f a s t e r g r o w i n g l a m b s , i e . m a l e a n d / o r s i n g l e l a m b s , a n d n o t i n t w i n l a m b s . I r o n t r e a t m e n t d i d n o t i n f l u e n c e a v e r a g e w e i g h t g a i n i n T r i a l 2 , p o s s i b l y b e c a u s e o f t h e l a r g e n u m b e r s o f t w i n l a m b s . The v e r y l o w i n c i d e n c e o f d i s e a s e d u r i n g t h a t p a r t i c u l a r t r i a l may a l s o h a v e b e e n a f a c t o r . P r e l i m i n a r y i n f o r m a t i o n was p r o v i d e d on t h e e f f e c t o f i r o n d e f i c i e n c y o n t h e l a m b p l a s m a p r o f i l e . S u r p r i s i n g l y , no c o m p r e h e n s i v e s t u d y o f t h e p l a s m a p r o f i l e i n i r o n d e f i c i e n c y h a s a p p a r e n t l y b e e n p u b l i s h e d f o r a n y s p e c i e s , i n c l u d i n g m a n . H o w e v e r , c o m p a r i s o n o f t h e d a t a w i t h t h e l i t e r a -t u r e on human m e d i c i n e i n d i c a t e s t h a t i r o n d e f i c i e n c y a f f e c t s p l a s m a m e t a b o l i t e s s i m i l a r l y i n l a m b s a n d h u m a n s . I n b o t h s p e c i e s , i r o n d e f i -c i e n c y i n c r e a s e s p l a s m a g l u c o s e a n d a l k a l i n e p h o s p h a t a s e , w h i l e d e c r e a s i n g p l a s m a i r o n a n d c h o l e s t e r o l . O t h e r c o m p a r a t i v e d a t a was l a c k i n g f o r h u m a n s . - 127 -I ron had a major impact on the plasma p r o f i l e i n T r i a l 2, and P j , glucose, c h o l e s t e r o l , t o t a l p r o t e i n , AP, AT, and plasma i r o n responded l i n e a r l y to i r o n dosage. Many parameters (glucose, BUN, c h o l e s t e r o l , TP, AP, and AT) were a l s o s i g n i f i c a n t l y c o r r e l a t e d with plasma i r o n . These r e s u l t s suggest that i r o n s t a t u s may broadly i n f l u e n c e metabolism through m u l t i p l e r o l e s i n t i s s u e enzyme systems. Many i r o n enzymes are reduced at an e a r l y stage of i r o n d e p l e t i o n , and t h e r e f o r e would be a f f e c t e d by even a mild degree of d e f i c i e n c y . Levels of i r o n s u f f i c i e n t for Hb maintenance may not provide s u f f i c e n t i r o n for other f u n c t i o n s , as Hb may have p r i o r i t y for i r o n . A weight response to i r o n supplementation may depend on the p r o v i s i o n of adequate i r o n for t i s s u e enzymes, which may e x p l a i n c o n f l i c t i n g r e s u l t s of i r o n on weight at dose l e v e l s of 150 and 300 mg Fe used i n e a r l i e r s t u d i e s . I ron and selenium supplementation a f f e c t e d plasma p r o t e i n f r a c t i o n s . As expected, i r o n d e f i c i e n c y r e s u l t e d i n s i g n i f i c a n t l y higher (P<0.01) 3- g l o b u l i n l e v e l s at 2 and 4 weeks of age, due to increased t r a n s f e r r i n p r o d u c t i n . Iron i n j e c t i o n increased (P<0.001) albumin at k weeks, and a l s o increased y - g l o b u l i n l e v e l s at 6 weeks of age. An e a r l i e r e f f e c t of i r o n may not be observed as y - g l o b u l i n production i s depressed i n the f i r s t weeks of l i f e ; t h e r e f o r e i t would be i n t e r e s t i n g to f o l l o w y - g l o b u l i n l e v e l s a f t e r 6 weeks. Selenium treatment s i g n i f i c a n t l y (P<0.05) depressed B- g l o b u l i n l e v e l s at 2 weeks, and the reason f o r t h i s was unknown. E f f e c t s of i r o n and selenium on disease r e s i s t e n c e were measured by s u s c e p t i b i l i t y to soremouth, and anti-CRBC hemagglutination t i t e r . Selen-ium treatment appeared to i n f l u e n c e s u s c e p t i b i l i t y of lambs to soremouth i n f e c t i o n . The response of lambs to a n t i g e n i c challenge from chicken RBCs - 128 -was apparently influenced by selenium as w e l l , even though Se status of the Se - i n j e c t e d lamb was suboptimal at time of challenge. Iron treatment had a l e s s e r i n f l u e n c e , p o s s i b l y because lambs were already eating creep-feed co n t a i n i n g adequate d i e t a r y i r o n during t h i s part of the t r i a l . Iron may be r e l a t i v e l y more important i n r e s i s t a n c e to such diseases as F£. c o l i scours, i n which phagocytosis i s more c r u c i a l than humoral immunity. Scours occurred i n most lamb crops, but the r e l a t i o n s h i p of scours to i r o n treatment, and i t s e f f e c t on growth, could not be measured. The data presentd i n t h i s study suggest that i r o n d e f i c i e n c y i n suck-l i n g lambs a f f e c t s the o v e r a l l metabolism, growth r a t e , and health of the lambs. Anemia per se does not appear to be the major concern i n i r o n d e f i -c i ency. Other e f f e c t s of i r o n d e f i c i e n c y include suboptimal f u n c t i o n i n g of many i r o n enzyme systems, impairment of various immune systems, and p o s s i -b l y malabsorption syndromes and/or g a s t r o i n t e s t i n a l l e s i o n s . Quantita-t i v e l y , these c o n d i t i o n s may be more important and more p e r s i s t e n t than the anemia. 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Res. 23: 966-971. - 147 -APPENDIX - 148 -APPENDIX 1. Composition of creep-feed A. Ingredient Composition Barley 80.0% Soybean Meal 10.0% B u t t e r f i e l d s 32% C a t t l e Supplement, Reg. No. 5273 10.0% B. N u t r i e n t Composition, As-fed Basis ( c a l c u l a t e d ) P r o t e i n 1 17.0% F i b e r 5 « 5 * Calcium 0.33% Phosphorus 0.20% Iron 9 5 PP m not more than 2% equivalent p r o t e i n from urea. APPENDIX 2. Procedures used in analysis of plasma constituents METABOLITE REFERENCE Calcium Inorganic Phosphate Glucose BUN C h o l e s t e r o l Total P r o t e i n Albumin A l k a l i n e Phosphatase L a c t a t e Dehydrogenase Aspartate Transaminase K e s s l e r , G. and Wolfman, M. 1964. C l i n . Chem. 10:686 Hurst, R.O. 1964. Can. 0. Biochem. 42:287 Bondar, R.J.L. and Mead, D.C. 1974. C l i n . Chem. 20:586. Marsh, W.H., Fingerhut, B. and M i l l e r , H. 1965. C l i n . Chem. 11:624. Levine, 3., Morganstern, S. and V l a s t e l i c a , D. 1967. Automation Anal. Chem., Technicon Symp. 1967. Skeggs, L.T. and Hochstrasser, H. 1964. C l i n . Chem. 10:918. Doumas, B.T., Watson, W.A. and Biggs, H.G. 1971. C l i n . Chem. Acta 31:87. Morganstern, S., K e s s l e r , G., Averbach, 3. F l o r , R.V., and K l e i n , B. 1965. C l i n . Chem. 11:876. Hochella, N.3. and Weinhouse, S. 1965. A n a l y t i c a l Biochem 13:322. Morganstern, S., Oklander, M., Averbach, 3., Kaufman, 3. and K l e i n , B. 1966. C l i n . Chem. 12:95. Plasma Iron Ca r t e r , P. 1971. A n a l y t i c a l Biochem. 40:450. APPENDIX 3. E f f e c t of i ron dextran i n j e c t i o n on lamb weight, hemoglobin and packed c e l l volume from b i r t h to 11 weeks ( T r i a l 1 ) . WEIGHT (kg ± SE) HEMOGLOBIN ( g / d £ ± SE) P . C . V . (% ± SE) Age C o n t r o l Iron Contro l Iron Contro l I ron n= 17 n= 18 n=17 n= 18 n=17 n=18 B i r t h 3 . 9 ± 0 . 2 4 . 2 ± 0 . 3 N S t 1 3 . 7 ± 0 . 6 1 4 . 5 ± 0 . 4 NS 3 7 . 8 + 1.5 4 0 . 2 ± 1 .2 NS 1 week 4 . 5 ± 0 . 3 5 . 3 ± 0 . 3 NS 1 2 . 3 ± 0 . 5 1 2 . 9 ± 0 . 2 NS 3 3 . 5 + 1.1 3 5 . 8 ± 0 . 8 NS 2 w e e k s 6 . 2 ± 0 . 4 7 . 4 ± 0 . 5 NS 1 0 . 9 ± 0 . 4 1 3 . 3 ± 0 . 2 *** 3 1 . 2 + 1.1 3 8 . 8 ± 0 . 8 *** 3 w e e k s 8 .1 ± 0 . 4 9 . 5 ± 0 . 6 NS 1 0 . 7 ± 0 . 4 1 3 . 5 ± 0 . 2 *** 3 0 . 9 + 1 .3 3 9 . 4 ± 0 . 8 *** 4 w e e k s 9 . 9 ± 0 . 5 1 1 . 4 ± 0 . 7 NS 1 1 . 1 ± 0 . 3 1 3 . 7 ± 0 . 3 *** 3 2 . 2 + 0 . 9 3 9 . 5 ± 0 . 7 *** 5 w e e k s 1 1 . 5 ± 0 . 6 1 3 . 5 ± 0 . 8 NS 1 1 . 4 ± 0 . 3 1 3 . 7 ± 0 . 2 *** 3 2 . 8 + 0 . 9 3 9 . 0 ± 0 . 7 *** 6 w e e k s 1 3 . 4 ± 0 . 6 1 5 . 6 ± 1 .0 # 1 1 . 9 ± 0 . 3 1 3 . 7 ± 0 . 3 *** 3 4 . 4 + 0 . 8 3 9 . 3 ± 0 . 7 *** 7 w e e k s 1 5 . 2 ± 0 . 7 1 7 . 9 ± 1.1 * 1 2 . 0 ± 0 . 2 1 3 . 2 ± 0 . 2 *** 3 4 . 9 + 0 . 6 3 8 . 3 ± 0 . 5 *** 8 w e e k s 1 7 . 2 ± 0 . 8 1 9 . 8 ± 1 .2 * 1 2 . 0 ± 0 . 2 1 2 . 8 ± 0 . 2 3 5 . 4 + 0 . 7 3 7 . 3 ± 0 . 6 * 9 w e e k s 1 8 . 8 ± 0 . 8 2 1 . 4 ± 1 .2 * 1 2 . 2 ± 0 . 1 1 2 . 6 ± 0 . 2 * 3 5 . 5 + 0 . 4 3 6 . 9 ± 0 . 7 * 10 w e e k s 2 0 . 5 ± 0 . 8 2 3 . 3 ± 1 .3 NS 1 2 . 3 ± 0 . 2 1 2 . 8 ± 0 . 2 * 3 6 . 0 + 0 . 7 3 6 . 8 ± 0 . 6 NS 11 w e e k s 2 2 . 3 ± 0 . 9 2 4 . 6 ± 1 .3 NS 1 2 . 0 ± 0 . 3 1 2 . 6 ± 0 . 2 * 3 5 . 2 + 0 . 7 3 6 . 9 ± 0 . 6 * TNS P > 0 . 0 5 ; * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 APPENDIX 4 . Effect of three levels of iron treatment on hemoglobin and packed c e l l volume between birth and weaning (Trial 2) Control TREATMENTS 250 mg Fe 500 mg Fe Significance of contrasts 1 H e m o g l o b i n ( g / c U ± SE ) 2 d a y s 16 d a y s 30 d a y s 4 4 d a y s P . C . V . {% i 2 d a y s 16 d a y s 30 d a y s 4 4 d a y s SE ) 20 2 4 2 4 1 3 . 6 + 0 . 4 1 2 . 9 + 0 . 4 1 3 . 3 + 0 . 4 1 0 . 6 + 0 . 3 1 3 . 2 + 0 . 3 1 3 . 5 + 0 . 2 1 0 . 3 + 0 . 3 1 3 . 5 + 0 . 4 1 4 . 5 + 0 . 2 1 1 . 8 + 0 . 2 1 2 . 4 + 0 . 2 1 3 . 7 + 0 . 2 3 8 . 0 + 1 .0 3 6 . 6 + 1 .0 3 7 . 4 + 1.1 2 9 . 3 + 1 .0 3 7 . 2 + 0 . 7 3 8 . 6 + 0 . 6 2 9 . 2 + 0 . 9 3 7 . 2 + 0 . 7 4 1 . 0 + 0 . 7 3 4 . 0 + 0 . 7 3 6 . 7 + 0 . 5 4 0 . 0 + 0 . 7 NS L 1 » L 2 *-1 2 NS C x * * * C ! * * * , C 2 * I P r e d e t e r m i n e d o r t h o g o n a l c o n t r a s t s w e r e a s f o l l o w s : C i = C o n t r o l v s . b o t h F e t r e a t m e n t s C 2 = 2 5 0 mg F e v s . 500 mg F e NS = n o t s i g n i f i c a n t , P > 0 . 0 5 ; * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 . APPENDIX 5. Regression analysis of Trial 2 Plasma Profile Data: Partial Correlation Coefficients Metabolite n=65 POTENTIAL INDEPENDENT VARIABLES Birth Weight Weight Gain Hb PCV Iron Glucose T.P. P i NS NS NS NS NS NS -G l u c o s e NS NS - 0 . 3 0 6 * - 0 . 2 5 3 * - 0 . 3 9 8 * * * - -B . U . N . 0 . 4 6 0 * * * NS NS NS 0 . 2 6 5 * - -C h o l e s t e r o l NS NS 0 . 3 5 9 * * 0 . 4 3 2 * * * 0 . 3 3 6 * * - 0 . 3 3 6 * * -T o t a l P r o t e i n NS NS - 0 . 3 2 8 * * - 0 . 2 9 2 * - 0 . 4 2 4 * * * - -A l b u m i n NS NS NS NS NS - NS A . P . - 0 . 4 7 6 NS - 0 . 4 2 7 * * * - 0 . 4 1 7 * * * - 0 . 4 6 8 * * * - -L . D . H . NS NS NS NS NS - -A . T . NS - 0 . 2 9 8 * NS NS 0 . 2 8 8 * - -I r o n 0 . 3 2 9 * * NS 0 . 4 8 4 * * * 0 . 4 7 7 * * * - - -* P < 0 . 0 5 * * P < 0 . 0 1 * * * P < 0 . 0 0 1 APPENDIX 6. Regression Analysis of Trial 2 Plasma Profile Regression Equations Data: METABOLITE1 REGRESSION EQUATION R-SQUARED F-PROBABILITY G l u c o s e Y = 9 8 . 1 2 7 4 + 0 . 6 9 8 1 W - 0 . 0 6 1 1 I 0 . 2 3 1 0 . 0 0 0 2 9 1 C h o l e s t e r o l Y = 6 0 . 8 9 4 9 - 1 .5701W + 2 . 1 8 7 1 P 0 . 2 5 8 0 . 0 0 0 0 9 4 T o t a l P r o t e i n Y = 5 . 8 6 0 6 - 0 . 0 0 2 4 1 0 . 1 8 0 0 . 0 0 0 4 2 7 A l k a l i n e P h o s p h a t a s e Y = 2 5 0 3 . 7 8 4 1 - 8 5 . 6 7 5 7 B - 6 7 . 5 2 3 6 H 0 . 3 4 9 0 . 0 0 0 0 0 2 A s p a r t a t e T r a n s a m i n a s e Y = 1 3 0 . 1 2 9 1 - 2 . 2 7 3 2 W + 0 . 1 2 1 1 1 0 . 2 0 0 0 . 0 0 0 9 9 3 I r o n Y = - 1 2 6 . 3 8 8 0 + 1 0 . 7 6 4 8 B + 1 7 . 1 7 4 5 H 0 . 2 9 7 0 . 0 0 0 0 1 8 w h e r e Y = d e p e n d e n t v a r i a b l e ( g l u c o s e , c h o l e s t e r o l , e t c . ) W = i n d e p e n d e n t v a r i a b l e w e i g h t g a i n ( w e i g h t a t s a m p l i n g - b i r t h w e i g h t ) I = i n d e p e n d e n t v a r i a b l e p l a s m a i r o n P = i n d e p e n d e n t v a r i a b l e p a c k e d c e l l v o l u m e B = i n d e p e n d e n t v a r i a b l e b i r t h w e i g h t H = i n d e p e n d e n t v a r i a b l e h e m o g l o b i n A l b u m i n a n d LDH n o t i n c l u d e d d u e t o l a c k o f s i g n i f i c a n t c o r r e l a t i o n s . APPENDIX 7. Effect of iron level on lamb weight (Trial 2). TREATMENTS Age Mean Control 250 mg Fe 500 mg Fe Significance of Contrasts and Main E f f e c t s 1 . 2 2 days 9 days 16 days 3 days 0 days 8 days 4 4 days 1 days n 4 . 4 2 ± 0 . 1 3 6 . 5 ± 0 . 2 8 . 5 ± 0 . 3 1 0 . 5 ± 0 . 3 1 2 . 0 ± 0 . 3 1 4 . 0 ± 0 . 4 1 6 . 0 ± 0 . 4 1 8 . 0 ± 0 . 5 6 8 X ± S.E. (kg) 4 . 4 ± 0 . 3 6 . 4 ± 0 . 3 8 . 3 ± 0 . 3 1 0 . 2 ± 0 . 5 1 1 . 8 ± 0 . 5 1 3 . 7 ± 0 . 6 1 5 . 6 ± 0 . 6 1 7 . 6 ± 0 . 6 20 4 . 4 ± 0 . 2 6 . 6 ± 0 . 3 8 . 8 ± 0 . 3 1 0 . 9 ± 0 . 5 1 2 . 6 ± 0 . 6 1 4 . 6 ± 0 . 7 1 6 . 5 ± 0 . 7 1 8 . 6 ± 0 . 8 2 4 4 . 4 ± 0 . 2 6 . 4 ± 0 . 4 8 . 3 ± 0 . 5 1 0 . 2 * 0 . 5 1 1 . 8 i 0 . 6 1 3 . 8 * 0 . 7 1 5 . 8 ± 0 . 9 1 7 . 7 * 0 . 9 2 4 Age***, B.Wt.*** Rear*, Age**, B.Wt.*** Rear**, Age*, B.Wt.*** Rear**, B.Wt.*** Rear***, B.Wt.*** Rear**, TrxSex*, Age**, B.Wt.*** Rear**, B.Wt.*** Rear**, B.Wt.*** C o v a r i a b l e s were age and birthweight; main e f f e c t s were treatment, breed, sex, r e a r i n g . 2 * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 . APPENDIX 8. E f f e c t o f i r o n and selenium on hemoglobin ( T r i a l 3). TREATMENTS S i g n i f i c a n c e S i g n i f i c a n t Age C o n t r o l SE FE SE + FE o f C o n t r a s t s 1 Main E f f e c t s 2 X ± S . E . ( g / d £ ) 31 30 31 2 9 2 d a y s 1 3 . 5 + 0 . 4 1 3 . 3 + 0 . 4 1 3 . 8 + 0 . 3 1 4 . 0 ± 0 . 3 NS NS 1 w e e k 1 1 . 4 + 0 . 4 1 1 . 7 + 0 . 3 1 3 . 0 + 0 . 3 1 2 . 7 + 0 . 3 NS 2 w e e k s 1 0 . 7 + 0 . 3 1 0 . 5 + 0 . 3 1 3 . 2 + 0 . 2 1 3 . 2 + 0 . 2 NS 3 w e e k s 1 0 . 9 + 0 . 3 1 0 . 6 + 0 . 5 1 3 . 6 + 0 . 3 1 3 . 4 + 0 . 2 p r r * * * NS 4 w e e k s 1 1 . 0 + 0 . 3 1 1 . 0 + 0 . 3 1 3 . 4 + 0 . 3 1 3 . 1 + 0 . 3 pp_*** NS 5 w e e k s 1 1 . 4 + 0 . 2 1 1 . 7 + 0 . 3 1 3 . 8 + 0 . 2 1 3 . 7 + 0 . 2 p p * * * NS 6 w e e k s 1 1 . 9 + 0 . 2 1 1 . 8 + 0 . 2 1 3 . 5 + 0 . 2 1 3 . 4 + 0 . 2 p p * * * NS 7 w e e k s 1 2 . 0 + 0 . 2 1 2 . 0 + 0 . 2 1 3 . 2 + 0 . 2 1 3 . 2 + 0 . 2 p p * * * NS 8 w e e k s 1 2 . 3 + 0 . 2 1 2 . 4 + 0 . 2 1 2 . 9 + 0 . 2 1 2 . 9 + 0 . 2 p p * * NS P r e d e t e r m i n e d o r t h o g o n a l c o n t r a s t s w e r e a s f o l l o w s : F E = C o n t r o l + Se v s . F e + ( S e + F e ) ; SE = S e + ( S e + F e ) v s . C o n t r o l + F e ; SE X FE = C o n t r o l + ( Se + F e ) v s . F e + S e ; N S > 0 . 0 5 ; * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 . ^ M a i n e f f e c t s t e s t e d w e r e r e p l i c a t e , t r e a t m e n t , b r e e d , s e x , a n d r e a r i n g . APPENDIX 9. Effect of iron and selenium on packed c e l l volume (Trial 3 ) . TREATMENTS Significance Significant Control SE FE SE + FE of Contrasts 1 Main Effects X ± S . E . (% PCV ) 31 30 31 2 d a y s 3 7 . 9 + 0 . 9 3 7 . 0 + 1 .0 3 9 . 0 ± 0 . 8 3 9 . 3 + 0 . 9 NS NS 1 week 3 1 . 0 + 0 . 7 3 1 . 7 + 0 . 8 3 6 . 2 ± 0 . 7 3 5 . 8 + 0 . 6 R e a r * * * 2 w e e k s 3 0 . 8 + 0 . 6 3 0 . 6 + 0 . 8 3 9 . 1 ± 0 . 6 3 8 . 5 + 0 . 7 p p * * * R e a r * 3 w e e k s 3 1 . 2 + 0 . 7 3 1 . 9 + 1 .0 4 1 . 1 ± 0 . 6 3 9 . 2 + 0 . 7 p p * * * NS 4 w e e k s 3 2 . 3 + 0 . 7 3 2 . 5 + 0 . 8 4 0 . 5 ± 0 . 6 3 9 . 4 + 0 . 6 p p * * * NS 5 w e e k s 3 4 . 1 + 0 . 7 3 4 . 7 + 0 . 7 4 0 . 2 ± 0 . 5 3 9 . 6 + 0 . 6 p p * * * R e p * 6 w e e k s 3 5 . 1 + 0 . 6 3 5 . 9 + 0 . 6 3 9 . 6 ± 0 . 5 3 9 . 0 + 0 . 6 p p * * * S e x * 7 w e e k s 3 6 . 1 + 0 . 6 3 6 . 1 + 0 . 5 3 8 . 8 ± 0 . 4 3 8 . 6 + 0 . 6 p p * * * S e x * 8 w e e k s 3 6 . 3 + 0 . 5 3 6 . 5 + 0 . 5 3 7 . 8 ± 0 . 5 3 7 . 4 + 0 . 5 F E * S e x * Predetermined o r t h o g o n a l s c o n t r a s t s w e r e a s f o l l o w s : FE = C o n t r o l + Se v s . F e + ( S e + F e ) ; SE = Se + ( S e + F e ) v s . C o n t r o l + F e ; S E x F E = C o n t r o l + ( S e + F e ) v s . F e + S e ; N S > 0 . 0 5 ; * P < 0 . 0 5 ; * * P < 0 . 0 1 ; * * * P < 0 . 0 0 1 . 2 M a i n e f f e c t s t e s t e d w e r e r e p l i c a t e , t r e a t m e n t , b r e e d , s e x , a n d r e a r i n g . - 157 -APPENDIX 10. Regression equations for hemoglobin versus packed c e l l volume (Trial 3). fAGE REGRESSION EQUATION *R' 2 d a y s Y = 0 . 4 8 5 1 + 0 . 3 4 1 9 X 0 . 8 6 2 4 4 d a y s Y = 0 . 4 4 2 9 + 0 . 3 7 3 8 X 0 . 8 8 2 9 6 d a y s Y = - 2 . 0 0 0 + 0 . 4 1 2 8 X 0 . 9 1 3 6 8 d a y s Y = - 0 . 0 6 2 5 + 0 . 3 6 0 9 X 0 . 8 4 3 6 2 w e e k s Y = 0 . 2 1 2 0 + 0 . 3 3 4 6 X 0 . 9 2 2 8 3 w e e k s Y = - 0 . 2 1 2 3 + 0 . 3 3 9 7 X 0 . 8 8 4 0 4 w e e k s Y = 0 . 6 8 0 1 + 0 . 3 1 8 7 X 0 . 7 8 8 2 5 w e e k s Y = 0 . 5 2 3 5 + 0 . 3 2 9 2 X 0 . 8 5 6 8 6 w e e k s A Y = 1 . 1 9 9 2 + 0 . 2 8 6 1 X 0 . 7 3 4 2 7 w e e k s Y = 3 . 4 1 7 + 0 . 2 4 6 7 X 0 . 5 6 1 4 8 w e e k s Y = 4 . 0 8 4 + 0 . 2 3 3 4 X 0 . 5 4 0 9 w h e r e Y = a + bX a n d Y = d e p e n d e n t v a r i a b l e ( H b ) a = c o n s t a n t , o r i n t e r c e p t b = r e g r e s s i o n c o e f f i c i e n t X = i n d e p e n d e n t v a r i a b l e ( PCV ) * R 2 , t h e c o e f f i c i e n t o f d e t e r m i n a t i o n , r e p r e s e n t s t h e p r o p o r t i o n o f t h e t o t a l t r e a t m e n t sum o f s q u a r e s a c c o u n t e d f o r by r e g r e s s i o n . t N u m b e r o f o b s e r v a t i o n s e a c h a t 4 d a y s a n d 6 d a y s = 4 3 l a m b s . A l l o t h e r p e r i o d s i n c l u d e d a t a f r o m a l l 121 l a m b s i n T r i a l 3 . APPENDIX 11 E f f e c t of i ron and selenium on lamb weight ( T r i a l 3). TREATMENTS _ — S i g n i f i c a n c e S i g n i f i c a n t Contro l SE FE SE + FE o f Contras t s 1 Main E f f e c t s 2 "X± S.E. (kg) n 31 30 31 29 2 days 3.9 ± 0.2 3.8 ± 0.1 4.1 ± 0.2 3.9 + 0.2 NS Rear*** 1 week 5.5 ± 0.2 5.4 ± 0.2 5.8 ± 0.3 5.7 + 0.3 NS Rep***, Rear*** 2 weeks 7.4 ± 0.3 7.2 ± 0.3 8.0 ± 0.3 7.8 + 0.4 FE* Rep***, Rear*** 3 weeks 9.1 ± 0.3 8.9 ± 0.3 9.9 ± 0.5 9.7 + 0.5 FE* Rep** 4 weeks 10.9 ± 0.4 10.6 ± 0.4 11.7 ± 0.5 11.8 + 0.6 FE* Rep*, Rear*** 5 weeks 12.6 ± 0.4 12.2 ± 0.4 14.0 ± 0.7 13.6 + 0.6 FE* Rep*, Rear*** 6 weeks 14.6 ± 0.5 14.0 ± 0.5 15.7 ± 0.7 15.6 + 0.7 FE** Rep*, Rear*** 7 weeks 16.7 ± 0.6 16.0 ± 0.5 17.9 ± 0.8 17.5 + 0.7 FE** Rep*, Rear*** 8 weeks 18.5 ± 0.6 18.1+ 0.5 19.9 ± 0.9 19.5 + 0.9 FE** Rep*, Rear*** P r e d e t e r m i n e d orthogonal c o n t r a s t s were as f o l l o w s : FE = Contr o l + Se vs. Fe + (Se + Fe); SE = Se + (Se + Fe) vs. C o n t r o l + Fe; FExSE = Control + (Se + Fe) vs. Fe + Se *P<0.05; **P<0.01; ***P<0.001. 2 M a i n e f f e c t s tested by An a l y s i s of Variance were r e p l i c a t e , treatment, breed, sex and r e a r i n g . - 159 -APPENDIX 12. T r i a l 3. Effect of iron, selenium and sex on hemagglutination t i t e r HA TITER (loc^ ± S.E. units) HA TITER ± S.E. (dilution units) 1 IRON TREATMENT -FE +FE -FE +FE AGE - 5 weeks 6 weeks 7 weeks 8 weeks 1.62 ± 0.18 2.82 ± 0.20 3.44 ± 0.15 3.68 ± 0.11 1.50 ± 0.22 2.71 ± 0.21 3.54 ± 0.11 3.80 ± 0.16 5.1 ± 0.5 16.7 ± 0.5 31.2 ± 0.4 39.5 ± 0.4 4.5 ± 0.6 15.0 ± 0.6 34.3 ± 0.4 44.7 ± 0.5 Number of lambs 22 21 22 21 SELENIUM TREATMENT -SE +SE -SE +SE AGE - 5 weeks 6 weeks 7 weeks 8 weeks 1.42 ± 0.19 2.75 ± 0.14 3.35 ± 0.13 3.60 ± 0.13 1.72 ± 0.20 2.78 ± 0.25 3.64 ± 0.13 3.88 ± 0.14 4.1 ± 0.5 15.6 ± 0.4 28.5 ± 0.4 36.6 ± 0.4 5.6 ± 0.6 16.2 ± 0.7 38.2 ± 0.4 48.6 ± 0.4 Number of lambs 23 20 23 20 SEX FEMALE MALE FEMALE MALE AGE - 5 weeks 6 weeks 7 weeks 8 weeks 1.24 ± 0.19 2.70 ± 0.20 3.40 ± 0.12 3.65 ± 0.11 1.77 ± 0.18 2.81 ± 0.20 3.56 ± 0.14 3.82 ± 0.16 3.4 ± 0.5 14.9 ± 0.6 30.0 ± 0.4 38.6 ± 0.4 5.9 ± 0.5 16.7 ± 0.5 35.2 ± 0.4 45.4 ± 0.4 Number of lambs 20 23 20 23 C o n v e r t e d from l o g 2 ( n a t u r a l log) of d i l u t i o n u n i t s . APPENDIX 13. T r i a l 3. Effect of selenium interactions with iron and sex on hemagglutination t i t e r . HA TITER ± S.E. (LOG9 UNITS) HA TITER ± S.E. (DILUTION UNITS)1 TREATMENT CONTROL AGE - 5 weeks 1 .49 ± 0.27 1, 6 weeks 2.85 ± 0.22 2, 7 weeks 3.28 ± 0.21 3, 8 weeks 3.55 ± 0.14 3, number of lambs TREATMENT 11 -SE FEMALE +SE 75 ± 0.27 78 ± 0.35 60 ± 0.20 78 ± 0.17 12 -SE MALE AGE - 5 weeks 1.42 ± 0.30 1.42 6 weeks 2.88 ± 0.23 2.68 7 weeks 3.39 ± 0.14 3.32 8 weeks 3.64 ± 0.25 3.58 number of lambs 9 0.24 0.19 0.20 0.15 11 C o n v e r t e d from l o g 2 ( n a t u r a l log) t i t e r s . +FE 1.35 2.64 3.41 3.64 0.27 0.20 0.16 0.21 11 +SE FEMALE 1.12 2.58 3.41 3.66 0.25 0.31 0.20 0.10 14 +SE+FE 68 78 70 4.01 0.38 0.41 0.16 0.25 +SE MALE 32 01 3.93 4.16 0.16 0.41 0.12 0.28 CONTROL +SE 4.4 17.3 26.6 34.7 0. 0. 0. 5. 16, 36, + + + 0.5 44.0 ± 0.7 0.9 0.6 0.5 11 12 -SE FEMALE -SE MALE 4.1 17.8 29.8 38.2 0. 0. 0. 0. 4.1 14.5 27.7 35.7 0.7 0.5 0.6 0.5 11 +FE 3 14 30 38 0.8 0.6 0.5 0.6 11 +SE FEMALE 3.1 13.1 30.2 38.8 : 0.7 : 0.8 : 0.6 : 0.4 14 +SE+FE 5.4 16.2 40.4 55.0 1.0 1.1 0.5 0.7 9 +SE MALE 10.2 ± 20.4 ± 50.9 ± 64.1 ± 0.5 1.1 0.5 0.8 

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