IRON AND SELENIUM SUPPLEMENTATION OF SHEEP

by

PAULA LEONE DUBESKI

B.Sc, The U n i v e r s i t y o f 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 o f Animal S c i e n c e )

We a c c e p t t h i s t h e s i s as conforming
to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

O c t o b e r 1983

© P a u l a Leone D u b e s k i , 1983
 In p r e s e n t i n g this thesis in partial f u l f i l m e n t of the
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 ABSTRACT

I r o n 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 s e l e n i u m

were investigated i n lambs raised 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 o f i r o n d e x t r a n t r e a t m e n t , 0 and 500 mg F e , u s i n g

35 lambs i n j e c t e d once a t b i r t h . T r i a l 2 i n v o l v e d 66 lambs and 3 l e v e l s o f

iron: 0, 250, and 500 mg. A third t r i a l was r e p l i c a t e d 3 t i m e s , u s i n g a

total o f 121 lambs, i n o r d e r t o determine i f t h e i r o n t r e a t m e n t response

was l i m i t e d by the m a r g i n a l Se s t a t u s o f 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 iron, and a plasma profile (Ca, P j , g l u c o s e , BUN, t o t a l protein,

a l b u m i n , AP, LDH and AT). Additionally, plasma Se, plasma p r o t e i n frac-

t i o n s and d i s e a s e r e s i s t a n c e were measured i n T r i a l 3.

Injection o f 500 mg Fe s i g n i f i c a n t l y (P<0.05) i n c r e a s e d hemoglobin

from 2 t o 11 weeks o f age i n T r i a l 1, and from 1 t o 8 weeks i n T r i a l 3.

While iron dosages of e i t h e r 250 o r 500 mg p r e v e n t e d the d e p r e s s i o n o f

hemoglobin from b i r t h t o 30 days, plasma i r o n and hemoglobin (P<0.05) were

significantly higher a t 4 weeks i n lambs receiving 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 p r o p o r t i o n of c o n t r o l lambs were anemic a t 3-4 weeks

o f age.

Preliminary information was p r o v i d e d on the e f f e c t of i r o n defi-

ciency and o t h e r factors (breed, sex, r e a r i n g , birth weight and growth

r a t e ) on t h e lamb plasma p r o f i l e a t 4 weeks. The d a t a i n d i c a t e t h a t iron

deficiency a f f e c t s plasma m e t a b o l i t e s s i m i l a r l y i n lambs and humans. P^,

glucose, cholesterol, total protein, alkaline phosphatase and a s p a r t a t e

 - i i i -

transaminase responded linearly t o 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 w i t h plasma i r o n .

The interaction of i r o n w i t h selenium was s i g n i f i c a n t (P<0.05) o n l y

for plasma s e l e n i u m levels. Plasma s e l e n i u m a t 4 weeks was i n c r e a s e d i n

lambs i n j e c t e d w i t h selenium and not i n j e c t e d w i t h 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),

w i t h 18 t o 20 lambs per t r e a t m e n t .

D i s e a s e r e s i s t a n c e was a s s e s s e d by s u s c e p t i b i l i t y o f lambs t o s o r e -

mouth; h e m a g g l u t i n a t i o n t i t e r t o a c h i c k e n RBC a n t i g e n ; and gamma g l o b u l i n

levels from 2 t o 6 weeks. Selenium but not i r o n treatment influenced

susceptibility of lambs t o soremouth. The response of lambs t o a n t i g e n i c

c h a l l e n g e from c h i c k e n RBC's was a l s o i n c r e a s e d (P<0.05) by Se treatment a t

birth, even though by t h e time of i n i t i a l c h a l l e n g e a t k weeks, plasma Se

was only slightly higher i n t h e S e - i n j e c t e d lambs (0.098 ppm v s . 0.086

ppm). I r o n had l i t t l e effect on t i t e r , except i n s e l e n i u m - t r e a t e d lambs.

Although iron treatment enhanced gamma g l o b u l i n p r o d u c t i o n a t 6 weeks o f

age, i r o n may be more c r u c i a l t o c e l l u l a r r a t h e r than humoral immunity.

This study consistently demonstrated a dramatic response of blood

hemoglobin t o i r o n t r e a t m e n t , but a l s o i n d i c a t e d t h a t o t h e r a s p e c t s of i r o n

deficiency may be more i m p o r t a n t than anemia. Marginal d e f i c i e n c i e s o f

both iron and selenium may a f f e c t lamb h e a l t h , and thus have an economic

impact on i n t e n s i v e sheep p r o d u c t i o n systems.

 iv

TABLE OF CONTENTS

Page

ABSTRACT i i

LIST OF TABLES v

LIST OF FIGURES vi

ACKNOWLEDGEMENTS v i i

INTRODUCTION 1

LITERATURE REVIEW 5
Iron 5
Selenium 29
I r o n and Selenium I n t e r a c t i o n s 43
MATERIALS AND METHODS 47
E x p e r i m e n t a l Design 47
S t a t i s t i c a l Analysis 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 I r o n 60
Plasma P r o f i l e 65
Weight 72
T r i a l 2 L e v e l of Iron Supplementation 73
Hb, PCV, Plasma I r o n 73
Plasma P r o f i l e 77
Weight 85
T r i a l 3 I r o n and/or Selenium S u p p l e m e n t a t i o n 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 o f Selenium on E p i d e m i o l o g y o f 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 o f i r o n t r e a t m e n t on plasma p r o f i l e a t 24-25

d a y s o f age ( T r i a l 1) 66

Table I I . E f f e c t o f t h r e e l e v e l s o f i r o n s u p p l e m e n t a t i o n on
plasma p r o f i l e a t 30-31 days o f age ( T r i a l 2) 79

Table III. E f f e c t o f r e p l i c a t e on plasma p r o f i l e a t 4 weeks

( T r i a l 3) 101

Table IV. E f f e c t o f i r o n and s e l e n i u m s u p p l e m e n t a t i o n 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 t o

l i t e r a t u r e values 110

Table VI. E f f e c t o f age and t r e a t m e n t on plasma proteins

( 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 o f t r e a t m e n t on plasma p r o t e i n s a t 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 o f s e l e n i u m t r e a t m e n t on e p i d e m i o l o g y o f
soremouth i n f e c t i o n 117
 vi

LIST OF FIGURES

Page

Figure 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 hemoglobin
( T r i a l 1) 62

Figure 2. 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 packed c e l l volume

(Trial 1) 63

Figure 3. E f f e c t o f 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 o f 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 o f i r o n and s e l e n i u m s u p p l e m e n t a t i o n on
hemoglobin ( T r i a l 3) 87

Figure 6. E f f e c t o f i r o n and s e l e n i u m s u p p l e m e n t a t i o n on

packed c e l l volume ( T r i a l 3) 88

Figure 7. Hemoglobin v e r s u s PCV a t 2 days ( T r i a l 3) 91

Figure 8. Hemoglobin v e r s u s PCV a t 4 weeks ( T r i a l 3) 92

Figure 9. Hemoglobin v e r s u s PCV a t 8 weeks ( T r i a l 3) 93

Figure 10. Hemoglobin v e r s u s PCV f o r a l l T r i a l 3 d a t a ,

5% o u t l i e r e x c l u s i o n 94

Figure 11. Hemoglobin v e r s u s PCV f o r a l l T r i a l 3 data 95

Figure 12. E f f e c t o f s e l e n i u m t r e a t m e n t a t b i r t h on plasma

s e l e n i u m a t 4 weeks 98
Figure 13. E f f e c t o f i r o n and s e l e n i u m s u p p l e m e n t a t i o n on
lamb weight 105

Figure 14. D e n s i t o m e t r i c t r a c i n g s o f plasma p r o t e i n s 109

Figure 15. E f f e c t o f i r o n on HA t i t e r 119

Figure 16. E f f e c t o f s e l e n i u m on HA t i t e r 119

Figure 17. E f f e c t o f sex on HA t i t e r 119

Figure 18. E f f e c t o f s e l e n i u m 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 o f 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
 vii

ACKNOWLEDGMENTS

Many people have been instrumental i n t h e development of t h i s

thesis. 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 o f hours

catching and blood sampling sheep with me, and a l s o i n such chores as

b l e e d i n g Dr. Fitzsimmon's c h i c k e n s ( w i t h a g u i l t y eye on the egg p r o d u c t i o n

sheets). However, t h e f r i e n d s h i p developed through this teamwork i s at

l e a s t as h i g h l y v a l u e d as the help and a d v i c e g e n e r o u s l y given. The o t h e r

members o f t h e committee - Dr. D i c k Beames, Dr. Wayne B u c k l e y , Dr. Bob

Fitzsimmons, Dr. Bruce Owen, and Dr. Oim S h e l f o r d - c o n t r i b u t e d both gui-

dance and encouragement a t many s t a g e s . My s i s t e r G l o helped o u t when I

could no l o n g e r tell a "button" from a " b l o b " o f red blood cells i n the

hemagglutination tests. G i l l e s Galzy came t o the rescue i n v a r i o u s emer-

gencies. Doug Loney and the o t h e r staff o f M a c M i l l a n L i b r a r y were always

friendly, helpful and t o l e r a n t . And 3 a n Howe spent many hours doing a

g r e a t job t y p i n g up the f i n a l result.

 - 1-

INTRODUCTION

Iron deficiency i s not considered to be of p r a c t i c a l importance i n

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 e f f e c t s

of iron deficiency i n livestock, other than on hemoglobin production, which

may be the most v i s i b l e but least important aspect of iron deficiency.

Various studies have documented the development of iron deficiency

anemia i n 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

al., 1975), and even i n beef calves on range (Raleigh and Wallace, 1962).

Iron deficiency anemia of pre-natal o r i g i n has also been demonstrated i n as

many as 30% of dairy calves at b i r t h , even though the dams have normal

hemoglobin l e v e l s (Hibbs jet _ a l . , 1963; Tennant ^ t _al., 1975b). Most of

these studies looked only at changes i n hemoglobin and red c e l l indices i n

blood, r e f l e c t i n g 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 p r a c t i c a l purposes c l i n i c a l l y
i r r e l e v a n t , except to the extent that a l i t t l e b i t of anemia
was good for most people" (Fielding, 1975).

Recently, interest i n the prevalence of iron deficiency i n . l i v e s t o c k

and possible e f f e c t s on health and mortality has been stimulated by current

findings on the role of iron i n disease resistance, tissue enzymes, tissue

morphology, and nutrient absorption. Most research on these subjects

concerns humans, as iron deficiency i s the leading worldwide n u t r i t i o n a l

 - 2 -

problem. Iron deficiency has occurred i n humans f o r thousands of years,

particularly in peoples with low-meat diets. While the early Greeks

believed that iron was able to impart strength and force to persons

suffering from weakness (Moore and Dubach, 1960), the connection between

dietary iron and iron-deficiency 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 r e c e n t , 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 first identified in

the 1920's i n p i g l e t s farrowed and raised i n confinement ( W i t t s , 1969, p.

4). Since then iron i n j e c t i o n of pigs has become a standard practice.

I r o n 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 v e a l c a l v e s , but is

otherwise believed to be unusual i n ruminants. Nonetheless, iron defi-

ciency may develop i n the rapidly 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 e l e p h a n t s (Morgan, 1980; Klos and

Lang, 1982).

Lambs may be susceptible to iron deficiency for three reasons: low

placental iron transfer ( H o s k i n s and Hansard, 1964), low milk i r o n content

(Underwood, 1966) and high growth rate. Iron deficiency i s primarily a

production disease, as high growth r a t 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 c o n d i t i o n s c o n d u c i v e to maximum growth

a r e most l i k e l y to be a s s o c i a t e d w i t h anemia ( S i l v e r m a n et a l . , 1970). The

increasing trend towards confinement housing of sheep, and improved manage-

ment p r a c t i c e s such as accelerated lambing s c h e d u l e s , may intensify iron

d e f i c i e n c y problems i n lambs.

This study was undertaken to investigate iron deficiency in lambs

raised under an intensive management system. In the first two trials,

 - 3 -

c o n t r o l lambs were compared w i t h lambs i n j e c t e d w i t h i r o n a t b i r t h . Hemo-

globin, h e m a t o c r i t , and weight were measured at b i r t h and weekly until

weaning, and plasma iron was measured a t 3-4 weeks. The impact of iron

deficiency on o v e r a l l metabolism o f the lamb was a s s e s s e d by measuring a

p r o f i l e o f plasma m e t a b o l i t e s . No i n f o r m a t i o n 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 o f i r o n s t a t u s on t h e plasma p r o f i l e . These experiments also

investigated on t h e i n f l u e n c e o f f a c t o r s such as growth rate, s e x , breed

and rearing.

The first trial compared t r e a t m e n t effects f o r 2 l e v e l s of i r o n , 0

and 500 mg, u s i n g 35 lambs. The second trial i n v o l v e d 66 lambs, which were

divided i n t o 3 t r e a t m e n t groups: 0, 250, and 500 mg Fe. A third trial,

which was r e p l i c a t e d three times, investigated the e f f e c t s of injecting

lambs w i t h 500 mg i r o n , w i t h or w i t h o u t 1.5 mg supplementary selenium.

As t h e e x p e r i m e n t a l f l o c k was m a r g i n a l i n s e l e n i u m s t a t u s , as shown

by t h e c h r o n i c but low i n c i d e n c e o f w h i t e muscle d i s e a s e (WMD), t h e v a l i d -

ity of the i r o n treatment results was questioned f o r several reasons.

Firstly, s e l e n i u m and/or v i t a m i n E may be r e q u i r e d 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 c o n s e q u e n t l y red c e l l turnover. Secondly, the

incidence o f WMD appeared t o be i n c r e a s e d by i r o n s u p p l e m e n t a t i o n i n the

f i r s t two e x p e r i m e n t s . S i n c e 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-

ally Se-deficient pigs, t h e question arose - was i r o n injection causing

muscle damage s i m i l a r t o and m i s t a k e n f o r WMD? Finally, t h e response t o

iron may have been l i m i t e d by a d e f i c i e n c y i n s e l e n i u m , and t h e r e f o r e an

additive or s y n e r g i s t i c response t o both iron and selenium might be

expected. I n o r d e r t o answer t h e s e q u e s t i o n s , t h e t h i r d trial tested the

effects of i r o n and s e l e n i u m , s e p a r a t e l y and combined, on a v a r i e t y of

 - k -

parameters: hemoglobin, PCV, plasma i r o n , selenium and m e t a b o l i t e s , plasma

p r o t e i n s , and d i s e a s e r e s i s t a n c e as measured by the h e m a g g l u t i n a t i o n test

and o b s e r v a t i o n s on soremouth.
 - 5 -

LITERATURE REVIEW

IRON

FUNCTIONS OF IRON

Iron proteins evolved out o f t h e n e 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 t o 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 t o the o x i d a t i o n o f

organic substances by m o l e c u l a r oxygen, and c o n s e q u e n t l y , t o t h e energy

metabolism o f a l l l i v i n g c e l l s ( F r i e d e n , 1974).

The iron proteins are a diverse group, structurally and f u n c t i o n -

ally. F o r convenience, they a r e u s u a l l y c l a s s i f i e d as heme and non-heme

iron compounds ( H a r r i s o n , 1969). Q u a n t i t a t i v e l y , t h e heme group o f i r o n

proteins dominates i r o n metabolism. The heme m o l e c u l e contains an i r o n

atom i n the center of a porphyrin ring, chelated to the p y r o l l e nitrogen

atoms. Heme can be s y n t h e s i z e d by a l l a e r o b i c mammalian cells except

normal RBC's ( H a r r i s and K e l l e r m e y e r , 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-

globin increases t h e oxygen c a r r y i n g capacity o f t h e blood about seventy

times that c a r r i e d by d i f f u s i o n ( H a r r i s o n , 1969). Myoglobin i s similar

structurally t o hemoglobin, but has o n l y one heme group i n s t e a d of four,

and i s l o c a t e d i n t h e sarcoplasm o f s k e l e t a l and heart muscle. As myoglo-

bin has a h i g h e r affinity f o r oxygen than does hemoglobin, i t accepts

oxygen r e l e a s e d from hemoglobin and a c t s as a t i s s u e r e s e r v o i r (Moore and

Dubach, 1960).

Heme p r o t e i n s i n c l u d e t h e cytochromes. These redox enzymes catalyze

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 t h e a b i l i t y o f t h e heme i r o n t o undergo

reversible oxidation. They t r a n s p o r t hydrogen t o m o l e c u l a r oxygen as p a r t

of the c e l l u l a r electron transport system (Malstrom, 1970; W r i g g l e s w o r t h

 - 6 -

and Baum, 1980). C y t o c h r o m e s ^ (cytochrome o x i d a s e ) , b, and c, a r e l o c a t e d

in the mitochondria cristae. 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 d e g r a d a t i o n of drugs and other

m e t a b o l i t e s , e s p e c i a l l y i n the l i v e r (NRC, 1979, p. 120).

The catalases and peroxidases are important heme enzymes which

d e s t r o y t o x i c oxygen d e r i v a t i v e s such as p e r o x i d e s . They a r e found i n the

cytoplasm of most animal cells, but c a t a l a s e i s a l s o found in organelles

such as peroxisomes. P e r o x i d a s e s a r e i n v o l v e d i n b i o l o g i c a l defence mech-

anisms. For example, m y e l o p e r o x i d a s e 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 d i s e a s e r e s i s t a n c e , and l a c t o p e r o x i d a s e i n m i l k and saliva

has a n t i b a c t e r i a l a c t i v i t y ( W r i g g l e s w o r t h 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 m e t a b o l i c r e a c -

tions. 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 s u c c i n a t e dehydrogenase, «-glycer-

ophosphate dehydrogenase, and NADA-dehydrogenase, a l l l o c a t e d i n mitochon-

dria, and monoamine o x i d a s e , x a n t h i n e o x i d a s e , and aldehyde dehydrogenase

in the cytoplasm (NRC, 1979, p. 120). Most of these are i r o n - s u l p h u r

proteins.

Among the major iron-dependent, non-heme i r o n enzymes which do not

c o n t a i n s u l p h u r a r e the p r o l y l and l y s y l h y d r o x y l a s e s ( c o l l a g e n s y n t h e s i s ) ,

phenylalanine hydroxylase (catabolism of p h e n y l a l a n i n e ) , t y r o s i n e hydroxy-

l a s e ( m e l a n i n and e p i n e p h r i n e s y n t h e s i s ) , and t r y p t o p h a n h y d r o x y l a s e ( s e r o -

tonin synthesis). Ribonucleotide reductase c a t a l y z e s an essential, con-

trolled s t e p 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 ( W r i g g l e s w o r t h and Baum, 1980).

 - 7-

IRON METABOLISM

Iron homeostasis i s achieved through control of iron a b s o r p t i o n as

iron excretion i s limited. Very l i t t l e i r o n i s excreted through the usual

routes of u r i n e , skin, hair and endogenous secretions into 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 i n t a k e o f i r o n r e p r e s e n t s o n l y a f r a c t i o n o f the amount

of i r o n c i r c u l a t e d through t h e plasma d a i l y and used i n s y n t h e s i s o f i r o n

compounds.

Maximal a b s o r p t i o n o f i r o n o c c u r s when t h e i r o n requirement i s high,

as i n young and g e s t a t i n g a n i m a l s . Percentage absorption g e n e r a l l y r i s e s

with decreasing i r o n c o n t e n t o f t h e f e e d , but t o t a l a b s o r p t i o n decreases.

Many f a c t o r s i n a d d i t i o n t o age and d i e t affect iron absorption i n

the ruminant. They include iron status; health; gastrointestinal condi-

tions such as m o t i l i t y , pH, mucosal t u r n o v e r and p a r a s i t i s m ; hypoxia; and

blood loss. Any c o 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 n o r m a l l y increase

i r o n a b s o r p t i o n ( K o l b , 1963; Underwood, 1971). Conversely, i n many d i s e a s e

states, the release of a leukocyte endogenous mediator from white blood

cells limits absorption regardless of iron status (Kampschmidt et, a l . ,

1973)

The most important site of i r o n absorption i s t h e duodenum and

jejunum, i n both man and a n i m a l s . Some i r o n can a l s o be absorbed through

the stomach, ileum, and c o l o n (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 specific,

g l y c o p r o t e i n , r e c e p t o r s i t e s i n t h e brush border. More r e c e p t o r s i t e s a r e

produced d u r i n g i r o n d e f i c i e n c y , a f t e r a l a g phase r e l a t e d t o turnover o f

mucosal c e l l s .
 - 8 -

The process of i r o n a b s o r p t i o n can be d i v i d e d i n t o two s t e p s : uptake

from the intestinal lumen into the mucosal cell, and t r a n s f e r from the

s e r o s a l s u r f a c e of the c e l l to the plasma.

The uptake step of i r o n a b s o r p t i o n i n v o l v e s e i t h e r an energy-depen-

d e n t a c t i v e t r a n s p o r t p r o c e s s , as reviewed by L i n d e r and Munro (1977), or a

p a s s i v e d i f f u s i o n p r o c e s s as d e s c r i b e d by May and W i l l i a m s (1980).

According to the active transport theory, ionic divalent iron

absorbed by receptors i n the brush borders i s moved into the cell in a

process r e q u i r i n g energy and intact protein synthesis ( L i n d e r and Munro,

1977). Consequently, cycloheximide, tetracycline, and other antibiotics

impair iron absorption by inhibition of protein synthesis ( F o r t h , 1974).

While the active transport mechanism has been the subject of numerous

reports, i t has not been u n e q u i v o c a l l y established. Much of the research

may be b e t t e r e x p l a i n e d i n terms of s i m p l e , passive diffusion of chelated

iron in equilibrium with various interacting pools of iron (May and

W i l l i a m s , 1980).

The passive diffusion theory proposes that iron is absorbed as

chelated complexes. The importance of c h e l a t i o n to iron absorption has

been g r a d u a l l y r e c o g n i z e d (Thomas, 1970). Low molecular weight, lipophilic

complexes of iron in either oxidation s t a t e are equally well absorbed.

Contrary to earlier work, F e + 3

i s absorbed as w e l l as F e + 2
(Christopher

et al. , 1974). The more lipophilic the iron 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 influence iron absorption are not

mediated by a s i n g l e r a t e - d e t e r m i n i n g step (May and Williams, 1980). In

t h e 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 p o o l , t h e s l o w l y exchangeable f e r r i t i n i r o n p o o l , and t h e t r a n s f e r r i n

in plasma. The rapidly exchangeable iron pool, possibly a protein or

p o l 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 saturation of t h i s pool regulate how incoming iron i s proportioned

between c e l l s t o r a g e and plasma t r a n s f e r r i n .

When the l a b i l e iron pool i s close to saturation, most iron is

diverted to f e r r i t i n synthesis. As t h i s p o o l i s o n l y s l o w l y exchangeable,

excess i r o n i s s e q u e s t e r e d i n t h i s form and l o s t d u r i n g normal c e l l exfoli-

ation, preventing iron overload. In iron deficiency, labile binding to

t h i s pool d i r e c t s most incoming i r o n immediately t o 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 i n v o l v e d i n t h e t r a n s p o r t o f i r o n a c r o s s t h e

mucosal c e l l , a d i s t a n c e 10,000 t o 20,000 times the diameter o f the i r o n

atom ( L i n d e r 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-

tein. The c o n c e n t r a t i o n o f t h i s p r o t e i n i s increased i n mucosal c e l l s o f

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 a r e g e n e t i -

cally incapable of t r a n s f e r r i n g adequate i r o n . S l a i s t h e gene involved

( B o t h w e l l et a l . , 1979, p. 2 7 4 ) .

The r e l e a s e of i r o n from t h e mucosal c e l l t o the plasma i s t h e f i n a l

s t e p of i r o n a b s o r p t i o n . During the i n i t i a l , rapid phase of i r o n release

60 t o 80% of the e v e n t u a l total may be t r a n s f e r r e d within 30 minutes.

Iron released i n t h i s phase o r i g i n a t e s from t h e r a p i d l y exchangeable iron

pool. The slow phase l a s t s 12-24 hours, as i r o n i s g r a d u a l l y r e l e a s e d from

ferritin ( B o t h w e l l et al^. , 1979, p. 2 7 2 ) .

The importance of i n d i v i d u a l dietary factors to iron absorption i s

u n c e r t a i n , even f o r m o n o g a s t r i c s . A n i o n s forming i n s o l u b l e or o n l y weakly

soluble s a l t s with iron limit i t s a b s o r p t i o n , as i r o n must be i n a soluble

 - 10 -

complex. C h e l a t i n g agents can improve or d e p r e s s i r o n a b s o r p t i o n . As pre-

viously mentioned, weaker c h e l a t i n g agents are n e c e s s a r y f o r i r o n absorp-

tion. They may improve iron availability by preventing the f o r m a t i o n of

i n s o l u b l e i r o n phosphates and h y d r o x i d e s , and a l s o by m a i n t a i n i n g t h e iron

i n a s o l u b l e , a b s o r b a b l e s t a t e (Conrad, 1970). O r g a n i c a c i d s and r e d u c i n g

agents such as a s c o r b a t e , c i t r a t e , l a c t a t e , pyruvate, succinate, cysteine,

histidine, lysine, and some sugars thus facilitate absorption (Thomas,

1970). HC1 i s of major importance as a complexing agent, i n a d u l t humans,

infants, and pigs ( B e u t l e r and F a i r b a n k s , 1980; B o t h w e l l _et ^1_., 1979, p.

267; and Hannan, 1971).

Strong intraluminal chelating agents depress iron absorption by

competing w i t h the c e l l u l a r a c c e p t o r s i t e for iron. Phytates, endotoxins,

a l k a l i n i z i n g a g e n t s , 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 o t h e r

endogenous and exogenous c h e l a t i n g agents depress i r o n a b s o r p t i o n in this

way (Thomas, 1970).

I r o n i s t r a n s p o r t e d by t r a n s f e r r i n between s i t e s of a b s o r p t i o n , stor-

age, utilization, and excretion, and in both plasma and extravascular

spaces (Aisen, 1980). Transferrin minimizes the loss of iron from the

body, by depositing surplus iron in tissues adapted for iron storage.

Transferrin i s also important i n d i s t r i b u t i n g iron i n proportion t o need

( B o t h w e l l et _ a l . , 1979, p. 2 9 3 ) .

Transferrin, 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 . Transferrin has

two metal binding sites which are capable of binding a wide variety of

bivalent and trivalent metals, but have the highest affinity for Fe + 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 delivery

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 transferrin, 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 for a l l t i s s u e s (Bothwell e_t

al., 1979, p. 293). The two iron-binding sites of t r a n s f e r r i n appear

functionally similar. 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-

ferentially donate iron to developing red blood cells and the placenta

(Jacobs, 1977a).

Najean et _al_. (1970) suggested a l a b i l e i r o n pool may be instrumental

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 transit pool

may be a c y s t e i n e - c o n t a i n i n g non-heme p r o t e i n (Najean et jal_, 1970), or a

low m o l e c u l a r weight complex ( J a c o b s , 1977b). E v i d e n c e f o r an intracellu-

lar transit i r o n pool has been o b t a i n e d for reticuloendothelial c e l l s , red

cell precursors, c u l t u r e d Chang c e l l s and liver. I r o n may e n t e r the t r a n -

sit pool from endogenous heme breakdown, mobilization of ferritin, and

exchange w i t h t r a n s f e r r i n ( J a c o b s , 1977b).

The main storage forms of iron are ferritin and hemosiderin.

Ferritin stores 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 m o b i l i z e d as

required, whereas hemosiderin iron is less available (Crichton, 1975).

F e r r i t i n has a l a r g e c a p a c i t y f o r i r o n , eg. 4500 Fe atoms per m o l e c u l e , but

u s u a l l y maintains a reserve c a p a c i t y of one t h i r d of t h i s ( H a r r i s o n et al_. ,

1980). F e r r i t i n c o n t a i n s on average 21% i r o n , s t o r e d as a f e r r i c - h y d r o x i d e -

phosphate c o r e 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 . I r o n passes f r e e l y i n

and out of the s h e l l through s i x c h a n n e l s ; 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 . Ferritin i s formed in a l l cells in response to

enlargement of the l a b i l e p r o t e i n p o o l . 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 -

Hemosiderin is insoluble, contains 25-33% Fe + 3

in addition to

Fe + 2
, and is much larger than ferritin (Kaneko, 1980). Hemosiderin is

considered t o be a breakdown product of f e r r i t i n (Harrison et^ j a l _ . , 1980).

IRON INTERACTIONS

Most trace minerals, including iron, are metals of the f i r s t transi-

tion series on t h e p e r i o d i c table. Many examples of iron interactions with

other minerals involve substitution or antagonism between minerals of

similar size and e l e c t r o n structure. Interactions can occur in the lumen

of the gut, at absorptive sites, and a t various metabolic levels.

The iron requirement is increased by high dietary levels of zinc,

cadmium, copper and manganese. These minerals can compete with iron for

the iron binding sites in the intestinal mucosa (Underwood, 1971).

Conversely, high levels of iron can induce C o , C u , Z n , Mn a n d S e deficien-

cies (Puis, 1981). Iron deficiency results in increased absorption of

heavy metals, mainly Fe, M n , Zn a n d N i , b u t n o t Cu ( B o t h w e l l et j d ., 1979,

p. 273). Possibly the site of interaction in this case is not the iron-

binding site, but transferrin, which is responsible for iron uptake from

the mucosa. The fact that the electron structure of Mn + 2

and Fe + 3
are

functionally identical (Thomas, 1970) and that transferrin is an important

protein for the transport of Zn (Brown, 1977) supports this interpreta-

tion. Furthermore, Z n may r e d u c e iron absorption by interfering with iron

incorporation into, or release from, ferritin (Underwood, 1971).

Biological substitution of iron with other minerals i s most likely to

occur between Mn + 2
and Fe + 3
, and Fe + 2
and Co + 3
. These ion pairs

share similar electron structures. When dietary manganese is high, or

 - 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 f o r iron

is greatly increased. Mn i s i n c o r p o r a t e d i n t o the heme m o l e c u l e , w i t h the

same r a t e of s y n t h e s i s and t u r n o v e r as the i r o n p o r p h y r i n (Thomas, 1970).

The availability of iron at the metabolic level is dependent on

copper enzymes ( F r i e d e n , 1974). Nearly a l l metabolic processes involving

i r o n depend on the i n t e r c o n v e r s i o n of f e r r o u s and ferric iron. 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 ferric iron. One of these is ceruloplasmin. Different

f e r r o x i d a s e s occur i n v a r i o u s s p e c i e s . I r o n accumulates i n the liver very

rapidly in response to even a mild copper deficiency, as a shortage of

ceruloplasmin impedes i t s m o b i l i z a t i o n (Grassman and Kirchgessner, 1974).

On the o t h e r hand, the copper content of the liver increases greatly as a

result of iron deficiency, indicating that iron is required for copper

utilization.

Iron interactions with phosphorus are important at high dietary

levels of iron. Too much iron in the diet interferes with phosphorus

a b s o r p t o n by forming an insoluble phosphate. R i c k e t s may then r e s u l t on an

otherwise adequate diet with a good phosphorus content. Studies with

piglets fed different levels of i r o n as ferrous sulfate indicate that the

amont of iron required to produce a toxicity depends on the amount and

s o u r c e of phosphorus i n the d i e t . In one e x p e r i m e n t , f e e d i n g 5000 ppm iron

reduced growth rate, serum inorganic phosphorus, and femur ash within 5

weeks, (0*Donovan ejt al_. , 1962). However, ruminants appear to be sensitive

to much lower levels of iron than are piglets, and 500 ppm should be

r e g a r d e d 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 c o n t a i n generous amounts o f i r o n . There i s u s u a l l y more

than 20 tonnes o f i r o n per a c r e i n t h e t o p 15 cm o f 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 t h e i r o n c o n t e n t o f f e e d s ( W r e t l i n d , 1968). In

the F r a s e r Valley of B r i t i s h Columbia, mean and ranges o f i r o n i n common

f e e d s were: g r a s s hay, 540 ppm (130-1370); a l f a l f a hay, 580 ppm (40-2185);

corn silage, 384 ppm (40-1490); grass silage 1373 ppm (40-5550); and

pasture 1076 ppm (40-5370), (Cathcart et a l . , 1980). Cereal grains are

poor s o u r c e s o f i r o n , containing 30-60 ppm i r o n ; o i l s e e d meals frequently

c o n t a i n 100-200 ppm i r o n (Underwood, 1981).

Solubility o f i n o r g a n i c i r o n s o u r c e s appears t o be a primary, but n o t

exclusive, determinant of t h e i r availability f o r ruminants. Ammerman et^

al. (1967) tested the a v a i l a b i l i t y of four inorganic iron sources f o r

ruminants. On the basis of t i s s u e Fe 5 9

retention, ferrous sulphate,

ferrous carbonate and f e r r i c c h l o r i d e were ranked i n decreasing order o f

availability, 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 in ferric

c h l o r i d e was 3 t o 4 times as a v a i l a b l e as t h a t i n f e r r i c oxide (Fe 0 ) to

2 3

iron-depleted calves. Ferrous sulphate and f e r r i c chloride a r e very

soluble, and f e r r o u s carbonate and f e r r i c oxide are s l i g h t l y o r non-

soluble.

The absorption coefficient of soluble iron as f e r r i c c h l o r i d e was

0.60 f o r young c a l v e s when t h e d i e t p r o v i d e d 30 mg i r o n daily, and 0.30

when t h e d i e t p r o v i d e d 60 mg i r o n d a i l y (Matrone e t a l _ . , 1957). Hemoglobin

s y n t h e s i s was used as t h e c r i t e r i o n of a v a i l a b i l i t y i n this study.

Iron i n plant products appears t o be l e s s a v a i l a b l e than iron i n

soluble iron salts. Most of t h e i r o n i n plants i s i n the f e r r i c (Fe + 3

)
 - 15 -

form i n o r g a n i c complexes (NRC, 1980, p. 2 4 3 ) . I r o n i n g r a s s was 48 t o 63%

as effective, and i r o n i n legumes 47 t o 57% as e f f e c t i v e , compared t o

ferric chloride f o r improvement o f hemoglobin (Raven and Thompson, 1959;

Thompson and Raven, 1959).

H o s k i n s 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 o f maize, soybean meal, and c o t t o n s e e d h u l l s was 0.29 f o r

pregnant ewes. The d i e t p r o v i d e d 19 ppm i r o n .

Milk iron availability was found t o be 26% f o r c a l v e s and 30% for

piglets (ARC, 1972). A similar value should p r e v a i l f o r t h e pre-ruminant

lamb. These values r e f l e c t t h e low i r o n content o f m i l k and t h e l a r g e

demand f o r i r o n .

IRON REQUIREMENTS

The i r o n r e q u i r e m e n t s o f sheep and c a t t l e a r e not w e l l defined. Few

e x p e r i m e n t s comparing levels of i r o n have been conducted on a l o n g - t e r m

basis; t h e requirement f o r maintenance and/or d e p o s i t i o n o f i r o n s t o r e s i s

g e n e r a l l y i g n o r e d ; and t h e 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 salt

more a v a i l a b l e than feed i r o n .

Work w i t h c a l v e s i n d i c a t e d t h a t t h e average d a i l y g a i n d u r i n g growth,

r a t h e r than body weight o f t h e a n i m a l , i s t h e major f a c t o r d e t e r m i n i n g t h e

amount of i r o n required. The requirement f o r maintenance i s low compared

to that f o r growth (Matrone _et j i l _ . , 1957). Mollerberg ejt al_. (1975a)

calculated that the iron requirement f o r 1 kg growth i n c a l v e s was 40-45

mg. Assuming 25% maximum d i e t a r y iron retention, the actual requirement

for 1 kg growth would be a t l e a s t 160-180 mg i r o n (Mollerberg et al.,

1975a).
 - 16 -

Subsequently, Suttle (1979) found that the iron c o n c e n t r a t i o n s of

lamb and c a l f c a r c a s s e s a r e s i m i l a r . I r o n c o n c e n t r a t i o n ranged from 52.6 -

75.1 mg/kg f r e s h c a r c a s s weight f o r lambs weighing 18 - 69 kg, d e c r e a s i n g

slightly w i t h age at s l a u g h t e r . The v a l u e of 55 mg iron/kg carcass gain

was taken to r e p r e s e n t the approximate net growth r e q u i r e m e n t , e x c l u s i v e of

iron storage. The v a l u e f o r c a l v e s was similar. Suttle (1979) c o n c l u d e d

that the t o t a l net requirement of ruminants f o r i r o n s h o u l d be d e f i n e d i n

terms of d i e t a r y i n t a k e r a t h e r than c o n c e n t r a t i o n because of the r e l a t i v e l y

l a r g e and c o n s t a n t c o n t r i b u t i o n of the growth component.

Most s t u d i e s s e t 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 u n d e r e s t i m a t e the t o t a l iron requirement.

Demands on dietary iron for hemoglobin s y n t h e s i s 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 in

myoglobin o c c u r r e d when d i e t a r y i r o n fed t o d a i r y c a l v e s was i n c r e a s e d from

24 mg/kg to 44 and 104 mg/kg d i e t (Bremner _et j i l _ . , 1976). Other studies

w i t h d a i r y c a l v e s i n d i c a t e a minimum i r o n requirement as high as 100 mg/kg

d r y matter (ARC, 1980, p. 236). This figure i s in line with a possible

requirement of 100-125 ppm iron for milk-fed baby p i g s ( H i t c h c o c k et^ a l . ,

1974; U l l r e y _et a l . , 1960), but much h i g h e r 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 iron requirements of the milk-fed lamb have not been studied

experimentally. In view of the work w i t h c a t t l e (Matrone et a j _ . , 1957;

M o l l e r b e r g _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

t h a t the i r o n requirement of the s u c k l i n g lamb i s h i g h e r on a dry matter

b a s i s than t h a t of the o l d e r , weaned lamb.

 - 17 -

Two experiments on t h e i r o n requirement o f weaned lambs were carried

out by Lawlor ejt a_l. (1965). A dietary l e v e l o f 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 diet. I n the second trial,

40 ppm i r o n met t h e requirements o f weaned lambs, but feed conversion

e f f i c i e n c y was poor compared t o the d i e t w i t h 70 ppm i r o n .

The iron requirement of a d u l t sheep i s s t a t e d t o be 30-50 ppm, o r

mg/kg, o f d i e t d r y matter (NRC, 1975, p. 4 7 ) . Iron deficiency is not

believed t o be a common problem of mature r u m i n a n t s . However, t h e lower

range of iron levels i n feeds were marginal f o r two Canadian surveys

( P e t e r s o n and Waldern, 1977; C a t h c a r t e t j a l . , 1980). While i r o n deficiency

is improbable, ruminant reproduction could be a d v e r s e l y affected by low

iron availability i n some roughages, as a high correlation 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 (Hidiroglou, 1979).

METHODS OF IRON SUPPLEMENTATION

Oral, parenteral and i n t r a v e n o u s methods of i r o n t h e r a p y a r e used.

Intravenous iron therapy has l i t t l e advantage i n rate and magnitude o f

response compared t o p a r 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 deficient human p a t i e n t s , especially as more risk is involved.

T h i s l e a v e s t h e o r a l and p a r e n t e r a l r o u t e s o f i r o n administration.

Orally, f e r r o u s fumarate, s u l p h a t e , s u c c i n a t e and g l u c o n a t e are among

the iron salts used successfully to treat iron deficiency 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 l a r g e amounts o f i r o n salts

cause d i a r r h e a and damage t h e i n t e s t i n a l mucosa. The treatment must be

repeated a t s h o r t i n t e r v a l s . I r o n s u p p l e m e n t a t i o n o f t h e feed o f s u c k l i n g

a n i m a l s i s i n e f f e c t i v e , due t o low consumption of s o l i d feed f o r t h e f i r s t

weeks o f l i f e .
 - 18 -

Parenteral iron administration has become the method of c h o i c e f o r

both commercial and experimental i r o n supplementation of l i v e s t o c k . It i s

convenient and dependable. Piglets are routinely injected with iron

d e x t r a n or i r o n dextrin within the first few days of life. The iron is

used efficiently f o r Hb synthesis: 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 ( T h o r e n - T o l l i n g , 175, p. 44) . Due to t h e

h i g h m o l e c u l a r weight of i r o n d e x t r a n complexes c i r c u l a t i n g n the plasma,

renal clearance i s minimal.

I r o n d e x t r a n , or Imferon, i s the most w i d e l y used of the p a r e n t e r a l

iron products. I t has e x t e n s i v e use world-wide i n human and animal iron

therapy. Others a r e 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 effects of i r o n d e x t r a n 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; B e r e s f o r d _et aL., 1957; M a r t i n et aK, 1955). An a c u t e i n f l a m m a t o r y

reaction develops at the i n j e c t i o n site. Lymphatic a b s o r p t i o n of i r o n i s

rapid, and the lymph nodes may act as temporary iron stores (Thoren-

Tolling, 1975). Most iron is removed through the local inflammatory

reaction; but iron which diffuses away from the site i s taken up and

r e t a i n e d by t i s s u e macrophages.

Most of the iron i s absorbed within t h r e e days, accompanied by a

sharp rise i n the plasma iron content. This greatly exceeds the iron-

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 t o the h i g h s t a b i l i t y of the

iron dextran complex. Iron dextran must be processed through the

reticulo-endothelial system to s p l i t the i r o n from the d e x t r a n (Szilagyi

and E r s l e v , 1970). As a r e s u l t , iron d e x t r a n t a k e s t h r e e days t o measur-

ably i n c r e a s e blood Hb, compared to less than a day for iron sorbitex

(McCurdy, 1970).
 - 19 -

Side effects of iron dextran injection a r e only well known i n

humans. Reactions such as f e v e r , dealyed arthralgia, local d i s c o m f o r t , and

skin staining bear little relationship to dosage. Rarely, both f a t a l and

n o n - f a t a l a n a p h y l a c t i c r e a c t i o n s a r e caused by a repeated dose l a t e r than 5

days after the i n i t i a l dose i n humans (McCurdy, 1970) and i n beef cattle

(Perry et a l . , 1967).

STAGES OF IRON D E F I C I E N C Y

Iron d e f i c i e n c y progresses through s e v e r a l stages between i r o n deple-

t i o n and a c t u a l i r o n d e f i c i e n c y anemia. Initially, a negative i r o n balance

is counteracted by s e v e r a l mechanisms. Iron i s mobilized from the body

s t o r e s of l i v e r , muscle, s p l e e n , and marrow i n order t o m a i n t a i n Hb produc-

tion, while iron absorption i s increased. Normal serum iron, and serum

iron saturation percentage, a r e maintained (Bothwell et a_l., 1979, pp.

44-45). Iron d e p l e t i o n i s sometimes c o n s i d e r e d a pathological condition,

as the t i s s u e c o n c e n t r a t i o n of some i r o n - c o n t a i n i n g enzymes i s diminished

( V e r l o o p _et JJ1_. , 1970). F o r example, l i v e r enzyme a c t i v i t y i n the pentose

phosphate shunt i s affected at an unexpectedly early stage i n iron defi-

cient rats (Jacobs, 1977a).

In l a t e n t iron d e f i c i e n c y , the i n c r e a s e d iron absorption s t i l l helps

maintain normal hemoglobin l e v e l s . The serum i r o n s a t u r a t i o n percentage i s

decreased, as serum iron i s reduced and/or transferrin production is

increased. Iron s t o r e s a r e absent. At t h i s stage, growth rate, general

w e l l - b e i n g or d i s e a s e r e s i s t a n c e may respond to iron supplementation.

Iron d e f i c i e n c y anemia, the f i n a l stage of d e f i c e n c y , results when

the normal hemoglobin level cannot be maintained. An e q u i l i b r i u m can be

 - 20 -

reached a t any l e v e l o f Hb below t h e norm, or Hb may c o n t i n u e to decline

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 a r e not uncommon. The e r y t h r o c y t e protoporphyrin

content i s elevated, denoting d e f e c t i v e heme s y n t h e s i s ( 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 w e 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 c h a r a c t e r -

ized by a l a r g e p r o p o r t i o n o f r e d blood cells which are small i n size

( m i c r o c y t i c ) and p o o r l y f i l l e d w i t h hemoglobin (hypochromic). As new c e l l

formation i s restricted, the r e t i c u l o c y t e count (immature red blood cells)

tends t o be low. I n i t i a l l y , t h e l e v e l o f blood hemoglobin may be depressed

more than either t h e r e d blood cell count or hematocrit ( H a r r i s and

Kellermeyer, 1970, p. 1 1 5 ) . Bone marrow examination also demonstrates

characteristic changes during iron deficiency, including normoblastic

hyperplasia ( B e u t l e r and F a i r b a n k s , 1980).

MEASUREMENTS OF IRON STATUS

Different techniques a r e used t o assess iron s t a t u s depending on

whether or not anemia i s p r e s e n t . When a m i c r o c y t i c , hypochromic anemia

can be demonstrated, t h e few p o s s i b l e causes o t h e r than i r o n l a c k can be

readily eliminated ( i e . inflammation, infection, copper deficiency, lead

p o i s o n i n g ) or a r e extremely rare. 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 o f hemoglobin o r h e m a t o c r i t t e s t s , though i t i s

desirable to confirm the iron d e f i c i e n c y by o t h e r measurements. The

u l t 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 o f blood hemoglobin t o i r o n

therapy ( H a r r i s and K e l l e r m e y e r , 1970, p. 1 2 0 ) .

 - 21 -

In iron deficiency anemia, the plasma iron concentration is low,

total iron binding capacity (transferrin) is high, and percentage

saturation of transferrin i s very low. These measurements can be used to

c o n f i r m 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 affected by a wide range of condi-

tions, and thus is not very specific for iron status. Plasma iron is

increased in association with decreased erythropoiesis, increased

hemolysis, or increased release of iron from body stores, while reduced

levels of i r o n are consistently seen d u r i n g a c u t e and chronic infections,

even i f body s t o r e s are high. The decrease i n plasma i r o n l e v e l i s a late

development in iron deficiency and may occur only a f t e r the mobile iron

r e s e r v e s are c o m p l e t e l y exhausted ( F i e l d i n g , 1980).

The percentage iron saturation of transferrin best measures the

supply of iron to the erythroid marrow. A saturation of less than 16%

depresses basal erythropoiesis. 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 a s s o c i a t e d w i t h i m p a i r e d p r o t e i n production, the

transferrin level i s decreased. Consequently, the quantity of Tr, also

known as the total iron-binding capacity (TIBC) should be measured as well

as the percentage saturation ( B o t h w e l l _et a^., 1979, pp. 50-56; Crosby,

1975; F i n c h , 1970).

Hemoglobin and hematocrit tests are the most rapid, accurate and

convenient tests, but cannot assess the full range of iron status except

f o r o b v i o u s anemias. The lower v a l u e s of normal hemoglobin and hematocrit

values also o v e r l a p the upper range of anemic v a l u e s (Garby and Killander,

1968). Transferrin measurements may also be ineffective in diagnosing

borderline cases of iron overload or deficiency (Beutler et^ a _ l . , 1954;

C r o s b y , 1975).
 - 22 -

In the pre-anemic stages of iron deficiency, the best tests for iron

status a s s e s s the level of iron stores. Marrow a s p i r a t i o n or the level of

absorption of a t e s t dose of i r o n have been the best methods of evaluating

iron depletion. Marrow aspiration is used to determine the presence of

hemosiderin, a storage form of iron, in the reticuloendothelial cells

(Beutler et a l . , 1954). This test i s laborious and unsuited for screening

purposes and nutritional trials. The iron absorption test is also not

appropriate for experiments with growing animals, e s p e c i a l l y as i t is not

valid in cases of increased erythropoiesis. The level of urinary iron

excretion a f t e r a t e s t dose of d e s f e r r i o x a m i n e i s supposed to be correlated

with the level of body stores, but is also inappropriate f o r most animal

research purposes. Consequently, the degree of i r o n d e p l e t i o n is difficult

to a s s e s s (Bothwell et a l _ . , 1979, pp. 88-104; H a r r i s and Kellermeyer, 1970,

p. 118).

A new t e c h n i q u e that will receive widespread c l i n i c a l application is

a rapid, 2-site radioimmune assay for serum ferritin. Serum ferritin is

highly c o r r e l a t e d with body i r o n s t o r e s in a l l states from iron deficiency

to i r o n overload (Jacobs, 1977c; Powell et a l . , 1975). The r o l e of serum

ferritin in iron transport and metabolism is uncertain (Worwood ^ t ^1_. ,

1975; Worwood, 1980). Should the serum ferritin test prove feasible for

nutritional studies i n animals, the measurement of both serum ferritin and

either hemoglobin or hematocrit would provide the optimum picture of iron

status.

EFFECTS OF IRON DEFICIENCY

There is increasing evidence that symptoms of ill-health, reduced

growth rate, and possibly decreased disease resistance can occur even in
 - 23 -

mild cases of iron deficiency. 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 cell s t r u c t u r e and f u n c t i o n may be more

i m p o r t a n t than the r e a d i l y r e v e r s i b l e anemia.

The susceptibility of c e l l s to iron deficiency i s determined by the

rate of cell turnover, and the rate of turnover of the iron-containing

compounds w i t h i n the c e l l ( F i e l d i n g , 1975). Rapidly proliferating tissues,

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 in

available iron. On the o t h e r hand, the higher the r a t e of t u r n o v e r of the

iron-containing enzymes and proteins w i t h i n the cell, the more r e v e r s i b l e

the deficiency condition. The cytochrome o x i d a s e a c t i v i t y i n the intes-

t i n a l mucosa reaches normal 48 hours a f t e r i r o n t r e a t m e n t , whereas s k e l e t a l

muscle cytochrome £ a c t i v i t y r e c o v e r s very s l o w l y (Dallman, 1971).

Iron deficiency causes a variety of enzymatic and morphological

defects in solid tissues. Many cytochromes and o t h e r enzymes are signifi-

cantly decreased by iron deficiency. The enzymes are differentially

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 ( J a c o b s , 1975; J a c o b s , 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 f o r the highly

significant reduction of various n o n - i r o n enzymes d u r i n g iron deficiency.

For example, the pentose phosphate shunt enzymes (phosphogluconate dehydro-

genase and glucose-6-phosphate dehydrogenase) suffer a major reduction

early i n iron deficiency. The disaccharidase activity i n the brush border

o f 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 ( J a c o b s , 1975), as i s glutathione

p e r o x i d a s e a c t i v i t y i n the e r y t h r o c y t e ( M a c d o u g a l l , 1972).

Defects are often observed in i r o n - d e f i c i e n t mitochondria. Many

workers have found evidence of increased mitochondrial fragility,

 - 24 -

swelling, vacuolation, and membrane breakdown in lymphocytes, intestine

and marrow c e l l s . Abnormal m i t o c h o n d r i a l 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 t r e a t m e n t , 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).

Epithelial 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 . All prolifer-

ating tissues are affected, especially the mucous membranes. Enzyme de-

f e c t s have been found i n the b u c c a l mucosa, stomach and small intestine but

as yet a cause and effect 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 ( V e r l o o p et j a l . , 1970).

Specific lesions of iron deficiency have been well documented in

humans. Most s t u d i e s have r e v e a l e d a high frequency of atrophic gastric

mucosal changes, i e . 85% (Beutler and F a i r b a n k s , 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

iron absorption, especially i n the young (Witts, 1966; Witts, 1969, pp.

39-44). Sores at the c o r n e r s 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, fissured skin are

other typical lesions (Heilmeyer and Harwerth, 1970; Harris and

Kellermeyer, 1970, p. 114). Similar l e s i o n s , e s p e c i a l l y of the gastroin-

testinal mucosa, have been observed in swine and other monogastrics but

have not been s t u d i e d i n ruminants.

In general, iron deficiency causes widespread metabolic changes, as

might be expected through the many functions of iron enzymes. Serum

triglyceride l e v e l s are elevated, folic acid levels depressed, and basal

m e t a b o l i c r a t e i s depressed ( B o t h w e l l _et a l . , 1979, p. 3 1 ) . A reduction in

the mitochondrial enzyme ^-glycerophosphate dehydrogenase in muscle

 - 25 -

tissue l e a d s to e x c e s s i v e p r o d u c t i o n of l a c t i c acid, i m p a i r i n g work capa-

city (Saltman et a_L., 1982). T h i s can be demonstrated in iron deficient

a n i m a l s even i n the absence of anemia ( B o t h w e l l et jal_., 1979, p. 3 1 ) . Iron

deficient animals v o l u n t a r i l y restrict physical activity, and appetite i s

reduced i n the e a r l y s t a g e s of anemia, at l e a s t i n c a l v e s (Bremner et a l . ,

1976). Consequently, i t i s d i f f i c u l t t o d i r e c t l y compare the e f f i c i e n c y o f

f e e d 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 a n i m a l s .

IRON AND DISEASE RESISTANCE

D i s e a s e r e s i s t a n c e i s depressed d u r i n g i r o n d e f i c i e n c y i n a l l s p e c i e s

studied. Enzymatic and m e t a b o l i c d e f e c t s a r e found i n many components o f

the immune system (Pearson and R o b i n s o n , 1976). The number of l e u k o c y t e s

and lymphocytes are o f t e n 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 , resul-

ting in the impairment of both phagocytic function and cell-mediated

immunity, respectively. The effect of i r o n status on tissue morphology,

resident b a c t e r i a l p o p u l a t i o n s ( 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

bacterial e x o t o x i n s and endotoxins (Weinberg, 1971; Oanoff and Zweifach,

1960) may a l s o be i m p o r t a n t .

Mortality i n farm livestock i s increased by iron deficiency. In

clinically 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%, m a i n l y from

s e p t i c e m i c and enteric infections (Tennant et a l . , 1975a). The incidence

of e n t e r i t i s was significantly less (P<0.001) i n i r o n - i n j e c t e d c a l v e s than

i n anemic c a l v e s fed a commercial v e a l c a l f m i l k r e p l a c e r c o n t a i n i n g 19 mg

Fe/kg diet ( M o l l e r b e r g jet a l _ . , 1975a). Comparable data i s not available

for lambs, but Holz et al_. (1961) noted that mortality was 30.5%, 38.6%,

and 13.5% for 72 lambs injected with 0, 150 and 300 mg iron at
 - 26 -

birth. Iron treatment of piglets resulted in a significant reduction of

infectious diseases, particularly diseases associated with F_. c o l i organ-

isms, such as scours. Mortality was 13% in the iron-treated piglets

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 susceptibility of i r o n - d e f i c i e n t animals to gastrointestinal

i n f e c t i o n s may involve a f a i l u r e to produce adequate numbers of myeloper-

oxidase-containing cells. Neutrophils, the most important phagocytic

cells, contain myeloperoxidase, an iron-containing enzyme which affects

bactericidal capacity. Iron d e f i c i e n t rats had fewer MPO-cells in the

l a m i n a p r o p r i a and submucosa, and were much more s u s c e p t i b l e t o challenge

with Salmonella typhimurium (Baggs and Miller, 1973). In another study,

the iron deficient rats responded very slowly compared to controls in

production of intestinal MPO, which was correlated with survival and

r e t e n t i o n of j>. typhimurium i n the gut (Baggs and Miller, 1974). Studies

with humans i n d i c a t e d that p h a g o c y t o s i s by neutrophils was unaffected in

iron deficiency, but intracellular bacterial killing was significantly

(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 deficiency significantly a f f e c t s the size, 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-

tion of antibody-forming spleen cells; reduction i n thymus weight and in

thymus mononuclear c e l l s (Chandra _et a l . , 1977). I r o n d e f i c i e n c y may be

most important during development of these tissues. Studies with rats

indicated that iron deprivation during gestation and l a c t a t i o n was more

serious than after weaning, as the subsequent disease resistance was

 - 27 -

reduced, even after a period of n u t r i t i o n a l rehabilitation (Baggs and

Miller, 1973).

While i r o n s u p p l e m e n t a t i o n o f young a n i m a l s would appear t o be bene-

ficial i n i m p r o v i n g d i s e a s e r e s i s t a n c e , K n i g h t 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 c o n f i n e m e n t - r e a r e d p i g s r e q u i r e d Fe s u p p l e m e n t a t i o n
t o prevent anemia, the data p r e s e n t e d here and the l a r g e
amount o f p r e v i o u s l y r e p o r t e d evidence from o t h e r s p e c i e s
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 t h a t the e f f e c t s o f
s u s c e p t i b i l i t y t o i n f e c t i o n be i n c l u d e d i n d e t e r m i n i n g t h e
optimum Fe s u p p l e m e n t a t i o n 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 t h e growth o f

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 piglets at

various intervals after injection. The iron treatment significantly

enhanced b a c t e r i a l growth i n v i t r o i n some c a s e s , 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 ( K n i g h t et a l . , 1983).

As i r o n i s a c r u c i a l t r a c e m i n e r a l f o r m i c r o b i a l growth, an i n c r e a s e

in plasma i r o n ( h y p e r f e r r e m i a ) i n humans may be a s s o c i a t e d w i t h suscepti-

bility t o b a c t e r i a l and f u n g a l pathogens (Weinberg, 1974). I t should be

r e c o g n i z e d , though, t h a t the h y p e r f e r r e m i a i s extreme and/or l o n g - t e r m , and

is typically caused by d i s e a s e c o n d i t i o n s such as v i r a l hepatitis, sickle

cell anemia, m a l a r i a and o t h e r s which c o u l d t a x t h e immune system regard-

less of i r o n level. Thus, a comparison between h y p e r f e r r e m i a - a s s o c i a t e d

diseases i n humans and the r i s k s of t h e 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 c o m p l e t e l y v a l i d .

Whether or not i r o n s u p p l e m e n t a t i o n increases disease s u s c e p t i b i l i t y

depends on s e v e r a l factors. I n order to neutralize the m i c r o b i o s t a t i c

action o f serum, enough i r o n must be p r o v i d e d t o s a t u r a t e a t l e a s t 60-80%

 - 28 -

of t h e 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 iron supplement as no more than 1-3% o f the i r o n can be d i r e c t l y

transferred 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). Iron administra-

t i o n during experimental i n f e c t i o n s o f r a t s has been shown t o dramatically

lower the L D 5 0 dose f o r a v a r i e t y of b a c t e r i a , provided the i r o n is in a

form a b l e t o d i f f u s e t o the s i t e o f b a c t e r i a l r e p l i c a t i o n , and i s a d m i n i s -

t e r e d by i v , i p or im r o u t e s . C o n s e q u e n t l y , i r o n d e x t r a n and i r o n dextrin

are inactive i n enhancing microbial growth, whereas ferric ammonium

citrate, i r o n s o r b i t a l c i t r a t e and hemoglobin a r e h i g h l y active (Weinberg,

1971).

Accordingly, iron supplementation of i r o n - d e f i c i e n t animals appears

to confer little risk, especially when i r o n i s provided as i r o n dextran,

while t h e improvement i n iron status allows optimal functioning o f many

a s p e c t s o f the immune system.

 - 29 -

SELENIUM

FUNCTIONS OF SELENIUM

Biological functions o f selenium include t h e maintenance o f muscle

and membrane (erythrocyte, vascular endothelium, and cell organelle)

i n t e g r i t y ; s t i m u l a t i o n of a n t i b o d y and u b i q u i n o n e s y n t h e s i s ; maintenance o f

essential enzyme systems, pancreatic function, and v i g o r and m o b i l i t y o f

sperm ( C a l v i n _et _ a l . , 1981; Combs and Bunk, 1981; Ganther jet a±., 1976;

H i d i r o g l o u e t a _ l . , 1968; S p a l l h o l z et a l . , 1975). Many o f these functions

involve glutathione peroxidase.

The selenoenzyme, glutathione peroxidase (GSH-Px, EC 1.11.1.9) i s

p a r t o f a complex mechanism, i n c l u d i n g t h e s u p e r o x i d e d i s m u t a s e s , c a t a l a s e ,

and vitamin E, which defends the c e l l against cytotoxic oxygen deriva-

tives. Due t o t h e i m p o s s i b i l i t y o f i s o l a t i n g t h e s e compounds i n v i v o , t h e

models proposed f o r t h e a c t i o n o f GSH-Px a r e 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 p e r o x i d e s and f r e e

radicals. GSH-Px p r o b a b l y c a t a l y z e s the reduction o f many h y d r o p e r o x i d e s

to t h e i r corresponding a l c o h o l s , or H 0 , i n t h e case o f H 0 .
2 2 2 H 0
2 2 isa

natural by-product o f many enzyme r e a c t i o n s , including those o f x a n t h i n e

oxidase and d-amino a c i d oxidase (Rotruck, 1981). Free r a d i c a l s may be

produced by m i t o c h o n d r i a l r e s p i r a t i o n , autooxidation, i r r a d i a t i o n damage or

e n v i r o n m e n t a l p o l l u t a n t s ( C s a l l a n y e t a _ l . , 1981) and by i n t e r a c t i o n between

H 0
2 2 and metal i o n s or 0 2 ( R o t r u c k , 1981). 0 2 is generated in large

amounts by many e l e c t r o n transport steps i n mitochondrial enzyme systems,

and catalyzes the peroxidation o f membrane polyunsaturated fatty acids

(Diplock, 1981).
 - 30 -

The intimate r e l a t i o n s h i p between v i t a m i n E and selenium can p a r t l y

be explained by their related functions. GSH-Px, located both i n the

cytosol and m i t o c h o n d r i a in a 70:30 r a t i o , reduces lipid peroxides to

nontoxic hydroxy fatty acids. Vitamin E, located within membranes,

p r e v e n t s or d e c r e a s e s the f o r m a t i o n 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

t h a t GSH-Px should not be c o n s i d e r e d the only biochemical f u n c t i o n o f Se.

While more than 9 d i f f e r e n t s e l e n i u m - c o n t a i n i n g proteins have been

found i n lamb t i s s u e s one of these was p r e s e n t o n l y i n heart and muscle o f

normal lambs and not i n lambs a f f e c t e d w i t h White Muscle D i s e a s e . Signifi-

cantly, heart and muscle t i s s u e a r e t h e t a r g e t s i t e s during Se/vitamin E

deficiency i n sheep. Furthermore, a link 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 t h e n a t u r e of t h i s p r o t e i n . It

i s a cytochrome c o n t a i n i n g a heme group i d e n t i c a l t o cytochrome c;, but has

an amino a c i d c o m p o s i t i o n and weight s i m i l a r t o cytochrome (Whanger et^

al., 1974).

It has been h y p o t h e s i z e d 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 s e l e n i d e i n a c l a s s o f non-heme i r o n p r o t e i n s p r e s e n t i n t h e

mitochondria and smooth endoplasmic r e t i c u l u m , and p r o t e c t e d from o x i d a t i o n

by v i t a m i n E ( D i p l o c k and Lucy, 1973; D i p l o c k , 1974; C a y g i l l and Diplock,

1973; G i a s u d d i n j^t _ a l . , 1975). Deficiency of v i t a m i n E would lead to a

replacement of the s e l e n i d e with a more stable, but l e s s catalytically

a c t i v e , sulphur group, as t h e s e l e n i d e i n modified non-heme Fe p r o t e i n s i s

much more v u l n e r a b l e t o o x i d a t i o n than t h e s u l p h u r group ( D i p l o c k , 1970).

Increasing e v i d e n c e s u g g e s t s a major f u n c t i o n of s e l e n i u m may be i n

electron transport, possibly involving the same protein investigated by

 - 31 -

Diplock (1970) and Whanger et a l . (1974). Levander et _ a l . (1974) were the

first to present evidence of a r o l e f o r Se in catalyzing electron trans-

fer. They p o i n t out that with adequate d i e t a r y Se, the Se could "short-

circuit" the respiratory chain, avoiding the H 0

2 2 generating step, by

directly t r a n s f e r r i n g e l e c t r o n s from GSH t o cytochrome _c.

SELENIUM METABOLISM

The dietary selenium sources of ruminants are mainly organic com-

pounds of Se in feedstuffs and s e l e n i t e or selenate salt 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 m o n o g a s t r i c s . Based on the i n s o l u b l e nature of fecal

Se and the known a b i l i t y of the a n a e r o b i c , highly reducing rumen e n v i r o n -

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 t h a t rumen

microbes reduce Se compounds to unavailable forms such as selenide or

elemental selenium. At l e a s t 50% of fecal 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 c o n c e n t r a t e Se at 2 to 78 times the

dietary concentration. These organic forms, i n c l u d i n g selenoamino acids

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

al., 1978a) . The spontaneous r e c o v e r y 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 p r o p o r t i o n of selenium absorbed i n c r e a s e s w i t h decreased dietary

selenium and/or d e f i c i e n t Se status, and i s not affected by vitamin E

level. 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 inversely propor-

tional to dietary selenium (Ku _et a l . , 1972; K i n c a i d _et a l _ . , 1977; Van

 - 32 -

Fleet, 1975). Sodium s e l e n i t e i s more e f f e c t i v e than s e l e n o m e t h i o n i n e i n

increasing 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, a t a d i e t a r y level of 1.0 ppm, s e l e n o m e t h i o n i n e 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 site of Se a b s o r p t i o n i s t h e 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 p a s s i v e t r a n s p o r t , while selenomethionine i s transported

against a concentration gradient. As m e t h i o n i n e and s e l e n o m e t h i o n i n e s h a r e

t h e same t r a n s p o r t system, m e t h i o n i n e can i n h i b i t s e l e n o m e t h i o n i n e uptake.

T h i s may p a r t l y e x p l a i n t h e p r o t e c t i v e e f f e c t o f high p r o t e i n d i e t s a g a i n s t

selenosis. M e t h i o n i n e may be e f f e c t i v e o n l y i n c o m b i n a t i o n w i t h adequate

l e v e l s of v i t a m i n E (Levander, 1976; White and Somers, 1977).

Selenium i s t r a n s p o r t e d by plasma p r o t e i n s ( P o r t e r ^ t a l . , 1979). It

is gradually taken up from the blood by the l i v e r and kidney c o r t e x , and

less readily by the s p l e e n , muscle, h e a r t and l u n g s . Organic forms of

s e l e n i u m tend t o be r e t a i n e d more t e n a c i o u s l y by t i s s u e s compared t o i n o r -

g a n i c forms ( M a r t i n and G e r l a c h , 1972).

Wright and B e l l (1964) suggest that the t i s s u e s with highest Se

c o n t e n t a f t e r d o s i n g have t h e g r e a t e s t a b i l i t y t o s y n t h e s i z e organoselenium

compounds from inorganic selenium. Subsequently, these compounds a r e

redistributed, especially t o t h e muscle which i s v u l n e r a b l e t o Se defi-

c i e n c y i n sheep. Surprisingly, Se 7 5
s t u d i e s by Wright and B e l l (1964) and

others, indicate that 48 hours after 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 -

S e l e n i u m i s e x c r e t e d 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 r o u t e s . Fecal

excretion i s more i m p o r t a n t t o ruminants than m o n o g a s t r i c s , due to lower

r a t e s of a b s o r p t i o n . At h i g h l e v e l s of d i e t a r y Se, s e l e n i u m d e t o x i f i c a t i o n

by GSH-dependent m e t h y l a t i o n becomes important. Dimethyl selenide and

t r i m e t h y l s e l e n o n i u m i o n are the major pulmonary and u r i n a r y Se m e t a b o l i t e s ,

respectively (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 p r o t e i n s (hemoglo-

bin, cytochrome c;, myoglobin), enzymes (myosin, aldolase, urokinase,

f i b r i n a s e ) and n u c l e o p r o t e i n s . P r e v i o u s l y , Se was thought to be a contam-

i n a n t mistaken f o r S due to i t s c l o s e c h e m i c a l s i m i l a r i t y . While s e l e n o -

methionine is "accidentally" incorporated into proteins in place of

methionine (McConnell et a^., 1970), s e l e n o c y s t e i n e i s now known to form

the active c e n t e r of v a r i o u s s e l e n o p r o t e i n s which have been investigated

(Wilhelmsen et a l . , 1981).

Selenium metabolism, while s h a r i n g some pathways with sulphur, i s

othewise unique (Levander, 1976). Animals readily reduce such forms of

inorganic Se as s e l e n a t e or selenite, 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 . R e d u c t i o n i s c r u c i a l t o 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 oxidation

state. Ganther and Hsieh (1974) have described a probable biochemical

mechanism.

SELENIUM INTERACTIONS

Selenium interactions with a variety of nutrients have been docu-

mented, most commonly w i t h a r s e n i c , cadmium, coper, l e a d , mercury, silver,

tellurium, zinc and sulphate. Selenium metabolism is influenced by

 - 34 -

selenium's tendency t o complex w i t h heavy m e t a l s . These m i n e r a l s reduce

the t o x i c i t y o f h i g h l e v e l s o f d i e t a r y s e l e n i u m , and a l s o h i g h l e v e l s can

induce a deficiency when dietary selenium i s marginal (Lee and Jones,

1976). Selenium also interacts, directly and i n d i r e c t l y , w i t h v i t a m i n E,

p r o t e i n , c e r t a i n c a r b o h y d r a t e f r a c t i o n s , and o t h e r f a c t o r s which remain t o

be identified.

Dietary sulphur at l e v e l s found naturally i n feeds a f f e c t s selenium

a b s o r p t i o n and r e t e n t i o n significantly. High l e v e l s of s u l p h a t e i n c r e a s e

the Se requirement i n sheep (Ekermans and S c h n e i d e r , 1982). Major changes

in 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 . Increasing dietary s u l p h u r i n c r e a s e s u r i n a r y Se

secretion. On a high s u l p h u r d i e t , t h e i n c r e a s e d p o p u l a t i o n o f Desulpho-

vibrio bacteria i n t h e rumen may reduce more selenium t o H Se

2 through

s u l p h a t e r e d u c t i o n pathways. H S e i s thought
2 t o d i s s o c i a t e t o 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 e l e m e n t a l s e l e n i u m or h i g h l y i n s o l u b l e metal

s e l e n i d e s (Pope e t a_l., 1979).

The selenium content o f rumen m i c r o o r g a n i s m s , and p o t e n t i a l l y t h e

availability o f Se, i s reduced by s u l p h u r deficiency (Whanger e t a_l. ,

1978a). S u l p h u r d e f i c i e n c y a l s o i n c r e a s e s s e l e n o s i s a t h i g h Se l e v e l s , as

s u l p h u r i n g l u t a t h i o n e and g l u t a t h i o n e r e d u c t a s e i s needed f o r t h e forma-

t i o n of e x c r e t o r y p r o d u c t s such as d i m e t h y l s e l e n i d e and t r i m e t h y l s e l e n o n -

ium i o n (Pope e t a l . , 1979).

C o n f l i c t i n g e v i d e n c e o f t h e e f f e c t o f s u l p h u r on i n c r e a s i n g t h e i n c i -

dence of white muscle d i s e a s e may be r e s o l v e d by t h e o b s e r v a t i o n t h a t

s u l p h u r competes w i t h s e l e n i u m o n l y when p r e s e n t as t h e s e l e n i u m analogue.

Thus, " i n o r g a n i c s u l p h u r may a l t e r t h e metabolism of i n o r g a n i c selenium

more than o r g a n i c s e l e n i u m " (Whanger et a l _ . , 1969a).

 - 35 -

Protein source may be a major i n f l u e n c e on t h e a b s o r p t i o n o f s e l e n -

ium. S t u d i e s w i t h beef cattle indicated that dietary 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 protein. 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 , cattle. The degree of p r o t e i n deposition

may be a f a c t o r ( T r e v i s , 1979). Feeding a h i g h p r o t e i n d i e t , on t h e o t h e r

hand, has been recommended i n cases of Se t o x i c i t y (Ekermans and S c h n e i d e r ,

1982).

Linseed o i l meal has l o n g been known t o have a p r o t e c t i v e effect

against chronic selenosis. Two cyanogenic glycosides i n linseed o i l meal

release CN- which interacts with metabolic selenium t o form SeCN-. This

i n t e r a c t i o n c o u l d ,be d e t r i m e n t a l i n a n i m a l s m a r g i n a l l y d e f i c i e n t i n s e l e n -

ium (Palmer, 1981).

Dystrophogenic f a c t o r s i n feeds may reduce t h e a v a i l a b i l i t y o f s e l e n -

ium o r v i t a m i n E. The v i t a m i n E i n a l f a l f a may not be c o m p l e t e l y a v a i l a b l e

for c a l v e s or c h i c k s . A compound extracted i n ethanol from alfalfa i n -

creased excretion of a - t o c o p h e r o l from both alfalfa and added sources

( P u d e l k i e w i c z and M a t t e r s o n , 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 h i g h l e v e l s i n d y s t r o p h o g e n i c feeds (Diplock,

1970).

DIETARY SELENIUM AND TOCOPHEROL

Selenium deficiency occurs naturally i n large parts of the world,

including the P a c i f i c Northwest; Midwest, Southeast and N o r t h e a s t North

America. I n Canada, low s e l e n i u m soils a r e p r e v a l e n t i n c e n t r a l B.C.,

w e s t - c e n t r a l A l b e r t a , N o r t h e r n O n t a r i o , t h e A t l a n t i c p r o v i n c e s and p a r t s o f
 - 36 -

Quebec (Ullrey, 1981). Forages sampled i n t h e F r a s e r V a l l e y o f B.C. were

also consistently inadequate i n selenium (Cathcart e t a K , 1980). The

availability of Se t o p l a n t s i s i n c r e a s e d i n w e l l - a e r a t e d , a l k a l i n e soils

and decreased i n a c i d , water-logged soils. Studies i n t h e 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

soil t y p e s , which were more highly correlated with soil pH and m o i s t u r e

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 cultural p r a c t i s e s , as shown by a d r a m a t i c increase

in WMD i n New Zealand after 1957. Increasing yields by fertilization,

seeding of more productive species, i r r i g a t i o n (and a s s o c i a t e d l e a c h i n g )

seems t o d i l u t e t h e amount o f selenium i n t h e f e e d , and heavy c r o p p i n g may

remove more selenium than i s recycled. Intensive stocking may also

decrease the a v a i l a b i l i t y o f Se t o p l a n t s , as much f e c a l selenium i s tied

up i n elemental and o t h e r insoluble forms o f selenium (Counsins and

Cairney, 1961).

Tocopherol c o n t e n t o f f o r a g e s does not seem t o be a f f e c t e d by s e l e n -

ium l e v e l . However, i t i s s e n s i t i v e t o stage o f m a t u r i t y , d r y i n g l o s s e s i n

hay, rain, processing and s t o r a g e losses (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 a p p r o x i m a t e l y 0.1 ppm

(NRC, 1975, p. 4 7 ) . The form of dietary selenium, the c r i t e r i a used t o

assess adequacy, and the d i e t composition including vitamin E, will

 - 37 -

i n f l u e n c e the requirement. L e v e l s o f 0.11 t o 0.12 ppm Se were r e q u i r e d i n

order t o m a i n t a i n t i s s u e PSH-Px l e v e l s i n sheep f e d a p r a c t i c a l - t y p e ration

(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 p r e v a l e n t as d i e t a r y Se f a l l s below 0.08 mg /kg D.M. Dietary

selenium levels between 0.03 and 0.05 ppm a r e m a r g i n a l (ARC, 1980, p.

243). Under B.C. c o n d i t i o n s , 0.2 ppm i s c o n s i d e r e d adequate, and <0.2 ppm

may be m a r g i n a l ( P u i s , 1981, pp. 76-87).

The d i e t a r y v i t a m i n E may be i m p o r t a n t o n l y a t " m a r g i n a l " i n t a k e s o f

Se (ARC, 1980, p. 244-251). I t was suggested t h a t t h e d i e t a r y Se r e q u i r e -

ment f o r a l l s p e c i e s i s 0.1 - 0.15 ppm when v i t a m i n E i s s u f f i c i e n t , b u t

may be as h i g h as 0.5 - 1.0 ppm when v i t a m i n E i s low (Hoffman and La

Roche, 1971).

The selenium requirement may be markedly i n c r e a s e d when dystropho-

genic feeds such as c u l l kidney beans a r e i n c l u d e d i n t h e r a t i o n . The

addition o f 0.17 ppm Se t o a k i d n e y bean and hay r a t i o n d u r i n g lactation

did not s i g n i f i c a n t l y reduce clinical NMD i n lambs; t h e a u t h o r s suggested

that a level of 1.0 ppm added Se may not be c o m p l e t e l y e f f e c t i v e under

these c i r c u m s t a n c e s ( H i n t z and Hogue, 1964).

SELENIUM DEFICIENCY AND WHITE MUSCLE DISEASE

Numerous d i s e a s e s have been i d e n t i f i e d as r e s p o n s i v e t o s e l e n i u m and

v i t a m i n E. Some respond t o o n l y v i t a m i n E or o n l y s e l e n i u m , w h i l e o t h e r s

such as w h i t e muscle d i s e a s e (WMD) may respond t o e i t h e r depending on t h e

d e f i c i e n t n u t r i e n t and o t h e r n u t r i t i o n a l stresses. By f a r t h e most common

worldwide Se/Vitamin E deficiency i n ruminants i s w h i t e muscle d i s e a s e ,

also known as n u t r i t i o n a l muscular dystrophy or s t i f f lamb disease.

 - 38 -

Recently, retained placenta in dairy cows, lameness i n breeding stock,

"sawdust liver" in feedlot steers, and illthrift, peridontal disease,

reduced f e r t i l i t y and poor wool p r o d u c t i o n i n sheep have a l s o been a s s o c i -

ated w i t h 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 affects young c a l v e s and lambs between b i r t h and

weaning, or sometimes j u s t a f t e r weaning or o t h e r s t r e s s e s . Morbidity may

be 65% or g r e a t e r , a l t h o u g h immediate S e / V i t a m i n E treatment usually helps

prevent severe losses. Seasonally, WMD i s most common in spring, and

frequently, lambs and ewes are on lush pasture, presumably rich in

a-tocopherol.

Symptoms of WMD are similar f o r lambs and calves. Mortality from

uncomplicated WMD invariably 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

p r o p e r l y diagnosed when c l i n i c a l s i g n s do not precede d e a t h . In p i g s the

disease i s called mulberry h e a r t d i s e a s e because e x t e n s i v e c a r d i a c hemor-

rhage r e s u l t s in a r e d d i s h - p u r p l e appearance. WMD affects heart f u n c t i o n

even b e f o r e h i s t o p a t h o l o g i c a l l e s i o n s d e v e l o p . Deficient calves display a

marked d e c r e a s e i n h e a r t r a t e a t the same time as i n i t i a l s i g n s of m u s c u l a r

dystrophy. Differences i n ECG t r a c i n g s were observed between c a l v e s on

normal and dystrophogenic d i e t s ( S a f f o r d ejt a _ l . , 1954). Similar abnormali-

ties have been seen i n vitamin E d e f i c i e n t lambs as well ( B a c i g a l u p o ejt

al., 1953). More i n f o r m a t i o n on the r o l e of Se i n h e a r t f u n c t i o n may come

from current s t u d i e s on Keshan d i s e a s e , a cardiomyopathy a f f e c t i n g thou-

sands of people i n China but now controlled by l a r g e s c a l e Se supplementa-

tion (Chen e t ^ a l . , 1981; Shamberger, 1981).

 - 39 -

The first s i g n of s t i f f lamb d i s e a s e i s r e l u c t a n c e t o walk, and then

a definite stiffness, especially of the back l e g s , and the characteristic

arched back s t a n c e . The muscles of the f r o n t and hind l e g s a r e a f f e c t e d

first and most s e v e r e l y , then those of the s h o u l d e r , rump, l o i n and neck

may be involved. In extreme cases lesions may affect the diaphragm,

intercostal muscles and tongue (Culik et al_., 1951). However, loss of

appetite i s rare.

Pathologically, WMD i s characterized by degeneration of striated

muscle, either skeletal or cardiac muscle, or both. Degeneration seems

closely related to the degree of muscle stress (Young and Keeler, 1962).

White or grey s p o t s and s t r e a k s 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 c a l c i u m p r e c i p i t a t i o n .

Two t y p e s of l e s i o n s - f i b e r and v a s c u l a r - a r e found i n WMD, and one

or both may occur in other Se/vitamin E responsive d i s e a s e s , such as

exudative diathesis i n chicks.

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 r u m i n a n t s . Van Fleet

et a l . (1977a) s t u d i e d ultrastructural changes i n f i b e r s of S e - v i t a m i n E

deficient swine, and found evidence of c o n c u r r e n t m y o f i b r i l l a r and mito-

chondrial changes, both probably initiated by cellular peroxidation.

S i m i l a r l y , Godwin et a l . (1974) observed an i n c r e a s e i n membrane lability

in organelles i n Se-vitamin E deficient tissues. The membrane damage

appears to affect intracellular fluid balance and energy production.

Damaged fibers eventually become mineralized, and the healed lesions

p e r s i s t as patches of s t r o m a l c o n d e n s a t i o n and fibrosis. Regeneration may

occur i n sheep skeletal muscle, but not i n cardiac muscle, at least in

swine (Van F l e e t et a l . , 1977a).

 - 40 -

Vascular damage i s f r e q u e n t l y found i n t h e h e a r t s and o t h e r tissues

of selenium or vitamin E deficient 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 skin as w e l l as t h e h e a r t i n swine (Van F l e e t e t a _ l . , 1977b),

developing independently of f i b e r lesions. The h e a r t o f lambs and c a l v e s

w i t h 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 t o

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 , tightly

joined endothelial cells lining t h e lumen o f normal v e s s e l s g i v e way t o

loosely attached, thickened but i n t a c t endothelium. The i n c r e a s e d perme-

ability allows leakage o f blood proteins into the v e s s e l wall, 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-

r h a g i n g may develop i n cases of spontaneous WMD.

SELENIUM AND DISEASE RESISTANCE

Since 1972 numerous r e p o r t s have been p u b l i s h e d on t h e e f f e c t s o f

selenium and v i t a m i n 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 selenite, however, o r g a n i c Se compounds

appear l e s s e f f e c t i v e than e q u i v a l e n t amounts o f Se as s e l e n i t e o r s e l e n i d e

(Spallholz, 1981). D e f i c i e n c i e s of selenium or v i t a m i n E a r e a s s o c i a t e d

with impaired immunity and i n c r e a s e d s u s c e p t i b i l i t y t o e x p e r i m e n t a l bacter-

i a l and f u n g a l i n f e c t i o n s , but s u p p l e m e n t a t i o n o f "normal d i e t s " l e a d s t o a

f u r t h e r increase i n antibody production.

Antibody production i n response t o v a r i o u s v a c c i n e s or SRBC-antigen

is enhanced in cattle, dogs, mice, r a b b i t s , and c h i c k s simultaneously

i n j e c t e d w i t h 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 a r e a l s o effective. The number o f p l a q u e - f o r m i n g c e l l s i n spleen of

mice were i n c r e a s e d 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 i n c r e a s e d from

0 t o 1.25 ppm ( S p a l l h o l z et a l . , 1973). (Plaque-forming cells produce

antibody.) D i e t s c o n t a i n i n g 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 t h e normal requirement, potentiated the synthesis o f 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 , t h e amount o f Se r e q u i r e d t o enhance t h e p r i m a r y immune

response i n mice was not much greater than the estimated daily Se

r e q u i r e m e n t ( S p a l l h o l z jet a l _ . , 1975).

Some e f f e c t s of selenium and v i t a m i n E on the immune system may be

interchangeable. The 2-week-old c h i c k r e q u i r e s both Se and v i t a m i n 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 function (Baumgartner, 1979). Selenium apparently can d u p l i c a t e

some o f t h e e f f e c t s o f v i t a m i n E on t h e immune system, which may i n v o l v e a

role i n u b i q u i n o n e and p r o s t a g l a n d i n synthesis ( H e i n z e r l i n g jet a d . , 1974;

Tengerdy and N o c k e l s , 1975; Tengerdy e t a l . , 1978). A l t e r n a t i v e l y , some o f

t h e immunostimulatory e f f e c t s o f Se and v i t a m i n E may i n v o l v e p r o v i s i o n o f

the proper biochemical environment for cellular i n t e r a c t i o n s , and may be

replaceable by s y n t h e t i c a n t i o x i d a n t s . Some o f t h e n o n s p e c i f i c mitogenic

f a c t o r s of macrophages a r e a n t i o x i d a n t s , and i t i s suggested t h a t antioxi-

dants such as Se and v i t a m i n E have a s i m i l a r adjuvant effect, i e . they

a t t r a c t macrophages (Baumgartner, 1979).

Defective microbicidal activity 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 o f n e u t r o p h i l s and i n g e s t i o n

o f b a c t e r i a l or f u n g a l c e l l s i s not i m p a i r e d , but t h e a b i l i t y t o k i l l these

c e l l s i s g r e a t l y impaired f o r both r a t s and c a t t l e , among o t h e r s (Serfass

 - kl -

and Ganther, 1975; Boyne and A r t h u r , 1978). The selenium deficiency

reduces neutrophil GSH-Px, and c o n s e q u e n t l y the a b i l i t y to metabolize

H 0 .
2 2 The H 0 2 2 accumulation results i n destruction of the -generating

system, which i s not a l t e r e d by v i t a m i n E status, at l e a s t i n rats (Baker

and Cohen, 1983). In contrast, Bass _et a l . (1977) compared b a c t e r i c i d a l

activity of n e u t r o p h i l s from species varying greatly i n normal GSH-Px

c o n t e n t , and c o n c l u d e d t h a t p o s t - p h a g o c y t i c o x i d a t i v e responces and b a c t e r -

ial killing "were not compromised by complete absence o f GSH-Px, even i n

species w i t h the h i g h e s t n a t u r a l l e v e l s o f t h i s enzyme".

Sex d i f f e r e n c e s i n a n t i b o d y response t o Se dosage have been o b s e r v e d

by several groups working w i t h chicks, 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 levels i n slight excess o f t h a t required to

prevent deficiency diseases, antibody titer i s s i g n i f i c a n t l y depressed i n

male but not female c h i c k s (Marsh et a l . , 1981; Baumgartner, 1979).

 - 43 -

IRON AND SELENIUM INTERACTIONS

Three p o s s i b l e areas of m e t a b o l i c interaction 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 life-

span, indirectly increasing iron 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

utilization of i r o n f o r heme s y n t h e s i s . Finally, iron status influences

l e v e l s of the selenoenzyme, GSH-Px i n red blood cells.

SELENIUM DEFICIENCY AND ANEMIA

S e l e n i u m and/or v i t a m i n E d e f i c i e n c i e s a r e a s s o c i a t e d w i t h hemolytic

anemia i n swine, monkeys and o t h e r animals. Both v i t a m i n E and selenium

a r e important i n maintaining RBC membrane i n t e g r i t y , but o n l y s e l e n i u m , as

GSH-Px, i s e f f e c t i v e a g a i n s t Hb o x i d a t i o n , due t o the membrane localization

of vitamin E (Hoekstra, 1974). Cells containing o x i d i z e d Hb, or Heinz

bodies, break down prematurely. Similarly, an inherited deficiency of

reduced glutathione (GSH), the s u b s t r a t e f o r GSH-Px, also reduces t h e

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 s y n t h e s i s and c a t a b o l i s m suggests a

role f o r selenium, distinct 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 s y n t h e s i s and c a t a b o l i s m was found i n l i v e r

but not i n s p l e e n of S e - d e f i c i e n t r a t s ( C o r r e i a and Burk, 1976). Further

investigations revealed that selenium i s e s s e n t i a l f o r normal utilization

of heme i n r a t l i v e r , and selenium d e f i c i e n c y l e a d s t o "wasted" heme; that

 - kk -

the effect 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 o f t h e element i n v o l v e d ; and t h e n u t r i t i o n a l selenium requirement o f

the r a t i s lower f o r maintenance o f h e p a t i c heme u t i l i z a t i o n than f o r main-

tenance o f h e p a t i c GSH-Px l e v e l s (Burk and C o r r e i a , 1981).

Whanger et a l _ . (1977) reported the f i r s t data on t h e e f f e c t s o f

selenium on o v i n e hepatic microsomal heme p r o t e i n s . Hepatic microsomal

cytochrome P^Q and t o t a l heme c o n t e n t were significantly lower i n WMD

lambs, but cytochrome b 5 content was not a f f e c t e d . Hepatic mitochondrial

heme p r o t e i n c o n t e n t 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 m i t o c h o n d r i a l

heme p r o 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 o f both v i t a m i n E and Se was necessary to alter microsomal

heme compounds i n t h e o v i n e liver.

The r o l e o f v i t a m i n E i n t h e r e g u l a t i o n of heme s y n t h e s i s has been

i n v e s t i g a t e d by N a i r et a l . (1972), w h i l e a d e t a i l e d study on t h e e f f e c t o f

v i t a m i n 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

also 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 v i t a m i n E and heme metabolism i s not y e t f u l l y understood.

SELENIUM AND HEME SYNTHESIS

Erythropoiesis

Selenium and/or vitamin E deficiency may also interfere with

erythropoiesis. An i n d i r e c t e f f e c t of selenium in stabilizing heme enzyme

systems may o c c u r , as was observed i n l i v e r (Whanger jet a l . , 1977).

Bone marrow a b n o r m a l i t i e s - erythrocyte hyperplasia and m u l t i n u c l e -

ated precursor cells - were found i n Se-E d e f i c i e n t swine (Niyo jst a l . ,

 - 45 -

1980). Multinucleated erythrocyte precursor cells are associated with

delayed erythrocyte maturation, which c o u l d e v e n t u a l l y be m a n i f e s t e d by low

b l o o d hemoglobin l e v e l s . Hemoglobin g r a d u a l l y decreased i n v i t a m i n E d e f i -

c i e n t lambs ( C u l i k et j r L . , 1951).

Baustad and Nafstad (1972) observed changes i n swine hematology con-

s i s t e n t w i t h impairment of h e m a t o p o i e s i s d u r i n g v i t a m i n E d e f i c i e n c y . The

reticulocyte count in vitamin E-treated piglets was significantly higher

than i n u n t r e a t e d l i t t e r m a t e s at 2 weeks of age (6.95 v s . 3.26% of e r y t h r o -

cytes). T o t a l Hb, PCV, and RBC count were a l s o h i g h e r i n v i t a m i n E - t r e a t e d

piglets. A d d i t i o n a l l y , bone marrow a b n o r m a l i t i e s typical of those des-

cribed by N a f s t a d (1973) were found i n v i t a m i n E d e f i c i e n t p i g l e t s o f any

age from newborn to 5 weeks.

Fontaine et _ a l . (1977a, 1977b) demonstrated that selenium, but not

v i t a m i n E, may have a s p e c i f i c role in erythropoiesis. Much work remains

t o be done i n t h i s a r e a . So f a r , the l i m i t e d i n f o r m a t i o n on Se and ery-

thropoiesis i n sheep i s confusing, as two papers reported that selenium

treatment depressed Hb levels.

Buchanan-Smith et c Q . (1969) observed a d e p r e s s i o n i n PCV, and more

slowly i n Hb, i n 4 month o l d lambs supplemented w i t h selenium compared t o

selenium-deficient controls. Horton et a l . (1978) compared f o u r methods o f

Se/vitamin E supplementation, finding the g r e a t e s t d e p r e s s i o n i n red cell

count and Hb w i t h the most e f f e c t i v e methods of Se supplementation.

IRON AND SELENIUM UTILIZATION

Iron s t a t u s a f f e c t s the c o n c e n t r a t i o n of GSH-Px i n red blood cells,

i n d i c a t i n g an u n a n t i c i p a t e d r o l e f o r i r o n i n selenium metabolism. Studies

 - 46 -

in humans demonstrate t h a t r e d blood c e l l GSH-Px i s s i g n i f i c a n t l y decreased

in iron deficiency anemia ( C e l l e r i n o ejt a i l . , 1976; M a c d o u g a l l , 1972).

The effect was not dependent on anemia per s e , as GSH-Px was e i t h e r

u n a f f e c t e d or i n c r e a s e d i n o t h e r types o f 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 iron levels.

Red c e l l GSH-Px was a l s o reduced i n i r o n deficient calves (Horber et a l . ,

1980), and i r o n - d e f i c i e n t rabbits (Rodvien j3t a l . , 1974). The d e c r e a s e i n

GSH-Px observed with iron deficiency may result from an inability to

utilize dietary Se f o r GSH-Px s y n t h e s i s (Ganther jst _ a l . , 1976). However,

this theory would not e x p l a i n why GSH-Px a c t i v i t y i n Se-deficient animals

might be i n f l u e n c e d by h i g h l e v e l s o f 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 s e l e n i u m i s

deficient. A single study was done u s i n g rats (Lee e t a l . , 1981). High

levels of d i e t a r y iron failed to influence RBC GSH-Px when Se was ade-

quate. However, GSH-Px was h i g h e r i n Se- and E - d e f i c i e n t r a t s f e d 1255 ppm

i r o n than those f e d 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 o f i r o n was not c o n s i d e r e d .
 - 47 -

MATERIALS AND METHODS

EXPERIMENTAL DESIGN

In a l l trials, lambs were born over a 3-6 week lambing p e r i o d , and

randomly a l l o c a t e d a t b i r t h t o one o f t h e i n j e c t i o n t r e a t m e n t s . The number

of treatments varied according to the t r i a l .

Iron Supplementation ( T r i a l 1)

Trial 1 investigated the e f f e c t s of i r o n supplementation u s i n g 17

c o n t r o l and 18 i r o n t r e a t e d lambs. The i r o n dosages were 0 and 500 mg o f

iron, administered within 3 days of b i r t h . As breed (Finn or D o r s e t

s i r e d ) , sex and r e a r i n g (as s i n g l e o r t w i n ) , may markedly influence weight

gains and p o s s i b l y o t h e r parameters, these factors were i n c l u d e d i n the

experimental model. Consequently, the t r i a l was s e t up as a c o m p l e t e l y

randomized 2X2X2X2 d e s i g n .

Weight, hemoglobin and PCV were measured weekly from b i r t h t o 11

weeks of age. Plasma samples taken a t 24-25 days o f age were a n a l y z e d f o r

a blood p r o f i l e i n c l u d i n g c a l c i u m , i n o r g a n i c phosphate, g l u c o s e , blood urea

nitrogen, total protein, albumin, a l k a l i n e phosphatase, lactate dehydro-

genase, a s p a r t a t e transaminase and plasma iron. T h i s p r o f i l e was s e l e c t e d

t o a s s e s s t h e e f f e c t of i r o n t r e a t m e n t and/or anemia on o t h e r p h y s i o l o g i c a l

parameters. F o r example, glucose and c h o l e s t e r o l are affected 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 t o a n a l y z e a l l t h e T r i a l 1

data:
 - 48 -

Y i j k l = u + Tj^ + Bj + + Ri + Ti Bj + T j S ^ + T^Ri + B j S ^ + BjR^ +

S Ri
k + Wijki + E i j k i .

where Yj.jkl =
t h e dependent v a r i a b l e hemoglobin, PCV, e t c .

u = t h e o v e r a l l mean common t o a l l samples

T^ = t h e e f f e c t o f t h e i ' t h treatment

Bj = t h e e f f e c t o f t h e j ' t h breed

= t h e e f f e c t o f t h e k ' t h sex

Rl = the e f f e c t of the l ' t h r e a r i n g

T^B-j = t h e i n t e r a c t i o n o f t h e i ' t h treatment w i t h the

j ' t h breed

T^S^ = t h e i n t e r a c t i o n o f t h e i ' t h treatment w i t h t h e

k ' t h sex

T^R| = t h e i n t e r a c t i o n o f t h e i ' t h treatment w i t h t h e

l ' t h rearing

B-JSJ,. = t h e i n t e r a c t i o n o f t h e j ' t h breed w i t h t h e k ' t h

sex

BjRi = t h e i n t e r a c t i o n o f t h e j ' t h breed w i t h t h e l ' t h

rearing

S^Ri = t h e i n t e r a c t i o n o f t h e k ' t h sex w i t h t h e l ' t h

rearing

W i j k l = the covariable birth weight

E i j k l = t h e u n e x p l a i n e d r e s i d u a l e r r o r a s s o c i a t e d w i t h each
sample

The above model was a l t e r e d for analysis o f t h e plasma p r o f i l e data

by t h e a d d i t i o n of a hemolysis covariable f o r T r i a l s 1 and 2. Hemolysis

was ranked on t h e s c a l e o f 0 (no h e m o l y s i s ) t o 4 ( s e v e r e h e m o l y s i s ) as some

h e m o l y s i s was u n a v o i d a b l e .
 - 49 -

Level of Iron Supplementation ( T r i a l 3)

Trial 2 compared the effects of 3 l e v e l s of i r o n treatment: 0, 250

mg, and 500 mg iron. As b e f o r e , breed, sex and rearing were o t h e r sources

of variation, resulting i n a 3X2X2X2 d e s i g n . 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 ( T r i a l 3)

In T r i a l 3, i r o n and selenium t r e a t m e n t s were combined i n t o 4 t r e a t -

ment c o m b i n a t i o n s . These were c o n t r o l , 1.5 mg Se o n l y , 500 mg Fe o n l y , 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 p e r i o d s . Hb, PCV, weight and

plasma p r o f i l e data were c o l l e c t e d as b e f o r e . However, plasma s e l e n i u m and

plasma p r o t e i n data were o b t a i n e d from r e p l i c a t e s 2 and 3 o n l y , 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 o n l y .

The following l i n e a r model was used to measure the e f f e c t s of repli-

cate, treatment, breed, sex and rearing on Trial 3 Hb, PCV, weight and

plasma p r o f i l e data:

Y
ijklm = u + Pi + Tj + ^ + S X + + PiTj + PiBk + TjBk + +

T
j^n + + E
ijklm

where Yijklm = t n e
dependent v a r i a b l e Hb, PCV, etc.

u = the o v e r a l l mean common to a l l samples

Pi = the e f f e c t of the i'th replicate

Tj = the e f f e c t of the j ' t h t r e a t m e n t

B k = the e f f e c t of the k'th breed

Si = the e f f e c t o the l'th sex

R m = the e f f e c t of the m'th rearing

 - 50 -

Pjjj = 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 ' t h breed

TjB^ = the i n t e r a c t i o n of the j ' t h treatment with the

k ' t h breed

TjS| = t h e i n t e r a c t i o n o f t h e j ' t h treatment w i t h t h e

I ' t h sex

TjR m = t h e i n t e r a c t i o n o f t h e j ' t h treatment w i t h t h e

m'th rearing

E^k^m = t h e i n t e r a c t i o n o f t h e j ' t h breed w i t h t h e m'th

rearing

^ijklm = t n e
birthweight covariable

F-ijklm = t h e u n e x p l a i n e d r e s i d u a l e r r o r a s s o c i a t e d w i t h
each sample

Plasma p r o t e i n s were a n a l y z e d u s i n g a s i m p l i f i e d l i n e a r model, which

included total protein as a covariable:

Y
ijklm = u
+ T
i+ Bj + S k + R i + P 1^1 + E i ; j k l

where ^ijkl = t n e
dependent v a r i a b l e a l b u m i n , b e t a - g l o b u l i n , e t c .

u = t h e o v e r a l l mean common t o a l l samples

l"i = t h e e f f e c t o f t h e i ' t h treatment

Bj - t h e e f f e c t o f t h e j ' t h breed

S|< = t h e e f f e c t o f t h e k ' t h sex

Ri = the e f f e c t of the I'th rearing

F>ijkl = t h e t o t a l p r o t e i n covariable

F-ijkl = the unexplained r e s i d u a l error associated with

each sample

Plasma s e l e n i u m data were a n a l y z e d u s i n g t h i s model:

 - 51 -

Y
ijklm = 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 s e l e n i u m

u = the o v e r a l l mean common t o a l l samples

1^ = the e f f e c t of the i ' t h i r o n treatment

Xj = the e f f e c t o f the j ' t h s e l e n i u m treatment

B k = the e f f e c t of the k'th breed

Si = the e f f e c t of the I ' t h sex

R m = the e f f e c t of the m'th rearing

I^X-j = the i n t e r a c t i o n of the i ' t h i r o n t r e a t m e n t w i t h

t h e j ' t h selenium treatment

XjSi = the i n t e r a c t i o n of the j ' t h s e l e n i u m treatment

w i t h the I ' t h sex

F-ijklm = the u n e x p l a i n e d r e s i d u a l e r r o r a s s o c i a t e d w i t h
each sample

The model for analysis of the h e m a g g l u t i n a t i o n data was the same,

with the a d d i t i o n of the i n t e r a c t i o n s of s e l e n i u m w i t h r e a r i n g , i r o n w i t h

sex, and iron with rearing.

STATISTICAL ANALYSIS

Analysis of v a r i a n c e was done u s i n g UBC BMD:10V (1975), a General

L i n e a r H y p o t h e s i s packaged program. A major advantage of t h i s program was

its ability to m a n i p u l a t e unbalanced c e l l s and missing dtaa, although not

missing cells. A n a l y s i s of c o v a r i a n c e was used instead of ANOVA when a

concomitant v a r i a b l e , which c o u l d be measured but not c o n t r o l l e d , affected

a dependent v a r i a b l e . For example, d e s p i t e random a l l o c a t i o n of lambs t o

t r e a t m e n t s , mean b i r t h weight tended to be h i g h e r f o r some t r e a t m e n t s . As

 - 52 -

birth weight i s related t o weight g a i n , i t was v e r y u s e f u l t o be a b l e t o

a d j u s t means f o r t h e e f f e c t o f b i r t h weight on weight.

ANOVA with BMD:10V also enabled the t e s t i n g of s i n g l e degrees o f

freedom c o n t r a s t s . A_ p r i o r i , o r t h o g o n a l hypotheses were:

for a l l i r o n l e v e l d a t a , T r i a l 2:

1. C o n t r o l lambs do not d i f f e r from i r o n t r e a t e d lambs.

2. High l e v e l o f 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. I r o n t r e a t e d lambs do not d i f f e r from n o n - i r o n t r e a t e d lambs.

2. Selenium treated lambs do not d i f f e r from non-selenium treated

lambs.

3. I r o n and s e l e n i u m do not i n t e r a c t .

The BMD:10V program was run f o r a l l s e t s o f data c o l l e c t e d , changing

the model as w a r r a n t e d . 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 o b t a i n e d , t h e

SS 1
and d . f . ' s were added i n t o t h e e x p e r i m e n t a l e r r o r t o i n c r e a s e t h e pre-

cision, then t h e F's were recalculated by t h e program. Consequently,

three-way and four-way interactions were normally eliminated, and many

biologically meaningless two-way i n t e r a c t i o n s . The f i r s t model g i v e n was

adjusted in this manner, w h i l e t h e r e m a i n i n g models represent the f i n a l

versions o f t h e complete initial models. This frequently resulted 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 m e t a b o l i t e s and plasma i r o n , Hb,

PCV, weight g a i n , and b i r t h weight were i n v e s t i g a t e d u s i n g UBC TRP (1978),

a triangular regression package. They were of i n t e r e s t as c o v a r i a n c e

a n a l y s i s was not a p p r o p r i a t e f o r l o o k i n g a t c o r r e l a t i o n s , y e t t h e e x i s t e n c e
 - 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 d a t a . TRP used a forward

s t e p w i s e r e g r e s s i o n t e c h n i q u e to d e r i v e r e g r e s s i o n e q u a t i o n s .

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 t e d i o u s to measure both Hb and PCV, when they might be of equal value

i n assessing iron deficiency. R e g r e s s i o n a n a l y s i s d e r i v e d p r e d i c t i v e equa-

t i o n s f o r Hb from PCV a t d i f f e r e n t ages, and was used t o a s s e s s the close-

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 also plotted scat-

tergrams of Hb versus PCV, with and without a severe level of outlier

rejection of 5% of the d a t a . Simple r e g r e s s i o n e q u a t i o n s were c a l c u l a t e d

for a l l plots.

All means are g i v e n w i t h v a r i a t i o n expressed as the s t a n d a r d error,

and not as the s t a n d a r d deviation.

ANIMAL MANAGEMENT

Lambs were housed w i t h t h e i r dams i n sawdust-bedded group pens w i t h i n

an open-eaved unheated building on the University of British 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 a t 3-7 days o f age. M a l e s were not c a s t r a t e d .

Lambs were weaned a t an average age of e i g h t weeks.

Water and cobalt-iodized s a l t were a v a i l a b l e ^ d l i b . Ewes were fed

alfalfa cubes and a barley-based grain mixture twice d a i l y . Starting at

ten days of age, the lambs began e a t i n g s m a l l amounts of a creep-feed

ration having a calculated iron content of 95 ppm (Appendix 1). By six

weeks of age, lambs were consuming about 0.5 kg of c r e e p f e e d per head per

day.
 - 54 -

Health problems were never severe. Contagious pustular dermatitis

(soremouth) was endemic, appearing i n each lamb crop s e v e r a l weeks after

t h e s t a r t o f lambing. I s o l a t e d cases o f both J E . c o l i s c o u r s and Corynebac-

terium ovis joint abscesses occurred; the l a s t rarely affected growth

rate. White muscle d i s e a s e a f f e c t e d some lambs a t v a r i o u s ages between

birth and 4 weeks of age i n T r i a l s 1 and 2 o n l y . The a f f e c t e d animals

usually responded t o a combined selenium/vitamin E injection. Overall

mortality was low and v a r i e d from 0 t o 7%; main causes were premature/

difficult b i r t h s , t r a m p l i n g and pneumonia.

Lambs were t r e a t e d w i t h i r o n and/or selenium depending on t h e e x p e r i -

ment. Both i r o n and selenium were a d m i n i s t e r e d by i n t r a m u s c u l a r injection

between 0 and 3 days o f age. The products used were H a e m a l i f t and Dysto-

sel, both from t h e Rogar/STB division o f BTI P r o d u c t s , I n c . , 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-

ide 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 in a pail with a spring balance

( a c c u r a c y ± 0.1 k g ) . When lambs reached 15 kg, a l a r g e beam balance with

an a c c u r a c y o f ± 0 . 5 kg was used.

Blood Samples

All samples were obtained by j u g u l a r v e n i p u n c t u r e using 20 gauge

needles. F i v e ml tubes c o n t a i n i n g a s m a l l amount o f sodium heparin were

 - 55 -

used to c o l l e c t s m a l l (2 ml) initial samples f o r hemoglobin and packed cell

volume d e t e r m i n a t i o n s . Subsequently, 10 ml heparinized vacutainer tubes

were used to c o l l e c t blood f o r Hb, PCV, plasma p r o f i l e , and m i n e r a l analy-

ses. Vacutainer tubes c o n t a i n i n g potassium o x a l a t e i n s t e a d of h e p a r i n were

used to collect samples for plasma protein electrophoresis. Plain or

s i l i c o n - c o a t e d v a c u t a i n e r s were used when serum was required.

Hemoglobin

Blood hemoglobin l e v e l s were measured by the cyanmethemoglobin t e c h -

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.

D u p l i c a t e a n a l y s e s were done f o r each sample w i t h i n 2k hours of collection;

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 i n v o l v e d c o n s i s t e d of Vanlab 20 mm (±1%) d i s p o s -

3

a b l e m i c r o p i p e t t e s , H y c e l No. 117 Cyanmethemoglobin C e r t i f i e d S t a n d a r d , and

a G i l f o r d Stasar I I spectrophotometer.

Packed C e l l 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 collection. Dade and Canlab heparinized microhematocrit

capillary tubes were t w o - t h i r d s filled with blood, capped w i t h Critoseal,

and centrifuged f o r 15 minutes in a Canlab International Microcapillary

C e n t r i f u g e w i t h Reader (Models MB and CR).

Plasma P r o f i l e

Plasma was e x t r a c t e d from 10 ml whole blood samples, f r o z e n and later

analyzed by a commercial laboratory (B.C. Biomedical Laboratories Ltd.,

 - 56 -

7845 Edmonds, Burnaby, B.C.). Eleven plasma c o n s t i t u e n t s were measured -

calcium (Ca), inorganic phosphate ( P ^ ) , glucose, blood urea nitrogen

(BUN), cholesterol, total protein, albumin, a l k a l i n e phosphatase ( A P ) ,

lactate dehydrogenase (LDH), aspartate transaminase ( A T ) , and iron.

References to a n a l y t i c a l procedures f o r each metabolite are given in

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

in vacutainer tubes c o n t a i n i n g potassium o x a l a t e . Heparin i s a u n s u i t a b l e

anticoagulant as i t may i n t e r f e r e w i t h various p r o t e i n bands i n e l e c t r o -

phoresis.

After thawing, samples were c e n t r i f u g e d and decanted as n e c e s s a r y t o

remove t u r b i d i t y caused by l i p i d s and denatured proteins. Hemolysis was

evident i n some samples but was not judged severe enough t o n e c e s s i t a t e

analysis o f Hb and use o f a correction factor. (The cyanmethemoglobin

technique i s not a p p r o p r i a t e f o r Hb c o n c e n t r a t i o n s l e s s t h a t 4 g/d£.)

However, hemolysis c o u l d w e l l be a source o f e r r o r i n both total protein

(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 .

Total protein. The b i u r e t method ( G o r n a l l ejt a l _ . , 1949; Cannon ejt

a 1., 1974) was chosen for total protein determination, as i t produces a

stable colour that obeys Beer's Law and i s u n a f f e c t e d by t h e r a t i o of

albumin to g l o b u l i n .

Protein fractions. Plasma protein fractions were separated by

electrophoresis on prepared agarose g e l i n the Corning Cassette Elector-

phoresis Cell System. The Corning procedure was f o l l o w e d ("Determination

 - 57 -

o f serum p r o t e i n s (Amido B l a c k 10B)", C o r n i n g M e d i c a l , C o r n i n g G l a s s Works,

Medfield, Massachusetts). In order t o maximize the r e s o l u t i o n of the pro-

tein fractions, various b u f f e r s were tested at varying pH's and ionic

strengths (Cannon et _al., 1974). The optimum combination seemed to be

sodium barbital buffer, ionic s t r e n g t h u=0.05, pH=8.6, i n p r e f e r e n c e to

C o r n i n g U n i v e r s a l B a r b i t a l B u f f e r c o n t a i n i n g EDTA. The electrophoretograms

were scanned i n the T r a n s i d y n e G e n e r a l 2980 Scanning Densitometer. Protein

fractions were c a l c u l a t e d as percentages of total p r o t e i n by calculating

t h e r e l a t i v e s u r f a c e area under each peak. A c t u a l v a l u e s of the 5 p r o t e i n

fractions were then calculated from the percentages using total protein

v a l u e s o b t a i n e d by the b i u r e t t e c h n i q u e .

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. L a y i n g 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 S c i e n c e s u p p l i e d the e r y t h r o c y t e s

for both antiserum production and testing. Using different birds each

time, ten or more b i r d s were b l e d three times weekly. Ten mis of blood

were withdrawn from a medial v e i n on the u n d e r s i d e of the wing (Garvey et

al., 1977, p. 3 1 ) . The equipment i n c l u d e d e t h a n o l , c o t t o n gauze, 21 guage

needles rinsed with concentrated sodium heparin solution, 12 ml syringes

c o n t a i n i n g 2-3 mis of A l s e v e r ' s s o l u t i o n p l u s sodium h e p a r i n , and collec-

tion flasks containing Alsever's solution. Heparin was necessary t o 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 syringe. 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 A l s e v e r ' s s o l u t i o n . C e l l s were washed by m i x i n g w i t h

 - 58 -

sterile citrate/saline solution (Garvey £t aK, 1977, p. 524), c e n t r i f u g e d

in s t e r i l e 40 ml tubes in a refrigerated centrifuge, then the supernatant

was decanted. These steps were repeated 4-6 times to remove plasma

p r o t e i n s and lipids.

The e r y t h r o c y t e c o n c e n t r a t i o n was s t a n d a r d i z e d by a d a p t i n g a method

for the p h o t o m e t r i c s t a n d a r d i z a t i o n of sheep e r y t h r o c y t e s (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 distilled water. The lysate was read against a distilled water

b l a n k at a wavelength of 520 nm, and a d j u s t e d to g i v e an o p t i c a l d e n s i t y of

0.500. The original suspension was then a d j u s t e d a c c o r d i n g l y by the a d d i -

tion of cells or buffer solution, resulting i n a 2.5% cell suspension

c o n t a i n i n g a p p r o x i m a t e l y 4 X 10 8
c e l l s per ml.

Antiserum p r o d u c t i o n . Sheep on a l l f o u r 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 w i t h 1 ml of t h e

freshly-prepared cell s u s p e n s i o n a t weekly intervals from 4 t o 9 weeks of

age. T h i s was done three times weekly at the usual sampling times to

minimize age variability within the sampling periods. Simultaneously,

blood samples were taken f o r serum. As every effort was made t o 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 s i d e e f f e c t s r e s u l t e d from the e r y t h r o -

cyte injections.

The sheep b l o o d samples were a l l o w e d t o c l o t a t room temperature, and

centrifuged. Serum was decanted and initially, refrigerated a t 4°C for

immediate a n t i s e r a t e s t n g u s i n g a p a s s i v e h e m a g g l u t i n a t i o n method. Because

of time r e s t r a i n t s , most samples were f r o z e n and t e s t e d 4-5 months later.

The initial serum samples were a l s o f r o z e n and retested.

 - 59 -

Hemagglutination Test

The passive hemagglutination t e s t was performed on t h e thawed, heat-

treated antiserum samples f o l l o w i n g the method d e s c r i b e d by Garvey et a l .

(1977, pp. 356-360). As t h e t e c h n i q u e 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

r b c ' s coated w i t h bovine serum albumin (BSA) as a n t i g e n , and anti-BSA from

rabbits, some m o d i f i c a t i o n s were necessary: 1) t h e a n t i g e n was chicken

r b c ' s , the a n t i s e r u m was sheep a n t i - c h i c k e n - r b c ; 2) the a n t i s e r u m required

a t most 1:2 and 1:10 d i l u t i o n , r a t h e r than 1:10,000 d i l u t i o n ; 3) e r y t h r o -

c y t e s r a t h e r than BSA were t h e a n t i g e n , therefore tannic acid 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 o b t a i n e d as d i l u t i o n u n i t s , and con-

verted to natural log units f o r 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

E f f e c t of Iron on Hemoglobin, PCV, and Plasma Iron

Iron supplementation o f lambs s u b s t a n t i a l l y a f f e c t e d t h e p a t t e r n o f

hemoglobin and h e m a t o c r i t changes d u r i n g the f i r s t 11 weeks o f l i f e . The

r a p i d d e c l i n e i n hemoglobin and PCV between b i r t h and t h r e e weeks o f age i n

c o n t r o l lambs was prevented by i r o n dextran injection. The s i n g l e injec-

tion of 500 mg i r o n at b i r t h significantly i n c r e a s e d Hb, PCV and plasma

i r o n from 2 t o 11 weeks o f age (P<0.05). Although few c o n t r o l lambs became

anemic, Hb, PCV, and plasma i r o n v a l u e s indicated t h a t 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 initial blood samples were taken a t v a r i o u s times between birth

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 v a l u e of 14.17 g/d& r e p o r t e d by H o l z et a l . (1961).

Mean i n i t i a l v a l u e s of both Hb and PCV were s l i g h t l y higher f o r the i r o n -

treated group than t h e c o n t r o l group, w i t h means o f 14.5±0.4 v s . 13.7±0.6

g/d£ Hb, and 40.2±1.2 v s . 37.8±1.5% PCV. T h i s d i f f e r e n c e was not s i g n i f i -

cant (P<0.05) due t o t h e high variability o f Hb and PCV a t t h i s age.

Contributing factors i n c l u d e polycythemia i n some lambs a t b i r t h , and a

rapid drop i n t o t a l red c e l l s d u r i n g t h e f i r s t 12 hours o f l i f e , followed

by a slower decrease i n Hb and PCV ( B l u n t , 1975). Consequently, Hb and PCV

v a l u e s change r a p i d l y d u r i n g t h e f i r s t t h r e e days o f l i f e .

The Hb l e v e l s o f both control and i r o n - t r e a t e d lambs c o n t i n u e d t o

fall until 1 week o f age (12.3±0.5 v s . 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, a t 2 weeks of age t h e 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 level i n the c o n t r o l group f e l l t o 10.7±0.4 g/d£ a t 3 weeks,

then i n c r e a s e d s l o w l y t o 12.0±0.2 g/d£ at 7 weeks and stabilized at that

l e v e l u n t i l the end of the t r i a l a t 11 weeks (Appendix 3 ) . In comparison,

the Hb level i n the i r o n - t r e a t e d group increased steadily from a low of

12.9±0.2 g/djj, a t 1 week t o 13.7±0.3 g/djj, a t 4 weeks, and remained near t h a t

l e v e l u n t i l 7 weeks. As shown i n F i g u r e 1, the Hb l e v e l d e c l i n e d slightly

from 8 t o 11 weeks but remained h i g h e r than i n t h e c o n t r o l s . The t r e a t m e n t

difference was still significant (P<0.001) at 7 weeks, and remained signi-

f i c a n t at 11 weeks (P<0.05).

Changes i n h e m a t o c r i t v a l u e s i n the two treatment groups paralleled

changes i n hemoglobin almost exactly, as shown i n F i g u r e 2. Treatment

effect was significant at 2 weeks (31.2+1.156 v s . 38.8±0.8% PCV, P<0.001).

S u b s e q u e n t l y , 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% a t 3

weeks of age, and increased steadily from 3 to 11 weeks, r e a c h i n g a mean

v a l u e of 36.0+0.7%. PCV levels i n the i r o n - t r e a t e d group i n c r e a s e d from a

low o f 35.8±0.8% a t 1 week t o a h i g h o f 39.5±0.7% a t 5 weeks. The treat-

ment e f f e c t was significant (P<0.001) from 2 t o 7 weeks of age, and r e -

mained s i g n i f i c a n t (P<0.05) at 11 weeks. Data i s g i v e n i n Appendix 3.

While actual Hb and PCV v a l u e s may v a r y from study t o s t u d y , t h e same

p a t t e r n of both minimum l e v e l s i n c o n t r o l s and maximum response t o 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 D u b e s k i , 1979). Ullrey et a l . (1965)

measured average Hb and PCV v a l u e s of 6.2 g/d£ Hb and 20.5% PCV i n 3 week

old control lambs, compared t o an i r o n - t r e a t e d group w i t h 10.8 g/djj, Hb and

15.0 A

Weeks of Age

Figure 1. Effect of iron supplementation on h e m o g l o b i n (Trial 1).

30 I 1 1

] 2 3 4 5 6 7 8 9 10 11
Weeks of Age
F i g u r e 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. I n another s t u d y , the minimum Hb l e v e l averaged 8.37 g/d& in 3

week o l d lambs (Holz et a l . , 1961). More r e c e n t l y , Wohlt (1982) found

l e v e l s o f 23% PCV and 9.4 g/d£ Hb i n 42 14-day o l d lambs t h a t had not been

docked. In the present study, the minimum Hb and PCV levels in controls

were much h i g h e r , at 10.7 g/d£ Hb and 30.9% PCV, yet i r o n treatment still

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 signifi-

cant o n l y at 2 and 3 weeks of age (P<0.05). Both parameters were slightly

higher in s i n g l e s compared to twins at birth (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 signifi-

cantly higher PCV and Hb levels than t w i n s , w i t h i n each treatment group.

T h i s i n d i c a t e s t h a t h i g h e r body r e s e r v e s o f i r o n present i n the s i n g l e lamb

a t 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 w i t h other species provides evidence that twinning leads to

reduced storage of iron i n each fetus. A study o f newborn d a i r y calves

revealed a significant incidence of severe neonatal anemia w i t h 15.8% of

s i n g l e c a l v e s , and 37.5% of t w i n c a l v e s 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 f e t u s e s r e s u l t e d from

unequal f u n c t i o n i n g of the two circulatory systems, as occurs i n human

twins. Twinning, low birthweight and prematurity predispose iron-lack

anemia i n human i n f a n t s ( B e t k e , 1970). The type and degree o f t r a n s p l a c e n -

tal i r o n t r a n s f e r would determine whether t w i n n i n g or low birthweight pre-

dispose anemia i n sheep. So f a r the l i m i t e d i n f o r m a t i o n on the s u b j e c t 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; H o s k i n s and Hansard, 1964).

Mean plasma i r o n at 4 weeks was 185 ug/d£. I r o n treatment signifi-

cantly (P<0.005) e l e v a t e d 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 t o 223±15 pg/d£ i n t h e i r o n - t r e a t e d lambs. Breed, sex and r e a r i n g

d i d 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 h i g h e s t

growth r a t e , and tended t o have e x t r e m e l y low i r o n levels. Thus, even

though single lambs a r e presumed t o have greater tissue iron stores at

birth, they may be most b e n e f i t t e d by supplementary i r o n due t o t h e i r high

growth r a t e .

Normal l e v e l s of plasma i r o n were r e p o r t e d t o be 159 yg/d£ i n 2 week

o l d lambs, and 228 yg/dj;, i n 6 week o l d lambs ( H i d i r o g l o u & 3 e n k i n s , 1971).

In o l d e r lambs normal v a l u e s were between 200 and 300 yg/d£, which i s much

h i g h e r than t h e normal l e v e l o f 115 yg/d£ i n humans ( B o t h w e l l et a_l., 1979,

p. 2 9 7 ) . I t i s not known i f t h e plasma i r o n t h r e s h o l d 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 s p e c i e s .

Plasma P r o f i l e

Variability was high f o r most m e t a b o l i t e s . I n s p i t e of minimizing

age v a r i a t i o n , and a c c o u n t i n g f o r breed, sex and r e a r i n g e f f e c t s , t h e h i g h

overall variability tended t o obscure subtle 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 o n l y m e t a b o l i t e significantly affected

by i r o n t r e a t m e n t (Table I ) . The a p p l i c a b i l i t y o f the plasma p r o f i l e test

t o n u t r i t i o n a l s t u d i e s has been q u e s t i o n e d (Rowlands, 1980). Large numbers

of a n i m a l s per t r e a t m e n t may be r e q u i r e d t o compensate f o r high innate

v a r i a b i l i t y i n the data.

Calcium

The mean plasma calcium value a t 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). Similarly,

Mitruka and Rawnsley (1977) gave a range o f 10.4-14.0 mg/d£ from t h e

 - 66 -

TABLE I.

Effect of iron treatment on plasma p r o f i l e at 24-25 days (Trial 1)

CONTROL IRON S.E.M.

n 17 18

C a l c i u m (mg/d£) 11.7 11.4 0.1

I n o r g a n i c Phosphate (mg/d£) 10.3 10.0 0.3

G l u c o s e (mg/d£) 95.3 93.9 2.8

Blood Urea N i t r o g e n (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 Protein (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

L a c t a t e Dehydrogenase (IU/£ ) 569 587 31

A s p a r t a t e Transaminase (IU/& ) 104.6 113.1 2.8

Plasma I r o n (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 t r e a t m e n t means i n t h e same row,

P<0.05.
 - 67 -

l i t e r a t u r e , w i t h a mean o f 11.4 mg/d£. Plasma c a l c i u m and phosphorus have

been observed t o vary w i t h age i n lambs (Long et a l _ . , 1965; Moodie, 1975)

as well as i n c a l v e s (Simesen, 1970, p. 3 2 0 ) . A comprehensive study o f

lamb hematology r e p o r t e d 2 week serum c a l c i u m l e v e l s o f 12.2+0.19 mg/d£,

f a l l i n g t o 11.5+0.14 mg/d£ a t 4 weeks (Long jet a l . , 1965).

I r o n 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 v a l u e s were 11.73±0.15 mg/d£ and 11.41+0.17 mg/dz f o r t h e 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 inorganic phosphate (P^) was 10.14+0.29 mg/d£.

V a l u e s i n the l i t e r a t u r e show g r e a t v a r i a b i l i t y because o f age and d i e t a r y

effects. Long e t a l . (1965) o b t a i n e d very s i m i l a r results t o the p r e s e n t

study. The mean from 2-4 weeks o f age was 11.0 mg/d£ . Weight gain

and feed intake are correlated with P^, resulting i n high variability

even among a n i m a l s f o t h e same age and d i e t (Moodie, 1975; L i t t l e et a l . ,

1977).

Plasma P^ i s more sensitive than plasma Ca to dietary factors.

Pi varies markedly with dietary phosphorus, and a l s o intimately linked

w i t h c a r b o h y d r a t e metabolism (Simesen, 1970). I r o n t r e a t m e n t d i d not s i g -

nificantly (P>0.05) a f f e c t P i # Plasma c o n t a i n e d 10.33±0.46 mg/d£ P j i n

the control group compared t o 9.97+0.37 mg/d£ Pj_ i n t h e i r o n - t r e a t e d

group.

Glucose

Blood glucose levels i n young ruminants and monogastrics are

comparable, but a r e s i g n i f i c a n t l y lower i n the adult ruminant. Blood

 - 68 -

g l u c o s e has been p o s i t i v e l y c o r r e l a t e d w i t h feed i n t a k e and weight gain i n

c a l v e s ( L i t t l e et a l . , 1977) and i n one year o l d sheep (Bensadoun et a l . ,

1962).

Literature values of ovine plasma glucose range from 55.0 t o 131

mg/djj, ( M i t r u k a and Rawnsley, 1977). H a c k e t t _et al_. (1957) r e p o r t e d a mean

of 53.5±2.3 mg/dJL, w i t h 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 . L i n d s a y and Leat (1975) l i s t e d

mean g l u c o s e levels o f 103.4 mg/d£ and 96.9 mg/d£ f o r 17-24 day o l d and

25-32 day o l d lambs, r e s p e c t i v e l y . These means a r e s i m i l a r t o t h e mean i n

T r i a l 1 of 94.6+2.8 mg/d£.

The blood g l u c o s e f a l l s steadily w i t h advancing age i n t h e growing

lamb, r e a c h i n g a d u l t l e v e l s at 6-9 weeks ( R e i d , 1953). In s p i t e of the

metabolic shift i n emphasis from glucose to v o l a t i l e fatty a c i d s , the

change i n blood glucose does not have a close relationship with rumen

development. V a r i o u s workers have a t t r i b u t e d t h e blood g l u c o s e changes t o

a s h i f t i n e r y t h r o c y t e metabolism, a s s o c i a t e d w i t h the replacement of f e t a l

erythrocytes with adult-type cells (Reid, 1953; Kappy, 1982). Adult

erythrocytes differ i n hemoglobin type, ionic composition, glucose meta-

b o l i s m and enzyme a c t i v i t y ( B l u n t , 1975).

Plasma g l u c o s e may not then be a p p r o p r i a t e f o r a s s e s s i n g energy sta-

t u s i n t h e young lamb. The response o f plasma FFA but not blood g l u c o s e t o

ovine growth hormone s u p p o r t s this viewpoint (Lindsay and L e a t , 1975).

Rowlands (1980) concluded t h a t i n sheep, u n l i k e i n c a t t l e , "FFA c o n c e n t r a -

t i o n s c o r r e l a t e b e t t e r w i t h energy i n t a k e than do g l u c o s e c o n c e n t r a t i o n s . "

As expected, plasma g l u c o s e was not a f f e c t e d (P>0.05) by i r o n treat-

ment (95.3±3.0 mg/d£ v s . 94.9±4.7 mg/d£ i n control and iron-treated

groups).
 - 69 -

T o t a l P r o t e i n , Albumin and BUN

Plasma total protein, a l b u m i n , and BUN (blood urea nitrogen) are

typically i n c l u d e d as i n d i c e s o f p r o t e i n metabolism i n t h e m e t a b o l i c pro-

file. R e s u l t s a r e f r e q u e n t l y ambiguous as these parameters a r e not s e n s i -

tive to small quantitative or q u a l i t a t i v e dietary changes. F o r example,

decreased serum p r o t e i n t u r n o v e r has been observed t o m a i n t a i n serum l e v e l s

f o r a n i m a l s on a low or z e r o - p r o t e i n d i e t . Serum a l b u m i n , t o t a l protein,

Hb and PCV a r e found t o v a r i a b l y and s l o w l y respond to protein deficien-

cies. Protein intake seems t o 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 s t u d y , t o t a l p r o t e i n , albumin and BUN were normal. There was

no reason t o a n t i c i p a t e s i g n i f i c a n t t r e a t m e n t responses.

Total protein v a l u e s averaged 5.98±0.09 g/d£, w i t h no significant

treatment e f f e c t a t P>0.05. T y p i c a l mean v a l u e s i n the l i t e r a t u r e average

5.81±0.54 g/d£ (Kuttler and M a r b l e , 1960) and 5.46 g/d£ (Irfan, 1967).

T o t a l p r o t e i n i n c r e a s e s w i t h age, m a i n l y because o f i n c r e a s i n g gamma g l o b u -

lins, whereas albumin d e c r e a s e s p r o p o r t i o n a t e l y less ( D i m o p o u l l o s , 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 o f t h e a n i m a l .

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£ ( M i t r u k a and Rawnsley, 1977).

The mean BUN was 16.0±0.8 mg/d£. C o n t r o l s and i r o n - t r e a t e d lambs d i d

not differ significantly (P>0.05), with means of 16.4±1.2 v s . 17.4±1.1

mg/d£ . BUN has been r e p o r t e d t o range from 15.0 t o 36.0 mg/djj, i n normal

sheep ( M i t r u k a and Rawnsley, 1977).

 - 70 -

Cholesterol

Sheep have a low plasma l i p i d c o n c e n t r a t i o n compared t o o t h e r rumin-

a n t s , 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 a r e a major compon-

ent, and w i t h a s i m i l a r amount o f p h o s p h o l i p i d , total from 70-80% of the

plasma lipid (Nelson, 1969; L i n d s a y and L e a t , 1975). Workers i n Germany

have measured serum cholesterol to predict several metabolic diseases i n

early lactation possibly associated with liver malfunction (Manston and

Allen, 1981), however i t s use i n o v i n e n u t r i t i o n studies remains t o be

clarified.

Mean plasma cholesterol was 137±7 mg/d£, which i s higher than t h e

mean of 57.8±8 mg/d£ r e p o r t e d by Smith jet al_. (1978) and 64.6±3.3 r e p o r t e d

by H a c k e t t et a_l. (1957), but w i t h i n the range o f 50.0-140 ( M i t r u k a and

Rawnsley, 1977). The high l e v e l o f c h o l e s t e r o l i s p r o b a b l y an age e f f e c t ,

as plasma c h o l e s t e r o l d e c r e a s e s when a d u l t rumen f u n c t i o n d e v e l o p s .

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 g r e a t e r i n the c o n t r o l lambs, w i t h a

mean of 755±40 IU/jl compared t o 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 o f 741±66 IU/a i n 28 lambs at 4

weeks of age.

Age, isoenzyme s o u r c e , 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 a r e among t h e major s o u r c e s o f v a r i a t i o n known t o a f f e c t alkaline

phosphatase a c t i v i t y . I n the c u r r e n t s t u d y , breed, s e x , and r e a r i n g were

not significant, while blood group was not i s o l a t e d as a source of

variation.
 - 71 -

The range i n AP a c t i v i t y may be maximum a t b i r t h . Serum AP a c t i v i t y

ranged from 220-8500 Will i n newborn lambs, a t which time v i r t u a l l y a l l

activity was o f s k e l e t a l origin (Healy, 1975b). AP a c t i v i t y peaks a t 2k

hours as i n t e s t i n a l AP e n t e r s the c i r c u l a t i o n . Serum a l k a l i n e phosphatase

activity tends t o d e c r e a s e w i t h i n c r e a s i n g age i n sheep. The isoenzymes

a l s o vary w i t h age. At 2 weeks o f age, isoenzyme a n a l y s e s showed t h a t 79%

of serum AP a c t i v i t y was s k e l e t a l t y p e , t h e remainder i n t e s t i n a l . The h i g h

AP activity i n lambs and the g r a d u a l fall with age a r e c o n s i d e r e d to

r e f l e c t t h e changing r a 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 c i a t e d w i t h skeletal

development. By m a t u r i t y , liver and/or intestinal isoenzymes predominate

(Healy, 1975a).

Many n u t r i t i o n a l factors affect plasma alkaline phosphatase. When

diets varying i n the r a t i o of wheat to a l f a l f a were f e d a t maintenance

l e v e l s , both t o t a l AP a c t i v i t y and the p r o p o r t i o n o f 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

D a v i s , 1975). Healy and Mclnnes (1975) a l s o observed t h a t 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 f e d t o g a i n a t d i f f e r e n t r a t e s on t h e

same d i e t , and i n s p i t e o f the absence o f isoenzyme s t u d i e s , c o n c l u d e d t h a t

t h e AP r e s p o n s e r e f l e c t e d t h e i n f l u e n c e of t h e 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 p i g s (Furugouri,

1972) and c a l v e s ( M i l l e r _et a l . , 1965) and c o u l d be u s e f u l i n t h e d i a g n o s i s

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-

t a s e a l s o responds q u i c k l y t o changes i n d i e t a r y phosphorus i n c a l v e s , w i t h

activity varying i n v e r s e l y t o serum P i (Wise jet ^1_., 1958). The rela-

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 A s p a r t a t e Transaminase

Plasma l a c t a t e dehydrogenase (LDH) and a s p a r t a t e t r a n s a m i n a s e (AT .or

GOT) a r e used t o d i a g n o s e s e l e n i u m and v i t a m i n E d e f i c i e n c y d i s e a s e s .

Mean plasma levels of lactate dehydrogenase (LDH) and aspartate

transaminase (AT or SGOT) were 578±31 IU/SL and 108.9±2.8 IU/£, respec-

tively. Smith et a l . (1978) r e p o r t e d a mean of 311±55 IU/£, o f LDH i n adult

ewes, and 71±26 IU/n o f AT. Data from Horton _et a l . (1978) were s i m i l a r ,

w i t h lamb serum c o n t a i n i n g h i g h e r amounts of LDH and AT. Mean LDH and AT

v a l u e s were 643 IU/£ and 35 IU/£ i n the lambs i n j e c t e d w i t h v i t a m i n E and

s e l e n i u m , and 869 IU/jj, LDH and 176 IU/x, AT i n the c o n t r o l lambs (Horton e t

al., 1978). The lambs i n the c u r r e n t study d i d not have AT and LDH levels

i n d i c a t i v e of s e l e n i u m and/or v i t a m i n E d e f i c i e n c y . I r o n t r e a t m e n t d i d 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 g i v e n i n Appendix 3. At the time of i n i t i a l treat-

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 t w i c e as f a s t as the

control group (1.1 kg v s . 0.6 kg). The weight d i f f e r e n c e continued to

i n c r e a s e 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 t r e a t m e n t e f f e c t from 6 weeks t o 9 weeks

of age.

Rearing significantly (P<0.01) a f f e c t e d lamb weight throughout t h e

study. At 2 days, s i n g l e lambs weighed 4.5 kg and t w i n s , 3.6 kg. At 11

weeks, singles weighed 56.5 kg and twins, 47.3 kg. D o r s e t s and Finns
 - 73 -

weighed 4.0 kg and 4.1 kg a t b i r t h , and 23.6 and 23.4 kg a t 11 weeks. A

s i g n i f i c a n t sex X r e a r i n g i n t e r a c t i o n was observed from 2 days t o 11 weeks

(P<0.05). Male s i n g l e s were on average h e a v i e r than female s i n g l e s , but

male t w i n s were l i g h t e r than female t w i n s .

TRIAL 2 LEVEL OF IRON SUPPLEMENTATION

E f f e c t of Iron Level on Hemoglobin, PCV, and Plasma Iron

Mean Hb and PCV v a l u e s d i d not d i f f e r i n t h e t h r e e groups o f lambs a t

2 days of age, j u s t prior to treatment. At 16, 30 and 44 days o f age

differences between the control and i r o n - t r e a t e d groups were significant

(P<0.001) f o r both Hb and PCV. Hb and PCV were h i g h e r (P<0.05) f o r t h o s e

lambs which r e c e i v e d 500 mg of i r o n compared t o those which r e c e i v e d 250

mg, a t 30 and 44 days o f age.

In t h e c o n t r o l group, Hb f e l l from 13.6 g/d£ a t 2 days t o a low o f

10.3 g/d£ a t 30 days, and then i n c r e a s e d t o 11.8 g/d£ a t 44 days (Figure

3). S i m i l a r l y , PCV decreased from 38.0% a t 2 days t o 29.2% a t 30 days, and

reached 34.0% a t 44 days ( F i g u r e 4 ) . I n c o n t r a s t , mean Hb and PCV v a l u e s

for both iron-injected groups i n c r e a s e d from 2 days t o 30 days, then

decreased s l i g h t l y . (Data a r e g i v e n i n Appendix 4.)

The maximum d i f f e r e n c e s between t h e c o n t r o l and h i g h i r o n (500 mg)

groups o c c u r r e d a t 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% h i g h e r respectively i n the high i r o n group.

Results i n Trial 1 and those reported i n the l i t e r a t u r e indicate that

minimum Hb and PCV v a l u e s a r e reached a t 3 weeks i n s u c k l i n g lambs. PCV

and Hb p r o b a b l y continued t o decrease t o 3 weeks, and then increased,

instead of p l a t e a u i n g from 16 t o 30 days o f age as shown i n F i g u r e s 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

F i g u r e 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
F i g u r e 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 c o u l d w e l l have been even lower a t 3 weeks

than observed a t the 2 and 4 week s a m p l i n g p e r i o d s .

Birth 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 analysis.

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 a t 16 and 44 days, and on PCV

at 16 and 30 days, i n d i c a t e s t h a t b i r t h weight i s r e l a t e d to i r o n stores.

T h i s appears t o be c o n f i r m e d by the r e l a t i o n s h i p between b i r t h weight and

plasma iron.

Plasma iron increased with iron dosage. Treatment means a t 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 h i g h

iron 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 v s . h i g h 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 iron levels than F i n n cross

lambs (P<0.05, 171±12 v s . 224±18 yg/d£), however more of the F i n n cross

lambs were e i t h e r t w i n s or t r i p l e t s and thus t h e i r slower growth r a t e c o u l d

have c o n t r i b u t e d t o t h i s e f f e c t .

The significance of b i r t h weight as a covariable (P<0.05) s u g g e s t s

that lamb b i r t h weight affects iron stores, as o c c u r s i n humans ( B e t k e ,

1970). Even though plasma i r o n was measured a t 30-31 days of age, birth

weight s t i l l had a major i n f l u e n c e on plasma i r o n , a c c o r d i n g t o the r e g r e s -

sion analysis. Significant partial correlation coefficients with plasma

iron (P<0.05) were 0.3289 for birth w e i g h t , 0.4839 f o r hemoglobin, and

0.4772 f o r PCV (Appendix 5 ) .

T h i r t e e n out of 65 lambs had plasma i r o n l e v e l s lower than 100 yg/d£

a t 30-31 days of age, i n s p i t e of the f a c t t h a t i r o n s t a t u s i s expected t o

improve from 21 days as lambs start t o 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 with

 - 77 -

250 mg of i r o n a t 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 t o 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 o f

between 250 and 500 mg iron i s warranted. I t was assumed t h a t 100 pg/d£

plasma i r o n i s a t or above the t h r e s h o l d 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 o l d lambs are

h i g h e r than 200 ug/d£ ( H i d i r o g l o u and J e n k i n s , 1971) and t h a t l e v e l may be

optimum.

Hemoglobin and PCV are more commonly used to a s s e s s anemia i n farm

animals than i s plasma i r o n . Schalm et j a l . (1975) have d e f i n e d anemia as

being c h a r a c t e r i z e d by a 20% r e d u c t i o n 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 b a s i s assuming normal v a l u e s 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 c o u l d be c o n s i d e r e d anemic

w i t h PCV v a l u e s below 28%. On the b a s i s of hemoglobin, 21% of the c o n t r o l

lambs c o u l d be c o n s i d e r e d anemic w i t h 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 f o r a s s e s s i n g anemia was caused by the a r b i t r a r y levels

set for "normal" Hb and PCV values, and not by the actual techniques.

A p p a r e n t l y , the normal l e v e l of 11.5 g/d£ Hb was too low f o r the UBC flock,

based on average v a l u e s f o r 8 week o l d lambs (12.4 g/d£ Hb and 36.4% PCV).

Plasma P r o f i l e

Plasma p r o f i l e data were a n a l y z e d 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 g a i n and b i r t h w e i g h t on the m e t a b o l i t e s

were of major interest, but both v a r i a b l e s could not be i n c l u d e d i n the

covariance model, due to confounding. Consequently, regression analysis

was used separately to investigate correlations between metabolites and

 - 78 -

birth weight, plasma iron, hemoglobin, PCV and most i m p o r t a n t l y , average

d a i l y g a i n t o 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 w i t h plane

o f n u t r i t i o n and growth r a t e i n c a t t l e (Kitchenham et^ , 1975; L i t t l e et

al., 1977; Kitchenham e t 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 w i t h growth r a t e 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 w i t h weight g a i n , whereas c h o l e s -

t e r o l and AT were n e g a t i v e l y c o r r e l a t e d w i t h weight g a i n (P<0.05). Results

of r e g r e s s i o n a n a l y s i s a r e g i v e n i n Appendix 5 and 6.

Covariance a n a l y s i s o f t h e plasma profile data indicated that iron

treatment had a s i g n i f i c a n t e f f e c t (P<0.001) on t e n out o f 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 o f T r i a l 2 compared t o

Trial 1 r e s u l t s may be r e l a t e d t o the h i g h e r overall growth rate, espe-

c i a l l y c o n s i d e r i n g t h e h i g h e r i n c i d e n c e o f t w i n s ; sampling a t 30 i n s t e a d o f

24 days o f age; much l a r g e r number o f e x p e r i m e n t a l u n i t s per treatment; use

of orthogonal c o n t r a s t s f o r means s e p a r a t i o n ; and v i r t u a l absence o f hemo-

l y s i s i n blood samples.

Calcium

Plasma c a l c i u m data were not a v a i l a b l e . The v a c u t a i n e r tubes were

apparently contaminated with disodium EDTA, o r m i s l a b e l l e d as c o n t a i n i n g

sodium h e p a r i n as a n t i c o a g u l a n t .

Phosphorus

The response of plasma P^ t o i r o n injection was s m a l l 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 I I .

E f f e c t of 3 levels of iron treatment on plasma p r o f i l e 1

at 30-31 days of age ( T r i a l 2)

TREATMENTS

Significance
0 mg Fe 250 mg Fe 500 mg Fe of Contrasts 2

X" ± SE
n 20 22 24

I n o r g a n i c Phosphate (mg/d£) 8.9 + 0.2 8.7 + 0.3 8.4 + 0.1 C1***, c * 2

G l u c o s e (mg/dx.) 104.9 + 2.5 101.3 + 2.6 92.9 + 2.0 p *** p ***

c
l ' ^2

17.0 + 0.8 15.6 + 0.8 18.4 + 0.7 p ***

BUN (mg/di) L
2
p *•** p *
Cholesterol (mg/d£) 96.1 + 4.5 108.5 + 3.1 123.5 + 5.8 n ' L
2

5.57 + 0.06 5.49 + 0.15 5.20 + 0.06 p •***• p ***

T o t a l P r o t e i n (g/d£ )

2.91 + 0.06 2.86 + 0.08 2.90 + 0.06 P *#* p *

Albumin (g/d£)
> ^2
1098 + 116 881 + 84 725 + 43 p •**# p *
A l k a l i n e Phosphatase (lU/l) L
l » 2 L

L a c t a t e Dehydrogenase (IU/z) 570 + 34 608 + 39 563 + 30 p *•**

A s p a r t a t e Transaminase (IU/£ ) 146 + 38 116 + 6 111 + 6 p * p *

p p *
Plasma Fe ( g/d£ ) u
132 + 22 , 204 + 18 230 + 14 n » *-2
1 C a l c i u m d a t a not a v a i l a b l e due t o manufacturer 's c o n t a m i n a t i o n of v a c u t a i n e r tubes w i t h disodium EDTA.

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 were as f o l l o w s :
C1 = C o n t r o l v s . both l e v e l s o f 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 ;
 - 80 -

control, low, and h i g h iron groups. tends to decrease during i n -

creased carbohydrate u t i l i z a t o n (Simesen, 1970, p. 3 1 9 ) . T h i s would indi-

cate more efficient carbohydrate utilization i n the i r o n - t e a t e d groups,

probably through increased s y n t h e s i s of iron-dependent enzymes. While

plasma i n o r g a n i c phosphate levels are "intimately related t o the i n t e r -

mediary metabolism of g l u c o s e " , these changes a r e s a i d t o occur indepen-

d e n t l y of blood g l u c o s e ( L a t n e r , 1975, p. 4 6 ) . Regression analysis con-

firmed that plasma was not s i g n i f i c a n t l y correlated with blood glu-

c o s e , nor w i t h weight g a i n , Hb, PCV or plasma i r o n , i n s p i t e o f t h e s i g n i -

f i c a n t response t o i r o n t r e a t m e n t .

Glucose

Plasma g l u c o s e means were 104.9±2.5, 101.3±2.6, and 92.9+2.0 mg/d£

f o r the c o n t r o l , low and high i r o n t r e a t m e n t s . The d i f f e r e n c e between t h e

control and both iron treatments, and t h e low and h i g h levels of iron

t r e a t m e n t , 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 iron and

glucose. As p r e v i o u s l y d i s c u s s e d i n T r i a l 1, plasma g l u c o s e changes appear

t o be r e l a t e d t o the s h i f t i n e r y t h r o c y t e 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 p r o d u c t i o n o f

a d u l t types of red c e l l s . T h i s h y p o t h e s i s i s supported by t h e s i g n i f i c a n t

correlation (P<0.05) of Hb, PCV, and plasma i r o n w i t h plasma g l u c o s e a t 4

weeks of age, w i t h p a r t i a l correlation coefficients of -0.3055, -0.2533,

and -0.3981 (Appendix 5). Alternatively, i r o n s u p p l e m e n t a t i o n may prevent

a s l i g h t e l e v a t i o n of blood g l u c o s e a s s o c i a t e d w i t h anemia. I n anemia, t h e

 - 81 -

transfer of g l u c o s e from blood t o 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 utilization i s involved (Latner, 1975, p. 6 5 ) .

K n i g h t jet _ a l . (1983) observed a significant depression i n fasting serum

glucose f o r i r o n - i n j e c t e d p i g l e t s compared t o c o n t r o l s .

Breed and r e 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 g l u c o s e . The p o s i t i v e relationship between i n t a k e and growth

r a t e w i t h plasma g l u c o s e e x p l a i n s t h e h i g h e r g l u c o s e l e v e l s i n s i n g l e com-

pared t o t w i n lambs (104.7 v s . 98.1 mg/d£). Means were 103.2 mg/d£ f o r t h e

D o r s e t s and 91.6 mg/dj, f o r t h e F i n n c r o s s lambs.

Total Protein

Total plasma p r o t e i n was s i g n i f i c a n t l y depressed w i t h i n c r e a s i n g iron

dosage. Mean t o t a l p r o t e i n v a l u e s were 5.57±0.06, 5.49±0.15, and 5.20±0.06

g/d£ f o r dosages o f 0, 250 and 500 mg or i r o n . The d i f f e r e n c e between

control and i r o n - t r e a t e d groups, and between t h e low and h i g h i r o n groups,

were both significant (P<0.001). Regression analysis confirmed a strong

negative c o r r e l a t i o n of TP w i t h plasma i r o n , and l e s s so w i t h hemoglobin.

Partial correlation coefficients were -0.4243 and -0.3276 (P<0.001 and

P<0.01). Iron may indirectly affect plasma t o t a l p r o t e i n s by enhancing

the s y n t h e s i s o f hemoglobin.

Albumin

Albumin was significantly depressed (P<0.001) by iron treatment.

Mean v a l u e s were 2.91±0.06, 2.86±0.08, and 2.90±0.06 g/d£ f o r the dosage

l e v e l s of 0, 250 and 500 mg i r o n . As t h e r e was no l i n e a r t r e n d w i t h treat-

ment, these results may not be m e a n i n g f u l , 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 protein. 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 w i t h plasma i r o n , nor w i t h 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 t r e n d 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 h i g h l y c o r r e -

lated with birth weight, w i t h a p a r t i a l correlation coefficient of 0.4595

(P<0.001); BUN was a l s o positively correlated with plasma iron, 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 o f 0.2651 (P<0.05).

Cholesterol

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 i n c r e a s e d 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 t o i r o n

may be e x p l a i n e d 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 w i t h 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 p r o p o r t i o n a t e t o the r i s e i n r e t i c u l o c y t e c o n c e n t r a -

tion. The b a s i s o f t h e 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 a s s o c i a t e d w i t h other changes i n plasma l i p i d s , i n c l u d i n g reduced

phospholipids ( L a t n e r , 1975, p. 142).

Cholesterol 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 n e g a t i v e c o r r e l a t i o n of

c h o l e s t e r o l w i t h weight g a i n (Appendix 5 ) . C h o l e s t e r o l was a l s o lower i n

D o r s e t lambs than i n F i n n c r o s s lambs (P<0.05, 105.9 v s . 119.5 mg/d£). The

breed d i f f e r e n c e may be g e n e t i c , r e f l e c t i n g t h e d i f f e r e n c e s i n l i p i d meta-

bolism between the F i n n breed and the v a r i o u s B r i t i s h breeds (Boylan e_t

al., 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 t o s e v e r a l of the f a c t o r s tested.

Breed affected AP (P<0.05). The 45 D o r s e t lambs had a mean plasma AP

activity of 987 IU/£, compared t o a mean of 680 IU/£ i n the 20 F i n n cross

lambs. I r o n t r e a t m e n t and b i r t h weight were a l s o major s o u r c e s of v a r i a -

tion. R e g r e s s i o n a n a l y s i s demonstrated a s i g n i f i c a n t negative correlation

(P<0.0005) of AP w i t h b i r t h w e i g h t , plasma i r o n , Hb, and PCV. B i r t h weight

and hemoglobin were the most important regression coefficients (Appendix

6).

The response of plasma AP t o i r o n s u p p l e m e n t a t i o n cannot be readily

explained. Means were 1098, 881, and 725 IU/£ for control, low and high

iron lambs. Clinically, an increase i n plasma AP reflects osteoblastic

proliferation or i n c r e a s e d 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 i n a d e q u a t e miner-

alization. T h i s o c c u r s i n r i c k e t s and o s t e o m a l a c i a , f o r example (Latner,

1975, p. 5 6 2 ) . Possibly iron deficiency a l s o a f f e c t s plasma AP through a

d i s t u r b a n c e i n bone m e t a b o l i s m . I r o n d e f i c i e n c y i s known t o cause s k e l e t a l

abnormalities i n both man and experimental animals, p r o p o r t i o n a t e to the

severity of the d e f i c i e n c y ( B e u t l e r and F a i r b a n k s , 1980). More commonly,

iron deficiency in children is frequently associated with temporary

e p i s o d e s of high blood a l k a l i n e phosphatase, presumably caused by a tempor-

a r y d i s t u r b a n c e of bone metabolism.

Isoenzyme a n a l y s e s o f plasma AP a r e n e c e s s a r y t o d e t e r m i n e t h e s o u r c e

o f the i n c r e a s e d 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 h e p a t i c , skele-

tal, or i n t e s t i n a l . If skeletal, i t i s important to d i s t i n g u i s h between

osteoblast and marrow s o u r c e s . AP i s known t o be p r i m a r i l y localized in

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, including

marrow, are thought t o 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 t o plasma AP levels

(Wolfe, 1970). One can s p e c u l a t e t h a t i n c r e a s e d AP activity i n marrow may

accompany the e r y t h r o i d h y p e r p l a s i a of anemia.

L a c t a t e Dehydrogenase and A s p a r t a t e 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 o r t h o g o n a l contrast comparing

the c o n t r o l treatment w i t h the combined i r o n - i n j e c t e d groups, was signifi-

cant (P<0.00001), yet there was no d i f f e r e n c e between low and high iron

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 w i t h

48 pg/d£ plasma i r o n , t o 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 significantly c o r r e l a t e d with weight gain, Hb or plasma iron using

linear regression a n a l y s i s . A l l in a l l , a true response of LDH to iron

treatment was not evident.

Aspartate transaminase a c t i v i t i e s v a r i e d markedly w i t h i n treatments,

apparently because of a s i g n i f i c a n t correlation with weight g a i n . 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. I n s p i t e of the apparent t r e n d , i t c o u l d be erroneous t o

c o n c l u d e t h a t i r o n reduced plasma AT. For i n s t a n c e , the maximum AT activ-

ity was 226 , i n a high iron lamb w i t h 245 yg/d£ plasma iron. The

minimum a c t i v i t y was 78 IU/£, i n a c o n t r o l lamb w i t h 31 g/d£ plasma p iron.

 - 85 -

Multiple linear regression i d e n t i f i e d weight g a i n and plasma iron as the

major regression c o e f f i c i e n t s . Plasma i r o n was positively related to AT,

whereas weight g a i n was n e g a t i v e l y r e l a t e d t o AT a c t i v i t y (Appendices 5 and

6). These results demonstrate the importance o f a c c o u n t i n g for unfixed

factors such as i n d i v i d u a l growth r a t e which a f f e c t plasma parameters.

WEIGHT

Age i n days, and actual birth weight, were covariables used in

a n a l y z i n g weekly weight data from 2 days t o 51 days. Exact age and birth

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) a t time of t r e a t m e n t 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) a t

t h e f i n a l sampling a t 7 weeks of age.

Initial weight and growth r a t e to 4 weeks were both higher in Trial

2, than i n T r i a l 1, i n s p i t e of the h i g h e r percentage of t w i n lambs (79%

and 54% t w i n s , respectively).

Rearing was the only significant main effect. Single lambs were

heavier (P<0.05) than twin lambs at 1 week, (8.0 kg compared t o 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) a t 30 days, a t which time singles

weighed 15.3 kg compared to 11.2 kg f o r t w i n s . The increased milk intake

of single lambs d u r i n g the f i r s t month of age accounts f o r t h i s effect;

after 30 days, s o l i d food consumption increases markedly and twin weight

g a i n b e g i n s to c a t c h up.

I r o n treatment had no effect on lamb weight (Appendix 7 ) . Average

w e i g h t s f o r 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

initial to f i n a l samplings. The l o w - i r o n group was o n l y 1 kg h e a v i e r than

the control 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 iron supplementation was observed inTrial 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 f o r t h e l a c k o f response

i n growth r a t e i n t h i s t r i a l cannot be r e a d i l y e x p l a i n e d .

TRIAL 3 IRON AND/OR SELENIUM SUPPLEMENTATION

E f f e c t of Treatment on Hemoglobin and PCV

Combined data from t h e t h r e e r e p l i c a t e d experiments in Trial 3 were

a n a l y z e d by a n a l y s i s o f v a r i a n c e (Appendices 8 and 9 ) . The f o u r treatments

were c o n t r o l , s e l e n i u m , i r o n and i r o n p l u s s e l e n i u m . 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 s e p a r a t i o n . The c o n t r a s t s

tested were iron effect, selenium effect, and i n t e r a c t i o n o f i r o n and

selenium. They were necessary as i r o n and selenium were not c o n s i d e r e d as

s e p a r a t e f a c t o r s i n the d e s i g n , but were combined t o g e t h e r under treatment.

F i g u r e s 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

for lambs from 2 days t o 8 weeks o f age. Hemoglobin and PCV decreased from

2 days to 3 weeks o f age i n c o n t r o l and selenium t r e a t e d lambs. The v a l u e s

then i n c r e a s e d s l o w l y from 3 t o 8 weeks o f age. I n lambs t r e a t e d w i t h iron

or iron plus selenium, the Hb and PCV v a l u e s d e c l i n e d from 2 days to 1

week, then i n c r e a s e d r e a c h i n g a p l a t e a u a t 3 weeks o f age.

As i n the previous trials, t h e maximum d i f f e r e n c e between t h e i r o n

treated lambs and those not t r e a t e d w i t h i r o n o c c u r r e d a t 3 weeks o f age.

Hb was 11.3% h i g h e r a t 1 week, 24.5% h i g h e r a t 2 weeks, 25.6% h i g h e r a t 3

weeks, and 20.5% h i g h e r a t 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 p l u s selenium t r e a t e d lambs r e s p e c t i v e l y . Corres-

ponding PCV v a l u e s 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

Figure 5. Effect of iron and selenium supplementation on hemoglobin (Trial 3).

F i g u r e 6. E f f e c t of i r o n and selenium s u p p l e m e n t a t i o n on b l o o d packed c e l l
volume ( T r i a l 3 ) .
 - 89 -

On t h e b a s i s o f 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 s e l e n i u m 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

c o u l d be c l a s s e d as anemic. As p r e v i o u s l y , t h e c r i t e r i a o f <9.2 g/d& Hb o r

<28% PCV were used t o a s s e s s t h e p r o p o r t i o n o f c o n t r o l lambs anemic a t any

time. Using this arbitrary Hb l e v e l , 22% o f c o n t r o l s were anemic a t 3

weeks, and 1196 a t 4 weeks. S i m i l a r l y , using the l e v e l o f <28% PCV as t h e

d e f i n i t i o n o f anemia, 25% o f c o n t r o l lambs were anemic a t 2 weeks, 2 3 % a t 3

weeks, and 17% a t 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 o r PCV a t any time between 2 days and 8

weeks, nor was an i n t e r a c t i o n w i t h i r o n e v i d e n t (P<0.05). The data t h e r e -

f o r e d i d not s u p p o r t t h e r e s u l t s o f Horton et al_. (1978) and Buchanan-Smith

et a l . (1969), who found s e l e n i u m s u p p l e m e n t a t i o n depressed Hb l e v e l s . The

use of r e g r e s s i o n a n a l y s i s of t h e i r data would have e l i m i n a t e d a growth

response t o Se as t h e reason f o r t h e Hb d e p r e s s i o n . The c u r r e n t study a l s o

failed t o C o n f i r m t h e e r y t h r o p o i e t i c response t o s e l e n i u m observed by Niyo

et a l . (1980) and by F o n t a i n e e t aJL (1977a, 1977b).

The absence o f a h e m a t o l o g i c a l response t o selenium i s not c o n c l u -

sive. I t only suggests t h a t the selenium s t a t u s o f t h e c o n t r o l lambs i n

T r i a l 3 was adequate f o r hemoglobin p r o d u c t i o n and/or r e d c e l l maintenance,

possibly because v i t a m i n E l e v e l s were good. On t h e o t h e r hand, blood

hemoglobin was p r o b a b l y a crude i f not i n a p p r o p r i a t e t e c h n i q u e t o a s s e s s

the importance o f Se i n heme metabolism. The e f f e c t o f selenium s t a t u s on

heme s y n t h e s i s and c a t a b o l i s m i n bone marrow has not y e t been t h o r o u g h l y

investigated, although l i m i t e d information i s available for liver, spleen

and muscle (Burk jet _ a l . , 1974; Burk and C o r r e i a , 1978; Whanger ejt al.,

1977).
 - 90 -

Hemoglobin versus Hematocrit

Much d i s c u s s i o n i n the literature on iron d e f i c i e n c y concerns the

comparative efficiency of hemoglobin and hematocrit for detection of

anemia. Different results are frequently obtained, depending on which

parameter i s used.

The hematocrit test i s cheaper and e a s i e r than the hemoglobin test,

but can be less effective than hemoglobin in identifying anemia. For

example, the hematocrit fails to d e t e c t 20-50% of c h i l d r e n who would be

c o n s i d e r e d anemic based on hemoglobin l e v e l s ( G r a i t c e r ejt a l _ . , 1981).

The l a r g e amount of data collected on lamb hematology i n t h i s trial

provided an opportunity to compare the Hb and PCV tests. Hemoglobin was

plotted against PCV, and regression equations were developed with and

without outlier rejection. A l l equations given were derived using 5%

outlier rejection.

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 a t 1 week, 0.96 at 2 weeks, 0.94 at 3 weeks, 0.89 at 4

weeks, 0.93 at 5 weeks, 0.86 a t 6 weeks, 0.75 a t 7 weeks, and 0.74 at 8

weeks. The r e g r e s s i o n equations f o r p r e d i c t i o n of Hb from PCV according to

these age groups are g i v e n i n Appendix 10.

The regression analyses showed t h a t 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 ( F i g u r e 7) and even c l o s e r at 4 weeks ( F i g u r e 8 ) .

As Figure 9 illustrates, the relationship between PCV and Hb was not as

t i g h t by 8 weeks.

When data from a l l sampling periods of Trial 3 were analyzed

t o g e t h e r , 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

 ... ARE USED 10 PLOT THE REGRESSION LINE; THE — IS USED XHEN * PLOT POINT COVERS DATA P O I N T S _
THE
17.(0
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F i g u r e 7. Hemoglobin v e r s u s PCV a t 2 days ( T r i a l 3 ) .

Y = 0.4851 + 0.3419X R = 0.8624 2
AND ••• ARE USED TO PLOT THE REGRESSION L I N E ; THE ••• I S USED WHEN A PLOT POINT COVERS DATA P O I N T S

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DISTANCE BETWEEN SLASHES ON THE X - A X I S I S 0 . 3 5 0 0

% Packed CelI VoIume

F i g u r e 8. Hemoglobin v e r s u s PCV a t k weeks ( T r i a l 3 ) .

Y - 0.2120 + 0.3346X R = 0.9228 2
 THE V AND ARE U S E D TO P L O T THE R E G R E S S I O N LINE; THE • • • IS USED WHEN A P L O T P O I N T COVERS DATA POINTS

1 1 1 1.

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DISTANCE B E T W E E N S L A S H E S ON T H E X - A X I S IS 0.1400

% Packed CelI Volume

F i g u r e 9. Hemoglobin v e r s u s PCV a t 8 weeks ( T r i a l 3 ) .

Y = 4.084 + 0.2334X R = 0.5409 2
 THC " . • » N O • • • ARE U S E O TO P L O T T H E R E G R E S S I O N L I N E : THE • • • IS U S E D WHEN A P L O T P O I N T C O V E R S D A T A P O I N T S
AN I N T E G E R " I " . B E T W E E N 1 AMD 9, R E P R E S E N T S A P P R O X I M A T E L Y 2*1 OATA P O I N T S ; ' O - R E P R E S E N T S 1 OR FEWER D A T A P O I N T S
17.20 - O 1 17.20
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DISTANCE BETWEEN SLASHES ON THE X-AXIS IS 0.3900

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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 = 0.8251 2
 • AND — A R E U S E O TO P L O T THE R E G R E S S I O N L I N E ; THE — I S U S E D WHEN A P L O T P O I N T C O V E R S DATA P O I N T S
THE
EGER I " . B E T W E E N 1 AND 9 . R E P R E S E N T S A P P R O X I M A T E L Y 2*I DATA P O I N T S ; ' O ' R E P R E S E N T S 1 OR FEWER DATA POINT*
AN I N T E G E R " I " , B E T W E E N 1 AND 9 , R E P R E S E N T S A P P H U X l l M I t L i J-i u . . . r u i » , , , „ .............
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% Packed CelI Volume

F i g u r e 11. Hemoglobin v e r s u s PCV f o r a l l T r i a l 3 d a t a .

Y = 1.613 + 0.2996X R = 0.6571 2
 - 96 -

outliers, 1081 complete pairs of o b s e r v a t i o n s were used i n the analysis.

The s i m p l e l i n e a r r e g r e s s i o n e q u a t i o n d e r i v e d 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 variable PCV.

The p l o t of Y v s . X and of the regression line i s shown i n F i g u r e

10. Figure 11 shows the same data plotted without e x c l u s i o n of the

outliers.

The hematocrit test appeared to be a good e s t i m a t o r of lamb iron

s t a t u s between b i r t h and 8 weeks. However, the hemoglobin t e c h n i q u e was

preferable for lambs over 6 weeks of age, depending on the accuracy

desired. 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 variables

deteriorated, 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. S i n c e the c o n c e n t r a t i o n of Hb i n the e r y t h r o c y t e i s

r e l a t i v e l y c o n s t a n t except d u r i n g anemia, t h i s may not o c c u r .

PLASMA SELENIUM

Plasma selenium data were o b t a i n e d 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 v a l u e s 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 significantly increased plasma Se levels at 4

weeks (P<0.05). Means were 0.086 ppm Se f o r the 38 control lambs, and

0.098 ppm Se f o r the 37 s e l e n i u m - i n j e c t e d lambs.

 - 97 -

The range i n plasma s e l e n i u m v a l u e s was l a r g e , e x t e n d i n g from defi-

c i e n t t o normal l e v e l s w i t h i n both t r e a t m e n t s ( F i g u r e 1 2 ) . The assumption

t h a t the f l o c k had a m a r g i n a l s e l e n i u m s t a t u s was j u s t i f i e d by plasma sel-

enium v a l u e s i n the c o n t r o l lambs. Control lambs had levels ranging from

0.016-0.118 ppm Se. Between 0.080 and 0.500 ppm Se i s c o n s i d e r e d adequate

for serum selenium l e v e l s i n sheep i n B.C. (Puis, 1981). Based on this

criterion, 25 out of 38 controls, or 66%, had adequate plasma s e l e n i u m

l e v e l s a t 4 weeks. The level of .08 ppm i s much h i g h e r than t h a t consi-

dered normal i n some o t h e r s t u d i e s ( i e . 0.02 ppm Se may be a d e q u a t e ) , 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-

r e s p o n s i v e WMD i n the UBC flock. However, no WMD o c c u r r e d i n any lamb used

in Trial 3.

C o r r e s p o n d i n g ranges f o r s e l e n i u m - i n j e c t e d lambs were 0.029-0.170 ppm

Se. A larger percentage of selenium-injected lambs had plasma levels

w i t h i n the nromal range. Out of 37 i n j e c t e d lambs, 30 a n i m a l s or 81% c o u l d

be c o n s i d e r e d t o have adequate plasma s e l e n i u m v a l u e s . Due t o t h e v e r y low

s e l e n i u m 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 o f

1.5 mg s e l e n i u m as sodium s e l e n i t e may not ensure adequate s e l e n i u m l e v e l s

in a l l t r e a t e d 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 s e l e n i u m l e v e l s r a n g i n g from 133-249

ng/mjj, , and the o t h e r having low l e v e l s ranging from 21-67 ng/m£. Blood

s e l e n i u m data from the p r e s e n t study appeared t o 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 rearing had no effect on plasma selenium levels

(P>0.05).
 - 98 -

50' Control Lambs

40'

30"

1
.o
20'

10-

— 1.02 —.04 .06 .08 .10 .12 .14 .16 .18

Plasma Selenium (ppm)

50

SeI e n i u m - i n j e c t e d Lambs

40

30
_a
E
fD
"20

10

J=L
.02 .04 .06 .08 .10 .12 .14 .16 .18
Plasma Selenium (ppm)

F i g u r e 12. E f f e c t o f s e l e n i u m t r e a t m e n t a t b i r t h on
plasma s e l e n i u m a t 4 weeks.
 - 99 -

The interaction of iron with selenium was significant (P<0.05).

Selenium levels were significantly higher i n those lambs injected with

selenium and not i n j e c t e d w i t h i r o n , than i n t h e o t h e r t h r e e treatment com-

b i n 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), w i t h 18 t o 20 lambs per treatment combination.

These d i f f e r e n c e s may reflect 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 levels. Selenium i s

incorporated into the e r 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 iron, which g r e a t l y s t i m u l a t e d 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 into the e r y t h r o c y t e fraction of +Se+Fe lambs than i n

+Se-Fe lambs. Analyses of Se and GSH-Px i n both plasma and r e d c e l l s would

provide valuable information.

I r o n d e f i c i e n c y , but not anemia per s e , may r e s t r i c t the s y n t h e s i s o f

GSH-Px (Rodvien et a l . , 1974). I r o n d e f i c i e n c y anemia i s a s s o c i a t e d with

decreased e r y t h r o c y t e GSH-PX a c t i v i t y , and i r o n s u p p l e m e n t a t i o n induces a

r a p i d i n c r e a s e i n GSH-Px. A study w i t h humans i n d i c a t e d t h a t t h e d e c r e a s e

i n GSH-Px was p r o p o r t i o n a l t o t h e d e c r e a s e i n Hb ( M a c d o u g a l l , 1972). How-

ever, a study with rabbits showed that erythrocyte GSH-Px activity 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 o f

hemoglobin (Rodvien et a l . , 1974).

The significant interaction i n the c u r r e n t study between i r o n and

selenium treatments on plasma selenium levels suggests a r o l e f o r i r o n i n

selenium metabolism. As GSH-Px does not c o n t a i n iron (Ganther jet a l . ,

1976), i t i s likely that i r o n - c o n t a i n i n g enzymes may be i n v o l v e d i n the

 - 100 -

synthesis 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 s e l e n i u m c o u l d be

decreased i n +Se+Fe lambs merely because of the i n c r e a s e d red c e l l mass

containing GSH-Px.

Plasma P r o f i l e

The plasma profile included the same parameters as i n t h e p r e v i o u s

two t r i a l s . The s t a t i s t i c a l analysis tested r e p l i c a t e , treatment, breed,

sex, rearing and i n t e r a c t i o n s , u s i n g orthogonal contrasts to i s o l a t e the

e f f e c t s o f i r o n and s e l e n i u m .

Replicate significantly affected every parameter, as can be seen i n

Table III. However, mean v a l u e s of each parameter were within normal

ranges. Replicate means were h i g h e s t f o r Pj_, BUN, LDH and AT in Repli-

cate 2, and l o w e s t i n R e p l i c a t e 1. Means were h i g h e s t f o r calcium, glu-

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. All

R e p l i c a t e 3 means were intermediate.

The data i n d i c a t e d t h a t R e p l i c a t e 2 samples s u f f e r e d 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 o f d e t e r i o r a t i o n .

Plasma samples were s t o r e d i n a freezer a t -10°C, but due t o temperature

f l u c t u a t i o n s the samples were o f t e n found t o be i n a s e m i - f r o z e n 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 s t o r e d f o r t h e l o n g e s t time and

t h e r e f o r e were t h e most a f f e c t e d .

While twenty of t h e most commonly measured plasma constituents,

including glucose, are s t a b l e when frozen f o r 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 t h e o n l y 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 ( T a b l e I V ) . As i n T r i a l s 1 and 2, t h e

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 o f

 - 101 -

TABLE I I I .

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)

Significant
METABOLITE X ± SEM R1 R2 R3 Main E f f e c t .
1 2

——————— = = = = = = = —— = = -

Calcium (mq/dl) 10.82 ± .12 11.56 10.41 10.60 Rep**, Rear*

P L (mg/d£) 11.27 ± .15 10.14 12.10 11.44 Rep**

G l u c o s e (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*, F e *

LDH (IU/£) 696 ± 2 1 578 756 775 Rep***, S e x *

AT (IU/£) 161 ± 10 109 188 180 Rep**

n (111) (33) (37) (41)

^ain e f f e c t s t e s t e d were r e p l i c a t e , t r e a t m e n t , breed, sex and r e a r i n g .

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 were as f o l l o w s :
FE. C o n t r o l + Se t r e a t m e n t s v s . Fe + Fe/Se t r e a t m e n t s
SE. C o n t r o l + Fe t r e a t m e n t s v s . Se + Fe/Se t r e a t m e n t s
FEXSE C o n t r o l + Fe/Se t r e a t m e n t s v s . Se + Fe t r e a t m e n t s

3
*P<0.05; **P<0.01; ***P<0.001
TABLE IV.

Effect of iron and selenium supplementation on plasma p r o f i l e ( T r i a l 3)

REPLICATE 1 REPLICATE 2 REPLICATE 3

PLASMA
METABOLITE Control Se Fe Se/Fe Control Se Fe Se/Fe Control Se Fe Se/Fe

Calcium (mg/d£) 11.76 11.70 11.71 11.06 10.82 10.50 10.13 10.13 10.06 11.02 10.85 10.40

Pi (mg/di) 10.44 10.22 9.97 9.96 11.94 12.57 11.67 12.23 11.85 11.26 11.28 11.39

Glucose (mg/d£) 97.0 93.5 94.8 93.0 19.5 12.0 25.8 15.1 40.2 43.4 41 .5 45.0

BUN (mg/d£) 17.2 15.6 18.4 16.2 22.2 21.2 23.6 23.7 18.3 17.0 20.1 16.6

Cholesterol (mg/d£) 148.0 135.7 129.4 134.5 111 . 3 94.0 114.2 105.9 120.8 123.8 133.0 128.4

TP (g/dji) 6.12 5.97 6.02 5.80 6.42 6.41 6.39 6.58 6.01 6.5 6.13 6.02

Albumin (g/d£) 3.26 3.29 3.23 3.18 3.95 3.97 4.08 4.01 3.84 3.94 3.78 3.94

AP (lU/i) 799 717 612 643 553 538 507 508 645 646 570 556

LDH (IU/£) 552 585 582 593 794 730 733 762 775 735 726 725

AT (IU/£) 110 100 112 115 262 150 172 159 176 172 204 160

n 8 8 9 8 10 9 9 9 10 10 12 9
 - 103 -

iron-treated lambs. Mean AP i n 56 control 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 t r e a t m e n t s were not a s s o c i a t e d with any changes i n plasma

parameters i n d i c a t i v e of w h i t e muscle d i s e a s e . The d e t e r i o r a t i o n of muscle

t i s s u e t h a t o c c u r s i n WMD i n d u c e s a host of b i o c h e m i c a l changes i n plasma

as w e l l as muscle tissue. For example, inorganic phosphate and alkaline

phosphatase are increased (Koval'skii and Ermakov, 1970), while serum

c a l c i u m and magnesium a r e u n a f f e c t e d (Godwin ejt a_l_., 1974). Plasma levels

of tissue enzymes a r e 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. C o n s e q u e n t l y , plasma malate dehydrogenase, a l a n i n e amino-

transferase, lactate dehydrogenase, and aspartate aminotransferase are

commonly used to diagnose WMD i n ruminants (Whanger et a i ^ . , 1969b; Boyd,

1973).

Except f o r plasma s e l e n i u m and GSH-Px, few plasma parameters respond

t o v a r i a t i o n s i n s e l e n i u m s t a t u s i n t h e absence of WMD. G l u c o s e and fatty

acid metabolism may be a f f e c t e d at v a r i o u s levels of s e l e n i u m a v a i l a b i l -

ity. Selenium s t a t u s i n f l u e n c e d r a t e of g l u c o s e metabolism, r a t e of f a t t y

acid metabolism, and tissue fatty acid c o n t e n t i n one study ( F i s c h e r and

Whanger, 1977). Supplementing d i e t a r y Se t o an adequate Se d i e t d e c r e a s e d

blood sugar, pyruvic acid, and P^, and increased tissue glycogen and

muscle ATP (Koval'skii and Ermakov, 1970, p. 68). On the o t h e r hand,

Whanger et aJL (1969b) were unable t o 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 t o 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 variability contributed by replicate and other s o u r c e s of

v a r i a t i o n may have obscured i r o n and s e l e n i u m t r e a t m e n t e f f e c t s . Contrary

 - 104 -

to e x p e c t a t i o n , Se did not significantly (P>0.05) affect LDH and AT

levels. The d i f f e r e n c e i n selenium s t a t u s a t 4 weeks between the control

and selenium t r e a t m e n t s may have been too narrow t o be r e f l e c t e d i n plasma

metabolites. The scanty literature on the s u b j e c t i n d i c a t e s that a sub-

clinical Se deficiency may be expected to have o n l y a s u b t l e effect on

plasma constituents. Hence plasma profile analysis, except for certain

muscle enzymes, may be of d o u b t f u l v a l u e f o r 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 e x p e r i m e n t a l c o n d i t i o n s .

Weight

The weight data in Trial 3 was taken from a total 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.

Replicate was a significant 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 . Initial weights

were 4.05±0.99, 3.84±0.88, and 3.90±0.98 kg i n Replicates 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 significantly affected weight throughout the trial

(P<0.001). The 46 single lambs were h e a v i e r than the 75 t w i n lambs from

b i r t h (4.47±0.97 v s . 3.59±0.77 kg) t o 8 weeks (21.8±3.9 v s . 17.3±3.1 k g ) .

Initial weights were 3.79±0.75 kg for 63 females, compared to

4.06±1.10 kg f o r 58 males. Final w e i g h t s a t 8 weeks averaged 17.9±3.2 kg

for f e m a l e s , and 20.2±4.5 kg f o r males, however the difference was not

significant (P>0.05).

I r o n treatment significantly affected lamb weight g a i n from 2 weeks

to the end o f the t r i a l (Appendix 11). As shown i n F i g u r e 13, the d i f f e r -

ence between iron-treated lambs and other categories increased slowly

throughout the t r i a l .
 - 106 -

The treatment i n t e r a c t i o n s w i t h sex and with rearing were not signi-

ficant (P>0.05), undoubtedly because iron and selenium combinations were

c o n s i d e r e d t o g e t h e r as t r e a t m e n t s . However, i f the e f f e c t s of s e l e n i u m are

ignored ( s e l e n i u m having no apparent i n f l u e n c e on weight g a i n at P>0.05),

some very interesting results emerge. The fastest gaining categories of

lambs - single lambs r a t h e r than t w i n s , and males rather than females -

appear to respond most f a v o u r a b l y to i r o n t r e a t m e n t .

The 29 iron injected 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 g a i n e d 15.1 kg. T h i s was a difference of 2.1

kg due to iron treatment of male lambs. In comparison, 31 female iron-

treated lambs gained 14.3 kg compared to 13.9 kg f o r 32 female c o n t r o l s , a

difference of o n l y 0.4 kg.

S i m i l a r l y , weight g a i n was improved by i r o n more markedly i n single

than in twin lambs. The 25 iron-injected single lambs gained 18.2 kg,

w h i l e the 21 control s i n g l e s gained 16.2 kg, a difference of 2 kg. The 35

i r o n - i n j e c t e d t w i n s gained 13.9 kg, which was o n l y 0.3 kg more than the 40

c o n t r o l twins gained.

These r e s u l t s indicate that fast-growing lambs may benefit most by

iron treatment, particularly single and/or male lambs. Under conditions

where i n t a k e 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 f o r growth. The

lambs i n t h i s study were D o r s e t and Dorset/Finn crosses. S u f f o l k or Hamp-

s h i r e lambs might demonstrate a g r e a t e r response to i r o n due to t h e i r h i g h

growth r a t e .

Iron s u p p l e m e n t a t i o n may enhance growth r a t e i n s e v e r a l ways. Logi-

cally, the availability of i r o n f o r hemoglobin, myoglobin and enzyme syn-

t h e s i s p r e v e n t s 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 , several deleterious

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 defi-

ciency i n young a n i m a l s o f most s p e c i e s so f a r s t u d i e d , including human,

dog, and p i g . A t r o p h y 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 a r e common i n i r o n d e f i c i e n t animals and may i m p a i r absorp-

t i o n of i r o n , vitamin A, and o t h e r n u t r i e n t s ( B e u t l e r and F a i r b a n k s , 1980;

Guha et a l _ . , 1968). This problem has not been studied i n t h e lamb.

Another side-effect of iron deficiency i s increased susceptibility to

scours and e n t e r i t i s , which could well contribute t o growth depresson

( L a r k i n and Hanran, 1983).

Plasma Protein Electrophoresis

The electrophoresis of plasma proteins yielded some interesting

r e s u l t s , a l t h o u g h the t e c h n i q u e i s not commonly used i n n u t r i t i o n s t u d i e s .

W h i l e serum p r o t e i n s a r e s e n s i t i v e t o 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 subtle 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 proteins have been d e s c r i b e d . However,

only five plasma p r o t e i n bands a r e o b t a i n e d from most s p e c i e s by agarose

gel electrophoresis. Thus a change i n a s p e c i f i c protein i s r a r e l y of

s u f f i c i e n t magnitude t o produce a c l i n i c a l change i n t h e a s s o c i a t e d protein

band ( L a t n e r , 1975, p. 2 0 0 ) .

As i n the plasma p r o f i l e samples, some s t o r a g e d e t e r i o r a t i o n was e v i -

d e n t , which may have caused some p r o t e i n d e n a t u r a t i o n . The t o t a l protein

v a l u e s were h i g h e r than those o b t a i n e d i n t h e 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 t o t h e exper-

imental error. Fibrinogen trails the b e t a - g l o b u l i n fraction (Kaneko,

 - 108 -

1975), which tends t o obscure t h e boundary between t h e beta- and

gamma-globulin bands when plasma i s used instead o f serum. In a useful

discussion o f agarose gel electrophoresis, Johansson (1972) suggested

adding heparin t o t h e plasma sample t o improve separation of beta

lipoproteins. Otherwise, use o f serum instead o f plasma might have

improved t h e r e s o l u t i o n o f t h e beta and gamma bands.

The densitometric tracings were very s i m i l a r t o those obtained by

Keay and Doxey (1982). The alpha-1 and alpha-2 globulin zones c o u l d be

e a s i l y subdivided, and the alpha-2 g l o b u l i n zone was much g r e a t e r quantita-

tively ( F i g u r e 14). I n t h e p r e s e n t s t u d y , the alpha-1 zone was even s m a l l -

e r , and i n some cases n o n - e x i s t e n t . The major d i f f e r e n c e was a n o t i c e a b l y

smaller gamma g l o b u l i n peak i n t h e c u r r e n t study. As Keay and Doxey (1982)

d i d not q u a n t i t a t e t h e i r r e s u l t s , t h e data c o u l d n ' t be compared. Compari-

son o f r e s u l t s w i t h other l i t e r a t u r e v a l u e s a r e summarized i n Table V.

Many o f t h e p r o t e i n f r a c t i o n s were h i g h l y correlated with the t o t a l

protein covariable (Table V I ) . Total 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 t o

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 Replicate 2 were a v a i l a b l e only from 4 week o l d lambs.

Resolution o f bands was r a t h e r poor, compared t o r e p l i c a t e 3, p o s s i b l y

because of longer time i n s t o r a g e . As a r e s u l t , treatment effects were

obscured. Data from R e p l i c a t e 2 i s shown i n Table V I I , b u t the discussion

will center around t h e r e s u l t s from lambs a t 2, 4 and 6 weeks o f 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. a t 2 weeks, and 0.10-0.62

g/d£ a t both 4 and 6 weeks o f 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 t o 0.37 g/d£, a t 6 weeks, r e f l e c t i n g the

 - 109 -

F i g u r e 14. D e n s i t o m e t r i c t r a c e s o f 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. c o m p a r a t i v e t r a c e from normal sheep, Keay
and Doxey (1982)
TABLE V.

Plasma protein electrophoresis results compared to l i t e r a t u r e values

REPLICATE 3 RESULTS 3
REPLICATE 2 a
MITRUKA & RAWNSLEY 6
IRFAN C

PROTEIN 2 weeks 4 weeks 6 iveeks 4 iveeks Normal Normal Normal 3 months

(g/d£) (40 ) d
(42) (39) (39) male sheep female sheep range (10)

TOTAL 7.83 + 0.16 7.70 ± 0.16 7.28 ±0.15 6.41 ± 0.15 6.80 ± 0.30 7.20 ± 0.31 5.70 - 9.10 5.46

ALBUMIN 4.37 + 0.12 4.52 ± 0.13 4.52 ± 0.10 3.66 ± 0.13 3.70 ± 0.35 3.81 ± 0.33 2.70 - 4.55 3.10

«-1 g l o b u l i n 0.41 + 0.03 0.38 ± 0.02 0.38 ± 0.02 0.43 ± 0.02 0.33 ± 0.08 0.38 ± 0.06 0.15 - 0.50 0.35

<*~2 g l o b u l i n 1.40 + 0.08 1.39 ± 0.07 1.26 ± 0.05 0.99 ± 0.06 0.96 ± 0.13 0.73 ± 0.12 0.45 - 0.12 0.48

B-globulin 1.02 + 0.06 0.93 ± 0.06 0.76 ± 0.07 0.77 ± 0.04 0.52 ± 0.10 0.91 ± 0.13 0.25 - 1.20 0.50

Y-globulin 0.69 + 0.04 0.43 ± 0.02 0.37 ± 0.02 0.54 ± 0.03 1.33 ± 0.20 i.37 ± 0.25 0.82 - 1.90 1.03

ALBUMIN/
GLOBULIN 1.30 + 0.05 1.50 ± 0.06 1.73 ± 0.07 1.36 ± 0.05 1.19 ± 0.20 1.12 ± 0.21 0.70 - 1.60 1.314

Variation expressed as S.E.

b
Mitruka, B.M. and Rawnsley, H.M. 1977. Data summarized from the l i t e r a t u r e ; variation expressed as S.D.

C
Irfan, M. 1967.

^Number of sheep samples

TABLE VI. Effect of age and treatment on plasma proteins ( T r i a l 3, Replicate 3)
Significance Significance of
Control Se Fe Se/Fe S.E.N. of Contrasts »1 2
Main Effects »
1 3

Protein Fraction (g/dt)

2 weeks TP*
Gamma globulin 0.73 0.77 0.64 0.57 NS NS
1.25 1.03 1.00 0.76 0.06 Se*, Fe** TP*
Beta Globulin TP*
Alpha-2 globulin 1.32 1.38 1.45 1.46 0.08 NS
0.40 0.31 0.48 0.45 0.03 NS NS
Alpha-1 globulin TP***
Albumin 4.42 4.29 4.44 4.29 0.12 NS
1.52 1.48 1.74 1.73 1.07 NS Sex*
Albumin/Globulin
Covar. (TP) 7.83 7.80 8.00 7.61 0.16

4 weeks TP*
0.40 0.50 0.43 0.41 0.02 NS
Gamma globulin Sex*
Beta globulin 1.17 1.02 0.86 0.57 0.06 Fe***
1.29 1.31 1.52 1.45 0.07 NS TP***, Sex*
Alpha-2 globulin NS
Alpha-1 globulin 0.37 0.46 0.34 0.34 0.02 NS
Albumin 3.84 4.50 4.93 4.89 0.13 Fe*** jp»#»
1.48 1.94 1.94 2.20 0.09 Fe* NS
Albumin/Globulin
7.12 7.76 8.09 7.82 0.16
Covar. (TP)

6 weeks
Gamma globulin 0.32 0.31 0.41 0.43 0.02 Fe* TP**
Beta globulin 0.78 0.71 0.80 0.71 NS NS
1.16 1.34 1.10 1.53 0.05 NS Rear*
Alpha-2 globulin Rear*
Alpha-1 globulin 0.41 0.35 0.34 0.43 0.02 NS
Albumin 4.43 4.56 4.47 4.67 0.10 NS T/p*#»

2.22 2.11 2.23 1.98 0.08 NS NS

Albumin/Globulin
Covar. (TP) 7.04 7.29 7.15 7.72 0.15

11 11 12 8

Covariable = Total Protein; Main Effects tested were treatment, breed, sex, rearing.
2
FE = 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.

E f f e c t of treatment on plasma proteins at 4 weeks T r i a l 3 ( T r i a l 3, Replicate 2).

Significant
Protein (g/di) Control Se Fe Se/Fe S.E.M. 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 globulin 1.01 1.02 1.08 0.88 0.66 1P*#*

Alpha-1 globulin 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 , s e l e n i u m , 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 o f 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 -

c a t a b o l i s m o f m a t e r n a l a n t i b o d i e s and the apparent i m m a t u r i t y o f t h e lambs'

lymphoid system.

Sheep serum contains 3 major immunoglobulins: I g A , IgG, and IgM.

IgG i s q u a n t i t a t i v e l y t h e most i m p o r t a n t , a c c o u n t i n g f o r 89% o f t o t a l serum

immunoglobulin, compared t o 1.5% and 9.5% f o r IgA and IgM r e s p e c t i v e l y

(Smith e t 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 c o n t a i n o n l y IgG, as IgA and IgM may extend i n t o t h e b e t a - g l o b u l i n band

(Laurell, 1972).

The plasma gamma-globulin c o n c e n t r a t i o n i n t h e young lamb may not be

related to neonatal n u t r i t i o n , except that t h e consumption, o f c o l o s t r u m

determines i n i t i a l levels. The young lamb i s born w i t h n e g l i g i b l e levels

of serum immunoglobulins, so t h e i n g e s t i o n o f c o l o s t r u m p r o v i d e s immunoglo-

bulins, mainly IgG, which are important f o r the f i r s t weeks of life

( C u r t a i n , 1975). These m a t e r n a l a n t i b o d i e s p r o b a b l y depress t h e endogenous

a n t i b o d y p r o d u c t i o n , as o c c u r s i n c a l v e s (Husband and L a s c e l l e s , 1975).

Selenium deficiency i s known t o depress gamma-globulin levels i n

sheep ( K e e l e r and Young, 1961). G i v e n t h e major c o n t r i b u t i o n of maternal

gamma-globulin t o lamb plasma l e v e l s , a response t o s e l e n i u m supplementa-

tion was not e x p e c t e d . Also, t h e dosage o f s e l e n i u m used was not s u f f i -

c i e n t t o ensure optimum s e l e n i u m l e v e l s i n a l l t r e a t e d 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 a t 2, 4 o r 6 weeks, a l t h o u g h v e r y

low gamma globulin levels 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 i n c r e a s e d g l o b u l i n l e v e l s a t 6 weeks

(P<0.05). Means were 0.43±0.2 g/d£, f o r t h e n i n e t e e n 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 w i t h iron.

 - 114 -

As expected, the beta-globulin f r a c t i o n was significantly lower in

i r o n - t r e a t e d lambs at 2 weeks (P<0.01) and 4 weeks (P<0.001). Transferrin

is a major component of the beta-globulin 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 a t 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 e x c e p t i o n 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 e x t r e m e l y r a r e (Kaneko, 1975).

The e f f e c t of iron status on plasma p r 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 s t u d y , i r o n very significantly (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

m a l a b s o r p t i o n syndrome (Naiman ejt a l . , 1964).

Very little information 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 , o t h e r than the d r a m a t i c i n c r e a s e i n c e r t a i n plasma enzymes

during white muscle disease. Keeler and Young (1961) found a marked

increase in alpha-globulin and a decrease in beta-globulin as well as

gamma-globulins in selenium-deficient sheep. Mice d e f i c i e n t i n s e l e n i u m ,

vitamin E and cystine had decreased fibrinogen and total plasma protein

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 fraction at

2 weeks by Se t r e a t m e n t was virtually 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£

for +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 variability t h i s e f f e c t was not significant (P<0.05).

I t i s p o s s i b l e t h a t a l a r g e r dosage of selenium might g i v e more conclusive

 - 115 -

results. If selenium treatment does depress beta-globulin levels, this

would c o n f l i c t w i t h the r e s u l t s of K e e l e r and Young (1961).

The influence of selenium cannot be explained at this point. A

measureable e f f e c t of s e l e n i u m on the b e t a - g l o b u l i n band would most likely

involve fibrinogen, transferrin, 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. Possibly a

major i n c r e a s e i n plasma enzymes might i n c r e a s e the beta f r a c t i o n i n s e l e n -

ium d e f i c i e n t lambs, but t h e r e was no e v i d e n c e 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 a t 4 weeks.

Selenium a l s o appeared t o a f f e c t many of the o t h e r p r o t e i n f r a c t i o n s ,

b u t 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 a t

6 weeks were 1.13±0.05 g/d£ f o r non-Se lambs, and 1.43+0.05 g/d£ for +Se

lambs, w i t h P<0.06.

The l i m i t a t i o n s of the electrophoresis technique, condition of the

samples, number of lambs compared to treatments, and the low selenium

dosage were some of the problems faced. The absence o f significant Se

treatment e f f e c t s f o r most plasma p r o t e i n s was therefore not conclusive.

More i n f o r m a t i o n i s needed on t h i s s u b j e c t , u s i n g a l a r g e r number of lambs

to compensate for the high variability in the data, and using clearly

differentiated 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 for the presence of soremouth lesions. Severity was evaluated

 - 116 -

subjectively on the scale of 0-5. Because of the subjective nature of

these o b s e r v a t i o n s a statistical 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 further i n v e s t i g a t i o n with a

g r e a t e r number of lambs i s w a r r a n t e d .

Soremouth i s an i n f e c t i o u s poxvirus disease of sheep and goats, in

which pustular lesions develop on the lips, o r a l mucous membranes, and

udder. Synonyms i n c l u d e contagious pustular dermatitis and orf. Trans-

mission o c c u r s through minor a b r a s i o n s or trauma. Pustules develop w i t h i n

k days of infection, scabs build up, and healing takes about 3 weeks.

Soremouth may be debilitating i n the case of severe secondary b a c t e r i a l

infections, or through i n t e r f e r e n c e w i t h e a t i n g and drinking (Mohanty and

Dutta, 1981).

I r o n d i d not appear t o have any e f f e c t on soremouth. The same number

of lambs t r e a t e d with i r o n were i n f e c t e d as those not treated. This was

contrary to expectation as mouth lesions are commonly observed in iron

d e f i c i e n t people ( F l e t c h e r et a l . , 1975) and also i n iron d e f i c i e n t calves

( B l a x t e r et a l _ . , 1957).

Selenium seemed t o i n f l u e n c e the i n c i d e n c e , d u r a t i o n and severity of

the disease (Table VIII). Selenium-injected lambs were less likely to

d e v e l o p soremouth, and appeared t o c o n t r a c t the d i s e a s e at a s l i g h t l y older

age, and recover faster.

The mean plasma s e l e n i u m level was greatest i n lambs which d i d not

d e v e l o p 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 t o .087 ppm i n soremouth-infected

+Se lambs. A d d i t i o n a l l y , i n +Se lambs w i t h soremouth l e s i o n s , selenium

level appeared to be negatively correlated with the severity of the

disease.
 - 117 -

TABLE V I I 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 Control

Number of lambs 20 23

Incidence
-2 weeks of age 10.0% 17.4%
-3 weeks 15.0% 34.8%
-4 weeks 20.0% 47.8%
-5 weeks 30.0% 56.5%
-6 weeks 15.0% 21.7%
-7 weeks 15.0% 17.4%
-8 weeks 5.0% 0.0%

Severity of disease
in infected lambs a

-2 weeks 2.00 2.25

-3 weeks 2.66 2.75
-4 weeks 2.00 2.45
-5 weeks 1.50 2.30
-6 weeks 1.30 1.60
-7 weeks 4.30 b
1.25
-8 weeks 1.00

Avg. duration of
disease in infected
lambs 2.3 weeks 2.9 weeks

Avg. age at infection 4.4 weeks 3.6 weeks

% lambs infected 50% (10/20) 70% (16/23)

Avg. 4 week plasma Se

in soremouth lambs 0.087 ppm 0.076 ppm

Avg. 4 week plasma Se

in healthy lambs 0.112 0.093

a
S e v e r i t y ranked on sale of 0 (no soremouth lesions) to
5 (severe l e s i o n s ) .
b
High score due to 2 lambs with +5 scores due to late
development of soremouth.
 - 118 -

While these r e s u l t s are far from conclusive, they do suggest that

selenium has an impact on the epidemiology of soremouth. P o s s i b l y a higher

dosage of selenium r e s u l t i n g i n optimum plasma selenium l e v e l s , could s i g -

n i f i c a n t l y a f f e c t the resistance of lambs to c e r t a i n diseases. The i n f l u -

ence of selenium on diseases of high morbidity and low m o r t a l i t y such as

scours or soremouth could explain the benefit of selenium treatment to

u n t h r i f t y f l o c k s and/or growth rate when obvious deficiency does not occur.

Hemagglutinaton Results

The hemagglutination t i t e r of lambs injected with chicken red blood

c e l l s (CRBC) from four to eight weeks of age was influenced by sex, dura-

tion of stimulus, interactions of selenium with sex and possibly iron.

Data are given i n 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

initial i n j e c t i o n at 4 weeks of age, the t i t e r was 0 . With progressive

i n j e c t i o n s , the mean hemagglutination t i t e r increased to 4.8±0.4, 15.9±0.4,

32.7±0.3, and 42.1±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 , it may be necessary to

continue the experiment for a longer period of time in order to accurately

assess treatment effects.

Iron and selenium did not s i g n i f i c a n t l y (P>0.5) affect t i t e r at any

time, as indicated by figures 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 45 weeks; 15.6+0.4 vs. 16.2±0.7 at

6 weeks; 28.5±0.4 vs. 38.2±0.4 at 7 weeks; and 36.6±0.4 vs. 48.6±0.4 at 8

 - 119 -

5 6 7 8
Weeks o f Age

F i g u r e 15. E f f e c t o f i r o n on HA t i t e r .

5 6 7 8
Weeks o f Age

F i g u r e 16. E f f e c t o f 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 o f t h e l a r g e s t a n d a r d e r r o r and a l s o t h e

l a c k o f e f f e c t o f selenium on female lambs, t h e e f f e c t o f selenium failed

to be s i g n i f i c a n t (P>0.05).

Breed and r e a r i n g d i d 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 in sig-

nificant effects (P<0.05) a t 5 and 7 weeks o f age ( F i g u r e 17). Male lambs

had higher hemagglutination titers at a l l times. Means were 3.4+0.5, and

5.9±0.5, f o r females and males a t 5 weeks. By 8 weeks, mean t i t e r i n

females was 38.6±0.4, compared t o 45.4±0.4 i n males. An interaction

between selenium and sex may account for this effect. Selenium seemed t o

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 i n c r e a s e d titer

in males (Figure 18). Males not t r e a t e d w i t h Se had t i t e r s s i m i l a r to

those of females. Thus t h e mean t i t e r was h i g h e s t f o r selenium-treated

males throughout the t r i a l .

While i r o n d i d 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 t h e experiment had been c o n t i n u e d . I r o n and

selenium a r e both i n v o l v e d 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 v i t a m i n E s i g n i f i c a n t l y raised the h e m a g g l u t i n a t i o n

titer t o sheep r e d blood c e l l s i n a study w i t h weanling swine ( P e p l o w s k i _et

al., 1980). Peplowski et a l . (1980) found t h a t an immediate source of

these nutrients, such as i n j e c t i o n s or a h i g h d i e t a r y c o n c e n t r a t i o n pro-

vided on a s h o r t - t e r m b a s i s , enhanced t h e immune response i n young pigs

marginally deficient i n Se and/or v i t a m i n E. A h i g h e r t i t e r was o b t a i n e d

w i t h high d i e t a r y Se than w i t h t h e 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 t o selenium between our r e s u l t s and those

of P e p l o w s k i et a j l . (1980) can be e x p l a i n e d by s e v e r a l f a c t o r s , mainly t h e

 - 121 -

5 6 7 8
Weeks o f Age

F i g u r e 18. E f f e c t of s e l e n i u m x sex i n t e r a c t i o n on
hemagglution t i t e r .
- 122 -
 - 123 -

dosage and t i m i n g of selenium use, i n a d d i t i o n to the presence of Se inter-

actions. In the present study 1.5 mg Se was injected at b i r t h , and CRBC

a n t i g e n was injected a t 4-4.5 weeks of age and weekly t h e r e a f t e r ; by this

time, while plasma selenium was still significantly higher in the +Se

group, many lambs i n both groups were m a r g i n a l l y d e f i c i e n t i n Se status.

I n c o n t r a s t , P e p l o w s k i e t a l . (1980) used weaned swine of t h e same age, the

same schedule of a n t i g e n injections using a similar a n t i g e n , but the Se

treatment simultaneously provided 0.50 ppm Se, which i s five times the

dietary 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 f o u r times the l e v e l we used f o r lambs of s i m i l a r size.

Work w i t h mice which demonstrated an immunologic response t o s e l e n i u m , a l s o

i n v o l v e d h i g h l e v e l s o f d i e t a r y selenium ( S p a l l h o l z et a l . , 1973; Spallholz

et a l . , 1974; S p a l l h o l z et a i l . , 1975).

The hemagglutination technique is very sensitive but important

limitations i n c l u d e the "occasional lack of reproducibility, qualitative

rather than quantitative nature, and occasional non-specificity"

( S t a v i t s k y , 1954). A c c o r d i n g t o 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

liability, i n that s m a l l amounts o f heterologous antibody or antigen i n

antisera or test antigen may confuse the e s t i m a t i o n of the major anti-

bodies.

The adaptation of the hemagglutination test for this experiment,

specifically 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 e q u i v a l e n t to f o u r t o s i x amino a c i d s or s i m p l e s u g a r s , the

potential 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 effect, then, a

 - 124 -

m u l t i t u d e of p o t e n t i a l a n t i g e n s , each a t 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 h e t e r o l o g o u s a n t i b o d i e s f r u s t r a t e d t h e i n t e r p r e t a t i o n

o f 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 -

tion between p o s i t i v e and n e g a t i v e r e a d i n g s , f r e q u e n t l y as many as 4 or 6

d i l u t i o n s i n sequence would appear i n t e r m e d i a t e between p o s i t i v e and nega-

tive. T h i s c o n t r i b u t e d t o poor r e p r o d u c i b i l i t y of the t e c h n i q u e .

 - 125 -

CONCLUSIONS

T h i s study has i n v e s t i g a t e d s e v e r a l a s p e c t s o f i r o n and s e l e n i u m sup-

p l e m e n t a t i o n o f newborn lambs. I r o n s u p p l e m e n t a t i o n had a profound influ-

ence on lamb metabolism, apparently affecting most blood metabolites

measured, as w e l l as b e t a - g l o b u l i n (transferrin), plasma iron, Hb, and

PCV. Iron supplementation unexpectedly depressed plasma selenium levels,

and a l s o enhanced gamma g l o b u l i n p r o d u c t i o n a t 6 weeks o f age, indicating

that lymphoid tissues may be p a r t i c u l a r l y v u l n e r a b l e t o preweaning iron

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 h e m a g g l u t i n a t i o n t i t e r except i n s e l e n i u m - t r e a t e d lambs.

Injection o f 500 mg Fe s i g n i f i c a n t l y (P<0.05) i n c r e a s e d hemoglobin

from 2 t o 11 weeks o f age i n T r i a l 1, and from 1 t o 8 weeks i n T r i a l 3.

While i r o n dosages o f e i t h e r 250 o r 500 mg prevented t h e d e p r e s s i o n o f Hb

and PCV from birth t o 30 days, plasma iron was s i g n i f i c a n t l y higher

(P<0.05) a t 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 r o p o r t i o n o f c o n t r o l lambs were anemic a t 3-4 weeks o f

age i n a l l s t u d i e s . The number o f lambs d e f i n e d as anemic v a r i e d a c c o r d i n g

to the t r i a l , and the parameter used. F o r example, 42%, 2 1 % and 50% o f t h e

control lambs were anemic i n Trial 2 based on t h e c r i t e r i a o f <28% PCV,

<9.2 g/d£ Hb, and <100 ug/d£ plasma iron. The assessment o f anemia s h o u l d

t h e r e f o r e be based on normal v a l u e s f o r t h e f l o c k r a t h e r than literature

values. PCV i s h i g h l y c o r r e l a t e d w i t h Hb i n lambs 0-6 weeks o f age; conse-

q u e n t l y , PCV can be measured i n s t e a d o f Hb t o a s s e s s i r o n status.

The two s e l e n i u m treatments d i d not a d e q u a t e l y contrast levels of

selenium s t a t u s . As plasma selenium l e v e l s i n both t h e s e l e n i u m - i n j e c t e d

 - 126 -

lambs and control lambs varied from marginal to adequate levels, the

effects of selenium treatment could not be considered conclusive. Probably

some selenium treatment effects were obscured.

Selenium treatment did not affect Hb and PCV values significantly

(P>0.05). While selenium may have a role in heme metabolism in the bone

marrow, the circulating hemoglobin concentration may not be affected except

at markedly deficient levels of Se.

A weight response to iron treatment, but not selenium treatment, was

observed in Trials 1 and 3. In Trial 3, iron-treated lambs were slightly

heavier than controls from 2 weeks (P<0.05) to 8 weeks (P<0.01) of age.

Trial 1 was continued to 11 weeks, and indicated that control lambs began

to catch up to iron-treated lambs after 8 weeks of age. A weight response

to iron treatment was consistently observed in the faster growing lambs,

ie. male and/or single lambs, and not in twin lambs. Iron treatment did

not influence average weight gain in Trial 2, possibly because of the large

numbers of twin lambs. The very low incidence of disease during that

particular trial may also have been a factor.

Preliminary information was provided on the effect of iron deficiency

on the lamb plasma profile. Surprisingly, no comprehensive study of the

plasma profile in iron deficiency has apparently been published for any

species, including man. However, comparison of the data with the litera-

ture on human medicine indicates that iron deficiency affects plasma

metabolites similarly in lambs and humans. In both species, iron defi-

ciency increases plasma glucose and alkaline phosphatase, while decreasing

plasma iron and cholesterol. Other comparative data was lacking for

humans.
 - 127 -

Iron had a major impact on the plasma p r o f i l e i n T r i a l 2, and Pj,

glucose, cholesterol, total protein, AP, AT, and plasma iron responded

linearly to i r o n dosage. Many parameters ( g l u c o s e , BUN, cholesterol, TP,

AP, and AT) were a l s o s i g n i f i c a n t l y correlated with plasma iron. These

results suggest that iron status may broadly influence metabolism

through multiple roles in tissue enzyme systems. Many i r o n enzymes are

reduced at an early stage of iron depletion, and therefore would be

affected by even a m i l d degree of deficiency. L e v e l s of iron sufficient

f o r Hb maintenance may not provide s u f f i c e n t iron for other functions, as

Hb may have p r i o r i t y f o r iron. A weight response to i r o n supplementation

may depend on the provision of adequate i r o n for tissue enzymes, which may

explain 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 a t dose l e v e l s of 150 and 300

mg Fe used i n e a r l i e r studies.

I r o n and selenium supplementation a f f e c t e d plasma p r o t e i n fractions.

As expected, iron deficiency resulted in significantly higher (P<0.01)

3-globulin levels at 2 and 4 weeks of age, due to increased transferrin

productin. I r o n i n j e c t i o n i n c r e a s e d (P<0.001) albumin at k weeks, and also

increased y-globulin l e v e l s at 6 weeks of age. An earlier effect of iron

may not be observed as y-globulin production is depressed in the first

weeks of life; therefore i t would be interesting to follow y-globulin

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.

Effects of i r o n and s e l e n i u m on disease resistence were measured by

susceptibility to soremouth, and anti-CRBC h e m a g g l u t i n a t i o n t i t e r . Selen-

ium treatment appeared to influence s u s c e p t i b i l i t y of lambs to soremouth

infection. The response of lambs to a n t i g e n i c c h a l l e n g e from c h i c k e n RBCs

 - 128 -

was a p p a r e n t l y i n f l u e n c e d by s e l e n i u m as w e l l , even though Se s t a t u s o f t h e

Se-injected lamb was s u b o p t i m a l a t time of c h a l l e n g e . I r o n t r e a t m e n t had a

lesser influence, possibly because lambs were a l r e a d y eating creep-feed

containing adequate d i e t a r y i r o n d u r i n g t h i s part of the t r i a l . Iron may

be relatively more i m p o r t a n t i n resistance t o such d i s e a s e s as F£. c o l i

scours, i n which phagocytosis i s more crucial than humoral immunity.

Scours o c c u r r e d i n most lamb c r o p s , but t h e r e l a t i o n s h i p o f s c o u r s t o i r o n

t r e a t m e n t , and i t s e f f e c t on growth, c o u l d not be measured.

The data p r e s e n t d i n t h i s study suggest t h a t 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 t h e o v e r a l l metabolism, growth r a t e , and h e a l t h of the

lambs. Anemia per se does not appear t o be t h e major concern i n i r o n d e f i -

ciency. Other e f f e c t s o f i r o n d e f i c i e n c y i n c l u d e s u b o p t i m a l f u n c t i o n i n g o f

many i r o n enzyme systems, impairment o f v a r i o u s immune systems, and p o s s i -

bly malabsorption syndromes and/or gastrointestinal lesions. Quantita-

t i v e l y , t h e s e c o n d i t i o n s may be more i m p o r t a n t and more p e r s i s t e n t than t h e

anemia. The d e m o n s t r a t i o n of anemia i n suckling lambs i s an i n d i c a t i o n

that l e s s v i s i b l e , c o n c u r r e n t i r o n d e f i c i e n c y syndromes may reduce produc-

t i v i t y and compromise h e a l t h .
 - 129 -

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- 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 (calculated)

Protein 1
17.0%
Fiber « *
5 5

Calcium 0.33%
Phosphorus 0.20%
Iron 9 5
PP m

not more than 2% e q u i v a l e n t p r o t e i n from u r e a .

 APPENDIX 2.

Procedures used i n analysis of plasma constituents

METABOLITE REFERENCE

Calcium K e s s l e r , G. and Wolfman, M. 1964. C l i n . Chem. 10:686

I n o r g a n i c Phosphate H u r s t , R.O. 1964. Can. 0. Biochem. 42:287

Glucose Bondar, R.J.L. and Mead, D.C. 1974. C l i n . Chem. 20:586.

BUN Marsh, W.H., F i n g e r h u t , B. and M i l l e r , H. 1965. C l i n . Chem. 11:624.

Cholesterol L e v i n e , 3., Morganstern, S. and V l a s t e l i c a , D. 1967. Automation A n a l . Chem.,

Technicon Symp. 1967.

Total Protein Skeggs, L.T. and H o c h s t r a s s e r , H. 1964. C l i n . Chem. 10:918.

Albumin Doumas, B.T., Watson, W.A. and B i g g s , H.G. 1971. C l i n . Chem. Acta 31:87.

A l k a l i n e Phosphatase 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.

L a c t a t e Dehydrogenase H o c h e l l a , N.3. and Weinhouse, S. 1965. A n a l y t i c a l Biochem 13:322.

A s p a r t a t e Transaminase Morganstern, S., Oklander, M., A v e r b a c h , 3., Kaufman, 3. and K l e i n , B. 1966.

C l i n . Chem. 12:95.

Plasma I r o n C a r t e r , P. 1971. A n a l y t i c a l Biochem. 40:450.

 APPENDIX 3.

Effect o f i r o n dextran i n j e c t i o n on lamb weight, hemoglobin and packed cell

volume from b i r t h t o 11 weeks ( T r i a l 1 ) .

WEIGHT (kg ± SE) HEMOGLOBIN ( g / d £ ± SE) P.C.V . (% ± SE)

Control Iron Control Iron

Age Control Iron

n=17 n= 18 n=17 n=18

n= 1 7 n= 1 8

NSt 13.7 ± 0.6 14.5 ± 0.4 NS 37.8 + 1.5 40.2 ± 1.2 NS

Birth 3.9 ± 0.2 4.2 ± 0.3

12.3 ± 0.5 12.9 ± 0.2 NS 33.5 + 1.1 35.8 ± 0.8 NS

1 week 4.5 ± 0.3 5.3 ± 0.3 NS

2 weeks 6.2 ± 0.4 7.4 ± 0.5 NS 10.9 ± 0.4 13.3 ± 0.2 *** 31.2 + 1.1 38.8 ± 0.8 ***

3 weeks 8.1 ± 0.4 9.5 ± 0.6 NS 10.7 ± 0.4 13.5 ± 0.2 *** 30.9 + 1.3 39.4 ± 0.8 ***

4 weeks 9.9 ± 0.5 11.4 ± 0.7 NS 11.1 ± 0.3 13.7 ± 0.3 *** 32.2 + 0.9 39.5 ± 0.7 ***

5 weeks 11.5 ± 0.6 13.5 ± 0.8 NS 11.4 ± 0.3 13.7 ± 0.2 *** 32.8 + 0.9 39.0 ± 0.7 ***

6 weeks 13.4 ± 0.6 15.6 ± 1.0 # 11.9 ± 0.3 13.7 ± 0.3 *** 34.4 + 0.8 39.3 ± 0.7 ***

7 weeks 15.2 ± 0.7 17.9 ± 1.1 * 12.0 ± 0.2 13.2 ± 0.2 *** 34.9 + 0.6 38.3 ± 0.5 ***

8 weeks 17.2 ± 0.8 19.8 ± 1.2 * 12.0 ± 0.2 12.8 ± 0.2 35.4 + 0.7 37.3 ± 0.6 *

9 weeks 18.8 ± 0.8 21.4 ± 1.2 * 12.2 ± 0.1 12.6 ± 0.2 * 35.5 + 0.4 36.9 ± 0.7 *

10 weeks 20.5 ± 0.8 23.3 ± 1.3 NS 12.3 ± 0.2 12.8 ± 0.2 * 36.0 + 0.7 36.8 ± 0.6 NS

11 weeks 22.3 ± 0.9 24.6 ± 1.3 NS 12.0 ± 0.3 12.6 ± 0.2 * 35.2 + 0.7 36.9 ± 0.6 *

TNS P > 0 . 0 5 ; *P<0.05; **P<0.01; ***P<0.001

 APPENDIX 4.

Effect of three levels of iron treatment on hemoglobin and packed c e l l

volume between birth and weaning ( T r i a l 2)

TREATMENTS
Significance of
Control 250 mg Fe 500 mg Fe contrasts 1

20 24 24

Hemoglobin (g/cU ± SE)

13.6 + 0.4 12.9 + 0.4 13.3 + 0.4 NS
2 days
+ 0.3 13.2 + 0.3 13.5 + 0.2
16 d a y s 10.6
10.3 + 0.3 13.5 + 0.4 14.5 + 0.2 L
1 » 2
L
30 d a y s
+ 0.2 12.4 + 0.2 13.7 + 0.2 *-1 2
44 days 11.8
I

P.C.V. {% i SE)
38.0 + 1.0 36.6 + 1.0 37.4 + 1.1 NS
2 days
+ 1.0 37.2 + 0.7 38.6 + 0.6 C ***
16 days 29.3 x

+ 0.9 37.2 + 0.7 41.0 + 0.7 C!***,C *

30 days 29.2 2

+ 0.7 36.7 + 0.5 40.0 + 0.7

44 days 34.0

Predetermined orthogonal contrasts were as follows:

Ci = Control vs. both Fe treatments
C2 = 250 mg F e v s . 5 0 0 mg F e
NS = not s i g n i f i c a n t , P>0.05; *P<0.05; **P<0.01; ***P<0.001.
 APPENDIX 5.

Regression analysis of T r i a l 2 Plasma P r o f i l e Data: P a r t i a l Correlation C o e f f i c i e n t s

POTENTIAL INDEPENDENT VARIABLES

Metabolite
B i r t h Weight Weight Gain Hb PCV Iron Glucose T.P.
n=65

NS NS NS NS NS NS -
Pi

Glucose NS NS -0.306* -0.253* -0.398*** - -

B.U.N. 0.460*** NS NS NS 0.265* - -
Cholesterol NS NS 0.359** 0.432*** 0.336** -0.336** -
Total Protein NS NS -0.328** -0.292* -0.424*** - -
Albumin NS NS NS NS NS - NS

A.P. -0.476 NS -0.427*** -0.417*** -0.468*** - -

L.D.H. NS NS NS NS NS - -
A.T. NS -0.298* NS NS 0.288* - -
Iron 0.329** NS 0.484*** 0.477*** - - -

*P<0.05
**P<0.01
***P<0.001
 APPENDIX 6.

Regression Analysis of T r i a l 2 Plasma P r o f i l e Data:

Regression Equations

REGRESSION EQUATION R-SQUARED F-PROBABILITY

METABOLITE 1

Y = 9 8 . 1 2 7 4 + 0.6981W - 0.0611 I 0.231 0.000291

Glucose

Y = 6 0 . 8 9 4 9 - 1.5701W + 2.1871P 0.258 0.000094

Cholesterol

Y = 5.8606 - 0.00241 0.180 0.000427

Total Protein

Y = 2503.7841 - 85.6757B - 67.5236H 0.349 0.000002

Alkaline Phosphatase

Y = 130.1291 - 2.2732W + 0.12111 0.200 0.000993

Aspartate Transaminase

Y = -126.3880 + 10.7648B + 17.1745H 0.297 0.000018

Iron

where Y = dependent variable (glucose, cholesterol, etc.)

W = independent variable weight gain (weight at sampling - b i r t h weight)
I = independent variable plasma i r o n
P = independent variable packed c e l l volume
B = independent variable b i r t h weight
H = independent variable hemoglobin

Albumin a n d LDH n o t i n c l u d e d due t o lack of significant correlations.

 APPENDIX 7.

E f f e c t of iron l e v e l on lamb weight ( T r i a l 2 ) .

TREATMENTS
Significance of Contrasts
Mean Control 250 mg Fe 500 mg Fe and Main E f f e c t s .
1 2

Age

X ± S.E. (kg)

4.4 ± 0.2 4.4 ±0.2 Age***, B.Wt.***

2 days 4.42 ±0.13 4.4 ± 0.3

6.6 ± 0.3 6.4 ±0.4 Rear*, Age**, B.Wt.***

9 days 6.5 ± 0.2 6.4 ± 0.3

8.3 ± 0.5 Rear**, Age*, B.Wt.***

16 days 8.5 ± 0.3 8.3 ± 0.3 8.8 ± 0.3

10.9 ± 0.5 10.2 * 0.5 Rear**, B.Wt.***

3 days 10.5 ± 0.3 10.2 ± 0.5

12.6 ± 0.6 11.8 i 0.6 Rear***, B.Wt.***

0 days 12.0 ± 0.3 11.8 ± 0.5

8 days 14.0 ± 0.4 13.7 ± 0.6 14.6 ±0.7 13.8 * 0.7 Rear**, TrxSex*, Age**, B.Wt.***

4 4 days 16.0 ± 0.4 15.6 ± 0.6 16.5 ± 0.7 15.8 ±0.9 Rear**, B.Wt.***

1 days 18.0 ± 0.5 17.6 ± 0.6 18.6 ± 0.8 17.7 * 0.9 Rear**, B.Wt.***

n 68 20 24 24

C o v a r i a b l e s were age and b i r t h w e i g h t ; main e f f e c t s were t r e a t m e n t , breed, s e x , r e a r i n g .

2
*P<0.05; **P<0.01; ***P<0.001.
 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
Significance Significant
Age Control SE FE SE + FE of Contrasts 1
Main E f f e c t s 2

X ± S.E. (g/d£)
31 30 31 29

2 days 13.5 + 0.4 13.3 + 0.4 13.8 + 0.3 14.0 ± 0.3 NS NS

1 week 11.4 + 0.4 11.7 + 0.3 13.0 + 0.3 12.7 + 0.3 NS

2 weeks 10.7 + 0.3 10.5 + 0.3 13.2 + 0.2 13.2 + 0.2 NS

3 weeks 10.9 + 0.3 10.6 + 0.5 13.6 + 0.3 13.4 + 0.2 prr*** NS

4 weeks 11.0 + 0.3 11.0 + 0.3 13.4 + 0.3 13.1 + 0.3 pp_*** NS

5 weeks 11.4 + 0.2 11.7 + 0.3 13.8 + 0.2 13.7 + 0.2 pp*** NS

6 weeks 11.9 + 0.2 11.8 + 0.2 13.5 + 0.2 13.4 + 0.2 pp*** NS

7 weeks 12.0 + 0.2 12.0 + 0.2 13.2 + 0.2 13.2 + 0.2 pp*** NS

8 weeks 12.3 + 0.2 12.4 + 0.2 12.9 + 0.2 12.9 + 0.2 pp** NS

Predetermined orthogonal contrasts were as follows: FE = C o n t r o l + Se v s . Fe + (Se + F e ) ; SE = Se + (Se +

Fe) vs. Control + Fe; SE X FE = C o n t r o l + (Se + Fe) v s . Fe + S e ; NS>0.05; *P<0.05; **P<0.01; ***P<0.001.

^Main effects tested were replicate, treatment, breed, s e x , and rearing.

 APPENDIX 9.

E f f e c t of iron and selenium on packed c e l l volume ( T r i a l 3 ) .

TREATMENTS
Significance Significant
Control SE FE SE + FE of C o n t r a s t s 1
Main E f f e c t s

X ± S.E. (% P C V )
31 30 31

+ 0.9 + 1.0 39.0 ± 0.8 39.3 + 0.9 NS NS

2 days 37.9 37.0

+ 0.7 + 0.8 36.2 ± 0.7 35.8 + 0.6 Rear***

1 week 31.0 31.7

+ 0.7 pp*** Rear*

30.8 + 0.6 30.6 + 0.8 39.1 ± 0.6 38.5
2 weeks

+ 0.7 pp*** NS
31.2 + 0.7 31.9 + 1.0 41.1 ± 0.6 39.2
3 weeks

+ 0.6 pp*** NS
32.3 + 0.7 32.5 + 0.8 40.5 ± 0.6 39.4
4 weeks

+ 0.6 pp*** Rep*

34.1 + 0.7 34.7 + 0.7 40.2 ± 0.5 39.6
5 weeks

+ 0.6 pp*** Sex*

35.1 + 0.6 35.9 + 0.6 39.6 ± 0.5 39.0
6 weeks

+ 0.6 pp*** Sex*

36.1 + 0.6 36.1 + 0.5 38.8 ± 0.4 38.6
7 weeks

+ 0.5 + 0.5 37.8 ± 0.5 37.4 + 0.5 FE* Sex*

8 weeks 36.3 36.5

Predetermined orthogonals contrasts were as 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 +

Fe) vs. Control + F e ; SExFE = C o n t r o l + (Se + Fe) v s . Fe + Se; NS>0.05; *P<0.05; **P<0.01; ***P<0.001.

2
Main effects tested were replicate, treatment, breed, s e x , and r e a r i n g .
 - 157 -

APPENDIX 10.

Regression equations f o r hemoglobin versus packed c e l l volume ( T r i a l 3).

f
AGE REGRESSION EQUATION *R'

2 days Y = 0.4851 + 0.3419X 0.8624

4 days Y = 0.4429 + 0.3738X 0.8829

6 days Y = -2.000 + 0.4128X 0.9136

8 days Y = -0.0625 + 0.3609X 0.8436

2 weeks Y = 0.2120 + 0.3346X 0.9228

3 weeks Y = -0.2123 + 0.3397X 0.8840

4 weeks Y = 0.6801 + 0.3187X 0.7882

5 weeks Y = 0.5235 + 0.3292X 0.8568

A

6 weeks Y = 1.1992 + 0.2861X 0.7342

7 weeks Y = 3.417 + 0.2467X 0.5614

8 weeks Y = 4.084 + 0.2334X 0.5409

where Y = a + bX
and Y = dependent v a r i a b l e (Hb)
a = constant, or intercept
b = regression coefficient
X = independent v a r i a b l e (PCV)

*R , 2
the coefficient of determination, represents the proportion of the
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 .

tNumber of observations each at 4 days and 6 days = 43 lambs. A l l other

periods include data from a l l 121 l a m b s in Trial 3.
 APPENDIX 11

Effect o f i r o n and selenium on lamb weight ( T r i a l 3 ) .

TREATMENTS
_ — Significance Significant
Control SE FE SE + FE of Contrasts1 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 o r t h o g o n a l 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 v s . Fe + (Se + F e ) ; SE = Se + (Se +

Fe) v s . C o n t r o l + Fe; FExSE = C o n t r o l + (Se + Fe) v s . Fe + Se
*P<0.05; **P<0.01; ***P<0.001.
2
M a i n e f f e c t s t e s t e d by A n a l y s i s o f V a r i a n c e were r e p l i c a t e , t r e a t m e n t , breed, sex and r e a r i n g .
 - 159 -

APPENDIX 12.

titer
T r i a l 3. E f f e c t of iron, selenium and sex on hemagglutination

HA TITER ± S.E. HA TITER ± S.E.

(loc^ units) (dilution units) 1

-FE +FE -FE +FE

IRON TREATMENT

1.62 ± 0.18 1.50 ± 0.22 5.1 ± 0.5 4.5 ± 0.6

AGE - 5 weeks
2.82 ± 0.20 2.71 ± 0.21 16.7 ± 0.5 15.0 ± 0.6
6 weeks
3.44 ± 0.15 3.54 ± 0.11 31.2 ± 0.4 34.3 ± 0.4
7 weeks 44.7 ± 0.5
3.68 ± 0.11 3.80 ± 0.16 39.5 ± 0.4
8 weeks

22 21 22 21
Number of lambs

-SE +SE -SE +SE

SELENIUM TREATMENT

1.42 ± 0.19 1.72 ± 0.20 4.1 ± 0.5 5.6 ± 0.6

AGE - 5 weeks
2.75 ± 0.14 2.78 ± 0.25 15.6 ± 0.4 16.2 ± 0.7
6 weeks
3.35 ± 0.13 3.64 ± 0.13 28.5 ± 0.4 38.2 ± 0.4
7 weeks
3.60 ± 0.13 3.88 ± 0.14 36.6 ± 0.4 48.6 ± 0.4
8 weeks

23 20 23 20
Number of lambs

FEMALE MALE FEMALE MALE

SEX

1.24 ± 0.19 1.77 ± 0.18 3.4 ± 0.5 5.9 ± 0.5

AGE - 5 weeks
2.70 ± 0.20 2.81 ± 0.20 14.9 ± 0.6 16.7 ± 0.5
6 weeks
3.40 ± 0.12 3.56 ± 0.14 30.0 ± 0.4 35.2 ± 0.4
7 weeks
3.65 ± 0.11 3.82 ± 0.16 38.6 ± 0.4 45.4 ± 0.4
8 weeks

20 23 20 23
Number of lambs

Converted from l o g 2 (natural l o g ) of d i l u t i o n u n i t s .

 APPENDIX 13.

T r i a l 3. E f f e c t of selenium interactions with iron and sex on hemagglutination t i t e r .

HA TITER ± S.E. (LOG UNITS) 9 HA TITER ± S.E. (DILUTION UNITS) 1

TREATMENT CONTROL +SE +FE +SE+FE CONTROL +SE +FE +SE+FE

AGE - 5 weeks 1 .49 ± 0.27 1, 75 ± 0.27 1.35 0.27 68 0.38 4.4 0. 5. + 0.7 3 0.8 5.4 1.0
6 weeks 2.85 ± 0.22 2,78 ± 0.35 2.64 0.20 78 0.41 17.3 0. 16, + 0.9 14 0.6 16.2 1.1
7 weeks 3.28 ± 0.21 3,60 ± 0.20 3.41 0.16 70 0.16 26.6 0. 36, + 0.6 30 0.5 40.4 0.5
8 weeks 3.55 ± 0.14 3,78 ± 0.17 3.64 0.21 4.01 0.25 34.7 0.5 44.0 ± 0.5 38 0.6 55.0 0.7

number o f lambs 11 12 11 11 12 11 9

TREATMENT -SE FEMALE -SE MALE +SE FEMALE +SE MALE -SE FEMALE -SE MALE +SE FEMALE +SE MALE

AGE - 5 weeks 1.42 ± 0.30 1.42 0.24 1.12 0.25 32 0.16 4.1 0. 4.1 0.7 3.1 : 0.7 10.2 ± 0.5
6 weeks 2.88 ± 0.23 2.68 0.19 2.58 0.31 01 0.41 17.8 0. 14.5 0.5 13.1 : 0.8 20.4 ± 1.1
7 weeks 3.39 ± 0.14 3.32 0.20 3.41 0.20 3.93 0.12 29.8 0. 27.7 0.6 30.2 : 0.6 50.9 ± 0.5
8 weeks 3.64 ± 0.25 3.58 0.15 3.66 0.10 4.16 0.28 38.2 0. 35.7 0.5 38.8 : 0.4 64.1 ± 0.8

number o f lambs 9 11 14 11 14

Converted from l o g 2 (natural log) t i t e r s .