9 1985 by The Humana Press Inc. AII fights of any nature whatsoever reserved. 0163-4984/85/0809-0145502.20

Selenium Utilization by Sheep Given Diets Differing in Sulfur and Molybdenum Content AHMED G. ABDEL-RAH1M,-~JOHN R. ARTHUR,~ AND COLIN F. MILLS Rowett Research Institute, Bucksbum, Aberdeen AB2 9SB, Scotland Received April 20, 1985; Accepted May 1, 1985

ABSTRACT The effects of differing dietary concentrations of sulfur (0.4, 1.8, and 3.9 g/kg diet) or Mo (0.3, 3, and 5 mg/kg diet) on Se utilization, were studied in Suffolk x Finn-Dorset ewes. Se concentrations and glutathione peroxidase (E.C.1.11.1.9) activity were measured in tissues of the ewes being repleted with Se or depleted of Se in the presence of the proposed antagonists. Evidence for an antagonism between dietary S and Se was found with an inverse relationship between dietary S concentrations and the Se concentrations of liver and wool, and glutathione peroxidase activity in the liver. Molybden u m (0.3, 3, or 5 mg) in diets of the sheep (0.01 or 0.03 mg Se/kg dry matter) had no significant effects on tissue Se concentrations or glutathione peroxidase activities. With an adequate supply of Se in the diet, there are probably no major effects of S on Se availability and metabolism in the sheep. Within dietary concentrations tested, dietary Mo does not influence utilization of Se by the sheep. Index Entries: Selenium, utilization in sheep; dietary S, effect on Se utilization; sulfur, Se interactions; molybdenum, Se interactions.

~Author to whom all correspondence and reprint requests should be addressed. ~Present address: Department of Physiologyand Biochemistry,Facultyof VeterinaryScience, Universityof Khartoum, Sudan. Biological Trace Element Research

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INTRODUCTION High dietary or environmental concentrations of a range of sulfur (S) compounds antagonize the uptake and for utilization of chemically similar selenium (Se) analogs by plants, microorganisms, and rats (1-8). However, evidence for a S/Se antagonism operating in ruminants is contradictory. Thus high dietary sulfate has been reported to increase the incidence of nutritional myopathy in sheep offered diets of low Se content (9-12), whereas other workers have been unable to produce evidence that dietary S contributed to the incidence of this disorder (13-17). In at least one investigation of the relationship of nutritional myopathy to dietary Se content (9), clinical symptoms were obtained at 0.1 mg Se/kg diet, a Se concentration normally considered to be adequate, thus implying other factors may be important in the etiology the disease. Some of the feedstuffs in these experiments had Mo concentrations as high as 3.86 mg/kg DM~ Studies with isolated segments of rat intestine have shown that molybdate competes during intestinal absorption for a transport system common to sulfate and selenate (18). In view of evidence of interrelationships between molybdenum and sulfur metabolism in ruminants, the experiments in this paper were designed to test whether nutritionally realistic dietary concentrations of molybdenum or sulfur influence the uptake or metabolism and incorporation of Se into the selenoen.zyme gluthathione peroxidase (GSHpx) in sheep. The potential of Mo and S as Se antagonists was enhanced by assessing their effects during periods of Se depletion and repletion (8).

METHODS Twelve Suffolk x Finn-Dorset ewes (12 mo of age/35-40 kg) were used in Experiment 1 (Sex S interaction), nine lambs of the same breed (4 mo of age/13-15 kg) were used in Experiment 2 (Se x Mo interaction). They were offered semisynthetic diets (Table 1) based on oat husks, Torula yeast (Type B, Chas. Tennant Co. Ltd. Glasgow), and urea, were housed individually in wooden pens with slatted floors, and were allowed free access to diet and deionized water in polythene buckets. Animal weights were recorded weekly and fooct offered and refused was recorded daily.

E x p e d m e n t 1: Effects of Dietary S on Se Utilization The 12 ewes initially consumed the low-Se basal diet (Table 1) for 18 wk to deplete tissue reserves of selenium. They were then randomized into three groups and offered the following diet (see Table 1): in Group A (0.4 S, containing 0.4 g S/kg) MgO replaced MgSO4-7HzO in the baSal diet (Table 1), Group B (1.8 S, containing 1.8 g S/kg) diet as in Table 1, and Group C (3.9S, containing 3.9 g S/kg) diet supplemented with Biological Trace Element Research

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TABLE 1 Composition of Basal Semisynthetic Diet Ingredient Whole oat husk Torula yeast Maize starch Sucrose Glucose (containing trace minerals a and vitamins b Major minerals c Urea Arachis oil

Amount/kg diet, g 300 105 318 125 63 48.91 30 10

"Trace minerals, g/kg diet FeSO4-7H20, 0.199; CuSO4-5H20, 0.036; ZnSO4"7H20, 0.114; MnSO4"4H20, 0.16 2; COC12"6H20, 0.004; KIO3, 0.016 g providing mg/kg diet Fe, 40; Cu, 3; Zn, 20; Mn, 40; Co, 1; I, 1o bVitamins g/kg diet A, 0.002; D3, 0.009; E, 0.04; providing IU/kg diet A, 1000; D 3, 360; E, 10. ~Major minerals g/kg diet KHCO3, 10.2 g; NaC1, 3.9; MgSO4"7H20, 9.2; CaHPO4"2H20, 22.01; CaCO3, 3.6 g. The Se content of this diet was within the range 0.008-0.015 mg/kg.

Na2SO~. The. Na2SO4 w a s c o n t a m i n a t e d with Se (1.2 m g Se/kg) a n d allowance was m a d e for this in the formulation of diets~ These m o d i f i e d diets were offered to ewes for 2 w k to p e r m i t adaptation of r u m e n microo r g a n i s m s to the c h a n g e d sulfur concentrations before adjusting the Se concentration of all diets to 0.1 m g Se/kg (with Na2SeO3) a n d offering these diets differing in sulfur content for a 17 wk period of Se repletion (Phase 1). O n e g r o u p A animal died, after 1 w k of repletion because of bloat. After 17 w k repletion, the Se c o n t e n t of the diets was d e c r e a s e d to 0.01 m g Se/kg DM for a further 10 w k to s t u d y effects of dietary S on the rate of Se d e p l e t i o n (Phase 2). Wool samples were obtained from the s h o u l d e r regions of ewes, wool from the same 5 x 5 cm area being taken on each occasion.

Experiment 2: Effects of Different Concentrations of Dietary Mo on $e Utilization Nine lambs were w e a n e d at 4 m o for a study lasting a total of 41 wk. Initially (pre-experimental period), they were offered the basal diet (Table 1) for 10 wk. For the r e m a i n d e r of the study, the low Se basal diet (Table 1) was s u p p l e m e n t e d with N a 2 S O 4 to give 3 g S/kg to p o t e n t i a t e the metabolic effects of Mo (19) a n d its Cu content was increased to 5 m g Cu/kg to restrict the d e v e l o p m e n t of Cu deficiency i n d u c e d by the h i g h e r S content. Animals were r a n d o m i z e d into three t r e a t m e n t g r o u p s a n d given diets with Mo content m o d i f i e d as follows d u r i n g the 22 w k Sedepletion p e r i o d of Phase 1: G r o u p A (0.3 Mo), no Mo a d d e d (basal conBiological Trace Element Research

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Abdel-Rahim, Arthur, and Mills

centration 0.3 Mo mg/kg); Group/3 (3 Mo), Na2MoO4.2H20 added to give 3 mg Mo/kg; Group C (5 Mo), as for B to give 5 mg Mo/kg. During the subsequent 9 wk period of Se-repletion (Phase 2), the above supplements were again given, but dietary Se was increased to 0.03 mg/kg. One animal from group C died during Phase 2 because of a prolapsed intestine. Blood samples were collected by jugular venipuncture and collected in heparinized vacutainers (Beckton, Dickinson, UK, Ltd.) At the end of Phase 2 in both experiments, the sheep were slaughtered and tissues removed and stored at -20~ until analyzed (8). Copper was determined by atomic absorption spectrophotometry and Mo by the method of Bingley (20) after digestion of samples in HNOs:HCIO4:H2SO4 (4:1:0.5 v:v:v). Sulfur was determined in diet samples after MgNO3 oxidation

(2t,22). Statistical analysis of results was by analysis of variance and Student's t-test. The F test was used to check whether the mean values of parameters from two animals in group C (Mo expt., Phase 2) were different from those of groups A and B.

RESULTS Experiment 1. Effects of S on Se ~letabolism Following the introduction of Se (0.1 mg/kg) into the previously Sedeficient diet at the start of Phase 1, there was a delay of 5 wk before whole blood GSHpx started to increase (Fig. 1). Neither this lag period nor the subsequent rates of increase in activity were influenced by dietary S content. Blood GSHpx continued to increase slowly even after withdrawal of Se supplements at the start of Phase 2, presumably because of the entry into the circulation of erythrocytes formed during the period of Se supplementation and the half life (approx. 140 d) of ovine erythrocytes in the circulation. Thereafter (wk 6, Se depletion, Phase 2) blood GSHpx declined at rates that were uninfluenced by dietary S (Fig. 1). Similarly the different S treatments did not influence Se concentrations in whole blood (Fig. 2). Plasma Se concentrations, animal feed intakes, and weight gains were unaffected by the S treatments (results not shown). Without exception the increases in S supplementation tended to reduce both the Se content and the GSHpx activity of all tissues examined (liver, muscle, kidney, spleen, heart, and skeletal muscle; Table 2). Although remarkably consistent, these effects were small and with the exception of observations made on liver tissue at the end of the period of Se depletion (Phase 2), differences attributable to S supplementation were not significant. The Se content of the wool of ewes given diet 0.4S was consistently and significantly greater than that of groups given higher dietary concenBiological Trace Element Research

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Sulfur, Molybdenum, and Selenium Use by Sheep Phase

149

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Fig. 1. Effects of differing concentrations of dietary S on whole blood GSHpx activity in sheep. Whole blood GSHpx activity (EU/mL) of groups 0.4S (G), 1.8S (F1),.and 3.9S (Q) during Phases 1 and 2 of the experiment. Animals and diets are described in the text. Results are mean values for three animals of group A and four animals of groups B and C. Vertical bars are SEMs. trations of S (Fig. 3). Although low dietary S decreased wool growth the quantity of Se incorporated into wool/cm 2 sampled was greater in the low S (0.4S) animals.

Experiment 2. Effects of Mo on Se Metabolism During Phase 1 (Se-depletion) of Experiment 2, whole blood GSHpx activities and Se concentrations of all the Iambs fell to less than 2% of their initial values (Figs. 4 and 5). Dietary Mo treatment had no significant effects on any of these changes. During the Se repletion phase of the experiment (Phase 2), whole blood Se concentrations and GSHpx activities rose in all groups, again with no significant effects of dietary Mo supplementation. The slow response of whole blood GSHpx activity to Se supplementation (Phase 2) was caused by the slow turnover of erythrocytes (see Phase 2, Expt. 1). Plasma Se concentrations, lamb feed intakes and weight gains were unaffected by the different Mo treatments (results not shown). Liver, kidney, spleen, heart, lung, and skeletal muscle Se concentrations and GSHpx activities at slaughter were unaffected by Mo treatment (Table 3). Despite the fact that the Cu content of the basal diet had been increased as a precautionary measure, the deBiological Trace Elernent Research

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150 Phase 1

Phase 2

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Fig. 2. Effects of differing concentrations of dietary S on whole blood Se concentrations in sheep. Whole blood Se concentration (mg/L) of groups 0.4S (A), 1.8S (r~), and 3.9S (0). Experimental details as in Fig. 1. creased liver Cu concentrations of groups 3 Mo and 5 Mo at slaughter indicated that Mo had affected Cu metabolism in the lambs (Table 3).

DISCUSSION In the investigations reported here, the significant effects of dietary S on Se metabolism in sheep were (i) decreased in liver GSHpx activity and tota! Se concentration caused by increased dietary S and (ii) increases in wool Se concentration w h e n dietary S was low. A l t h o u g h increasing dietary S had a small but detectable inhibitory effect on Se utilization it is evident that dietary S is unlikely to play a major role in determining susceptibility to Se responsive disorders in sheep. Thus significant interferences with hepatic retention of Se was evident only w h e n comparing data from a suboptimal S intake (Group 0.4S) with that of a high S intake (3.9S, Table 2). The S concentration in diet 1.8S is close to that commonly found in forages and changing dietary S concentration from 1.8 to 3.9 g/kg caused only a small decrease in liver Se concentration (Table 2). The Se concentrations in Group 3.9S tissues were not, however, as low as those found in sheep clinically affected by Se deficiency, e.g., 0.021-0.05 mg/kg DM in liver and 0.1-0.2 mg/kg DM in Biological Trace Element Research

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Selenium

Use by Sheep

151

TABLE 2 Effects of Dietary Sulfur on Tissue GSHpx Activities and Se Concentrations in Sheep Tissue Liver~ (End phase 1) Liver

Kidney

Spleen

Heart

Gastrocnemius muscle

Treatment

GSHpx EU/mg protein

Se mg/kg DM

0.4S 1.8S 3.9S 0.4S 1,8S 3.9S 0.4S 1.8S 3.9S 0.4S 1.8S 3.9S

0.044 0.041 0.040 0.027 0.023 0.021 1.67 1.67 1.60 0.320 0.278 0.275

___ 0.004 +_ 0.009 _ 0.002 +__ 0.001 +_ 0.001 + 0.001"* • 0.012 _ 0.013 • 0.001 • 0.018 ___ 0.021 • 0.012

0.982 0.949 0.875 0.523 0.450 0.410 4.60 4.41 4.34 0.71 0.64 0.63

• + • • • • • • •

0.088 0.137 0.041 0.003 0.02 0.04*** 0.31 0.09 0.28 0.070 0.061 0.10

0.4S 1.8S 3.9S 0.4S 1.8S 3.9S

0.652 0.574 0.542 0.130 0.121 0.113

• 0.022 • 0.062 • 0.033 ___ 0.027 ___ 0.004 +__ 0.009

0.68 0.64 0.58 0.20 0.17 0.18

• • -+ • • •

0.05 0.06 0.04 0.02 0.04 0.02

~Treatments and tissue analyses are described in the text; liver biopsies were taken from the sheep under general anesthesia at the end of Phase 1; dietary Se, 0.1 mg/kg. Other samples from the end of phase 2, dietary Se, 0.01 mg/kg. Results are mean +- SEM for three animals group 0.4S and four animals groups 1.8S and 3.9S. Significantly different from group 0.4S, **P < 0.01, ***P < 0.001. k i d n e y (23). The tissues m o s t f r e q u e n t l y affected b y Se d e f i c i e n c y in s h e e p are skeletal muscle a n d heart muscle. In E x p e r i m e n t I t h e Se conc e n t r a t i o n a n d s e l e n o e n z y m e G S H p x activity of these tissues at s l a u g h t e r w e r e n o t significantly affected b y d i e t a r y S concentration. A d d i t i o n a l l y the S t r e a t m e n t s h a d no effect o n w h o l e b l o o d c o n c e n t r a t i o n of Se or G S H p x activity, b o t h of w h i c h are f r e q u e n t l y u s e d to assess the Se s t a t u s of an animal. Sulfur s u p p l e m e n t a t i o n h a d no major effect on Se i n c o r p o r a t i o n w i t h G S H p x in s h e e p c o n s u m i n g an a d e q u a t e intake of Se. A role for S in the etiology of Se deficiency diseases m a y possibly be c o n f i n e d to an acceleration of r e s p o n s e s to a c u t e l y deficient intakes of Se. W h i t e a n d Somers (16) u s i n g diets c o n t a i n i n g Se as s e l e n o m e t h i o n i n e a n d S as sulfate h a v e s h o w n t h a t at low Se intakes (0.02 m g Se/kg diet) a d e c r e a s e in dietary S c o n c e n t r a t i o n from 2.0 to 0.7 g/kg can cause a small (20%) rise in p l a s m a Se c o n c e n t r a t i o n . No s u c h effect w a s d e m o n strable in E x p e r i m e n t 1, w h e n diets c o n t a i n e d 0.1 or 0.012 m g Se/kg a n d S varied f r o m 0.4 to 3.9 g/kg. H o w e v e r , in a g r e e m e n t with W h i t e a n d S o m e r s (16), w e h a v e d e m o n s t r a t e d increased incorporation of Se into Biological Trace Element Research

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Abdel-Rahim, Arthur, and Mills

152 0.8

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Fig. 3. Effects of differing concentrations of dietary S on wool Se concentration and wool growth in sheep. (i) Total wool weight (g) for groups A (0.4S), B (1.8S), and C (3.9S) during Phases 1 and 2 (time in weeks after start of Phase); area sampled, 25 cm 2. Results are mean -+ SEM (vertical bars). Significance of diffference between C and A or B, * P < 0.001. (ii) Total Se (~g) in the wool from sample areas (i) of groups A, B, and C. Results are mean -+ SEM (vertical bars). Significance of difference between A and B or C, * P < 0.001. (iii) Wool Se concentration (mg/kg) for groups A, B, and C. Results and statistical significance as (fi). w o o l of the e w e s c o n s u m i n g low S rations (0.4S, Fig. 3). It has b e e n i n f e r r e d by White a n d Somers (16) that s h e e p on high S rations w e r e in a b e t t e r Se status t h a n those c o n s u m i n g a low S diet b e c a u s e less Se w a s e n t e r i n g w o o l a n d t h u s m i g h t be available for incorporation into o t h e r tissues. This is not, h o w e v e r , consistent with results in this p a p e r ; dietary S t r e a t m e n t s did not significantly alter Se c o n c e n t r a t i o n s in k i d n e y , m u s c l e , heart, a n d spleen a n d c o n c e n t r a t i o n of the e l e m e n t w a s slightly d e c r e a s e d in the livers of e w e s c o n s u m i n g t h e - h i g h S diets. M o r e Biological Trace Element Research

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Fig. 4. Effects of differing concentrations of dietary Mo on whole blood GSHpx activity of sheep. Whole blood GSHpx activity (EU/mL) of groups 0.3 Mo (G), 3 Mo ( t ) , and 5 Mo (rT) during Phases 1 and 2 of the experiment. Animals and diets are described in the text. Results are mean values of three animals/ group Phase 1, and three animals/groups (0.3 and 3 Mo) and of two animals/ group (5 Mo) phase 2. Vertical bars are SEMs. For clarity GSHpx axis is expanded for results from Phase 2. Phase 1

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Time (weeks) Fig. 5. Effects of differing concentrations of dietary Mo on whole blood Se concentration of sheep. Whole blood Se concentration (rag/L) of groups 0.3 Mo (&), 3 Mo (Q), and 5 Mo (D) during Phases 1 and 2 of the experiment. For experimental details, s e e Fig. 4.

32

154

AbdeI-Rahim, Arthur, and Mills

TABLE 3 Effects of Dietary Molybdenum on Tissue GSHpx Activities Se Concentrations and Cu Concentrations in Sheep" Tissue

Treatment GSHpx EU/mg protein

Se mg/kg DM Cu mg/kg DM

Liver

0.3Mo 3Mo 5Mo

ND ND ND

0.194 _+ 0.007 31.34 -+ 0.46*** 0.199 + 0.003 19.00 --- 0.77 0.209 14.56

Kidney

0.3Mo 3Mo 5Mo

0.394 + 0.46 0.417 + 0.068 0.399

5.05 4.86 5.21

Spleen

0.3Mo 3Mo 5Mo

0.526 -+ 0.081 0.557 + 0.045 0.514

0.494 -+ 0.014 0.502 +- 0.013 0.509

----

Heart

0.3Mo 3Mo 5Mo

0.309 -+ 0.040 0.286 - 0.041 0.297

0.265 + 0.008 0.260 -+ 0.023 0.253

----

Lung

0.3Mo 3Mo 5Mo

ND ND ND

0.409 + 0.025 0.416 -+ 0.009 0.421

----

Gastrocnemius muscle

0.3Mo 3Mo 5Mo

ND ND ND

0.133 + 0.016 0.141 -+ 0,005 0.132

---

+ 0.36 + 0.25

----

'Treatments a n d tissue analyses are described in the text. Results are mean + SEM for three animals in groups 0.3Mo a n d 3Mo, and the mean for two animals in group 5Mo. ***Significantly different from groups 3Mo and 5Mo P < 0.00 1. ND-GSHpx activity less than 0.006 EU/mg protein.

" b i o c h e m i c a l l y " significant w a s the lack of effect of S t r e a t m e n t s on tissue G S H p x activities (Table 2), this e n z y m e r e p r e s e n t i n g t h e m a i o r k n o w n f u n c t i o n a l role for Se in the cell. W h e t h e r or n o t effects of d i e t a r y S on the m e t a b o l i s m of Se are detectable m a y well be i n f l u e n c e d by the r a n g e of S intakes selected for investigation. T h u s P a u l s o n et al. (13) f o u n d no e v i d e n c e of c h a n g e s in metabolism of 75Se g i v e n orally as selenate to s h e e p w h e n d i e t a r y S w a s i n c r e a s e d f r o m 2.8 to 5.0 g/kg. In c o n t r a s t the effects of S o n Se m e t a b o lism w e r e d e m o n s t r a t e d in the p r e s e n t w o r k a n d in the s t u d i e s of W h i t e a n d S o m e r s (16), W h i t e (17), a n d P o p e et al. (24) w e r e e v i d e n t w h e n res p o n s e s to S s u p p l e m e n t a t i o n w e r e c o m p a r e d w i t h t h o s e in w h i c h s h e e p w e r e g i v e n basal diets w i t h s u b s t a n t i a l l y lower S c o n t e n t s r a n g i n g f r o m 0.3 to 0.7 g S/kgo E v i d e n c e t h a t d i e t a r y S plays a relatively m i n o r role in d e t e r m i n i n g the o v i n e r e s p o n s e to Se s u p p l i e d as selenite (this s t u d y ) or as selenom e t h i o n i n e , W h i t e a n d Somers (16), d o e s n o t exclude the possibility t h a t e n v i r o n m e n t a l S m a y still influence susceptibility to Se d e f i c i e n c y disorders. C o m p a r i s o n of o u r results w i t h evidence of s t r o n g c o m p e t i t i v e inhibition of p l a n t u p t a k e of selenate Se by sulfate (3) s u g g e s t s t h a t S/Se Biological Trace Element Research

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a n t a g o n i s m s o p e r a t i n g at the soil/plant interface to r e d u c e h e r b a g e Se c o n t e n t m a y h a v e a m u c h greater relevance to the o c c u r r e n c e of Se deficiency in r u m i n a n t s . M o l y b d e n u m t r e a t m e n t s a l t h o u g h affecting C u m e t a b o l i s m in the s h e e p (Table 3) did n o t influence the utilization of Se. Elevated c o n c e n trations of S or M o t h a t can be f o u n d in practical rations are, therefore, u n l i k e l y to explain w h y S e - r e s p o n s i v e d i s o r d e r s occur in s o m e b u t n o t all a n i m a l s of a similar l o w Se status.

ACKNOWLEDGMENTS W e are grateful to the staff of the D u t h i e E x p e r i m e n t a l F a r m for care of t h e animals, Mr. I. M c D o n a l d for statistical analyses, a n d Mr. G. W e n h a m for p e r f o r m i n g liver biopsies.

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