Dietary Selenate Versus Selenite for Cattle, Sheep, and Horses' K. L. Podoll, J. B. Bernard, D. E. Ullrey2, S. R. DeBar, P. K. Ku, and W. T. Magee
Department of Animal Science, Michigan State University, East Lansing 48824
supplementation at .3 mg/kg of dietary DM. Serum Se concentrations and glutathione peroxidase (GSHPx) activities measured initially and periodically thereafter revealed no difference between Se forms in sheep and horses and only a small ( P e ,051 advantage for selenate in supporting serum Se concentration in dairy cattle. Selenium concentrations in skeletal muscle and liver of sheep were not different between Se forms. Serum Se, but not GSHPx, increased with time, and .3 mg of supplemental Se per kilogram of dietary DM from either sodium selenate or sodium selenite supported normal serum Se concentrations in sheep, dairy cattle, and horses.
Key Words: Selenium, Bioavailability, Cattle, Sheep, Horses
J. Anim. Sci. 1992. 70:1965-1970
Introduction Selenium (Se) is an essential nutrient (Schwarz and Foltz, 1957) with partially defined metabolic functions (Rotruck et al., 1973; Burk, 1991). Although Se is widely distributed in the earth's crust, its concentrations and availability in soil are extremely variable (McNeal and Balistrieri, 1989). Thus, there are regions in the United States (such as the Great Lakes area) where Se supplements must be used to ensure that domestic animals remain healthy and productive (NRC, 19831. Dietary supplements of Se not to exceed . I mg/kg of diet were approved by the U S . Food and Drug
'This research was supported by the Michigan Agric. Exp. Sta. We thank Julie h e l l , John Shelle, Paula Fulkerson. Herbert Bucholtz, Robert Kreft, Margaret Benson, George Good, and Rita House for their assistance. 2To whom correspondence should be addressed. Received September 26, 1991. Accepted February 3, 1992.
Administration in 1974 for swine and growing chickens, and up to .2 mg/kg diet for turkeys (Ullrey, 1980). A series of amendments (FDA, 1981a,b, 1982, 1987a,b) has extended approval of dietary Se supplements up to .3 mg/kg of diet for all major food-producing animals. No mention of Se supplements for horses appears in the regulations, presumably because horses are not used for human food in the United States. From first approval in 1974, either sodium selenate or sodium selenite could be used as the supplemental Se source, although the feed industry has used selenite primarily. Apparently, it was assumed that the two forms were of equal biopotency. Indeed, Mason and Weaver (19861found that, in the rat, 86% of a 75Se-labeled oral dose of either sodium selenate or selenite was absorbed. However, when sodium selenite was given orally to functional ruminants, most of the Se was excreted in the feces (Cousins and Cairney, 1961; Peterson and Spedding, 1963; Paulson et al., 1966; Wright and Bell, 1966; Lopez et al., 1969). Presumably, microorganisms and the reducing environ-
1965
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ABSTRACT: Food and Drug Administration regulations currently permit addition of .3 mg of Se per kilogram of diet for chickens, turkeys, ducks, swine, sheep, and cattle. However, field reports indicate that this level may not be adequate for ruminants in all situations. Because sodium selenite is the most common supplemental form and is known to be readily absorbed to particles or reduced to insoluble elemental Se or selenides in acid, anaerobic environments, studies were conducted with dairy cattle, sheep, and horses fed sodium selenate to determine whether Se from this source was more bioavailable than Se from sodium selenite. A 2-wk period of no Se supplementation was followed by 49 or 56 d of Se
1966
PODOLL ET AL.
Table 1. Basal diets fed to dairy cattle, sheep, and horses
Dairy cattle
Sheep
Experimental Procedures Experiments were conducted simultaneously with three herbivorous species. Two (dairy cattle and sheep) were chosen because they were ruminants but were managed quite differently. One (horses) was chosen because most of the microbial fermentation in the digestive tract occurs beyond the principal region of Se absorption. No unsupplemented controls were used throughout the study because Se deficiency has been adequately described in these species (NRC, 19831, and we preferred not to adversely affect productivity and health of the experimental animals.
Experiment 1 Eighteen lactating Holstein cows were randomly allotted from age groups to one of two treatments. Three cows in each treatment were in their first lactation, three or four were in their second, and the others were in their third to fifth lactation. All cows were housed in individual stanchions. They had ad libitum access to water, were fed twice a day, and were milked three times a day. Average daily milk production a t the beginning of the experiment was 35 f .09 kg, and average stage of lactation was 115 f .33 d. At the end of the experiment, average daily milk production was 27 k .09 kg. The basal diet (Table 1) was mechanically mixed before feeding and contained haylage, high-moisture corn, whole cottonseed, soybean meal, animal (bypass) protein, encapsulated fat, and supplemental vitamins and minerals without added Se. This diet was formulated to meet or exceed the nutrient requirements (NRC, 1989a) of the lactating dairy cow (except for Sel and was provided for 2 wk before initial collection of blood from the ventral coccygeal vein. Serum was used to establish baseline Se and glutathione peroxidase (GSHPx) values. Selenium was then added to the diet (as sodium selenate or sodium selenite) in a ground dry corn carrier in amounts to provide .3 mg of Se/kg of dietary DM. Blood serum was collected as before on d 3, 7, 10, 14, 28,
of DM
%
Species
Horses
Ingredient Haylage High-moisture corn Cottonseed Soybean meal (489'0 CP) Animal bypass) protein Encapsulated fat Mineralhitamin mix" Total Corn (80%l-Soybean meal (44% CP) (20?/01 Alfalfa-grass hay Trace mineral saltb Total Legume-grass pasture
DMI kg/d
43 30 9 13 2
1 2 100
24
66 33 1 100
1.5
100
8
"Provided in diet DM: ,3496 Ca, .05O/0 P, .09% Mg, ,496 NaCI. . l m g of I/kg, . l mg of Co/kg, 2 m g of CU/kg, 8 m g of Zn/kg , 2 0 mg of Fe/kg, 20 m g of Mn/kg. bContained 98% NaCI, 20 m g of I/kg, 20 mg of Co/kg, 200 m g of Cu/kg, 2,000 m g of Zn/kg.
42, and 49 for Se and GSHPx assays. Concentrations of Se in the diet before and after supplementation were also determined (Table 21.
Experiment 2 Twenty crossbred wether lambs, weighing a n average of 39 f .07 kg and approximately 3 mo old, were randomly allotted to one of two treatments. Lambs were housed in groups by treatment, had ad libitum access to water, and were fed twice a day. The basal diet (Table 1) was formulated to meet or exceed nutrient requirements (NRC, 19851, except for Se, and included alfalfagrass hay and a ground corn-soybean meal mix. Trace mineral salt without Se was provided for ad libitum intake for 2 wk. Blood was collected by jugular venipuncture, and serum was used to establish baseline Se and GSHPx values. Selenium was then added as sodium selenate or sodium selenite to the trace mineral salt a t 60 mg/kg of DM. Based on prior consumption of salt (about 15 g/d), this level was predicted to add .3mg of Se/kg of total dietary DM. Subsequently, blood serum was collected on d 3, 7, 10, 14, 28, 42, and 56 for Se and GSHPx assays. At 56 d, the lambs weighed 48 k .07 kg and were killed in a USDA-inspected slaughterhouse by stunning and exsanguination. Samples of skeletal muscle (common digital extensor) and liver (central lobe) were taken for Se assay. Selenium concentrations were also determined in the hay, corn-soybean meal mix, and trace mineral salt, before and after Se additions (Table 2).
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ment within the rumen promoted conversion of the selenite to less-soluble forms, such as elemental Se or selenides (Cousins and Cairney, 1961). Because field reports suggest that legal supplemental Se levels given orally as sodium selenite to ruminants may not meet requirements in all situations, this study was conducted to determine whether .3 ppm of supplemental Se from sodium selenite would support normal Se status or whether sodium selenate (which is more stable to reduction and less likely to be adsorbed to particles and excreted) would have superior bioavailability.
1967
SELENATE VS SELENITE IN LIVESTOCK DIETS
Table 2. Dry matter and selenium concentration [by analysis) in diets and diet ingredientsa DM, Species Dairy cattle Sheep
Yo
Basal mixed diet Mixed diet + .3 m g of Se/kg DM (atel Mixed diet + .3 mg of Se/kg DM (itel Corn (8Oo:ol-Soybean meal ( 4 4 % CPI (20%) Alfalfa-grass hay Trace mineral (TMI salt (no Se) TM salt + 60 m g of Se/kg DM (ate) TM salt + 60 mg of Se/kg DM (itel Legume-grass pasture Molasses [no Selb Molasses + 157 m g of Se/kg DM (ate) Molasses + 157 m g of Se/kg DM (itel
Se, mg/kg of DM
62 58 57 88 86 100 100 100
28 75 75 75
.076 .276 ,268 .068 ,330 ,000 59 58 .034 ,069 135 147
&Seadded as sodium selenate (atel or sodium selenite (itel. bWeighed 1.527 g/mL.
Expe Y imen t 3
Statistical Analyses
Twelve adult Arabian horses, 11 mares and 1 gelding, were randomly allotted to one of two treatments. All animals were kept on a legumegrass pasture with ad libitum access to water and a Se-free trace mineral salt block (Table 11, which provided all nutrient requirements (NRC, 198913) except for Se. No supplemental Se was provided for 2 wk. At the end of this period, blood was collected by jugular venipuncture, and serum was used to establish baseline S e and GSHPx values. Oral Se supplements, as sodium selenate or sodium selenite, in a molasses carrier were given in individual daily doses to supply the equivalent of .3 mg of Se/kg of total dietary DM, assuming a DMI of 2% of BW. Blood serum was collected on d 3,7, 10, 1 4 , 2 8 , 4 2 , and 56 for Se and GSHPx assays. Selenium concentrations also were determined in pasture samples and in molasses before and after Se additions (Table 2).
The serum data from each experiment were analyzed as a split-plot design; Se treatments (selenate or selenite) constituted the primary effects and days (sampling times) the subplot effects. Treatment effects were tested against animals within treatments; days were tested against the residual mean square. Regression equations (linear, quadratic, and cubic) were fit to the data when appropriate. Sheep tissue Se concentrations were compared with a n unpaired t-test (Gill, 1978). The level of significance was set a t P 5 .05.
Selenium and Glutathione Peroxidase Analyses Anhydrous sodium selenate and anhydrous sodium selenite were purchased from a commercial vendor (Alfa Division of Morton Thiokol, Danvers, MA) for use as Se supplements. Identity was qualitatively verified by selenite’s propensity for reduction to red, insoluble elemental Se when exposed in solution to ascorbic acid. Under the same conditions, selenate remained colorless and in solution. Selenium concentrations were determined in duplicate (serum) or in triplicate (feeds) according to the procedures of Whetter and Ullrey (1978). Serum GSHPx activities were determined in triplicate according to the method of Hafeman et al. (19741 within 8 h of collection (Zhang et al., 1986).
Results The effects of treatment over time on serum Se concentration and GSHPx activity are shown in Table 3. Serum Se concentrations rose ( P < ,011 during the study period in all species. This response to supplemental Se was cubic in dairy cows and quadratic in lambs and horses, but there were no differences between selenate and selenite except in dairy cows, in which selenate produced a slightly greater ( P < .05) serum Se concentration. Serum GSHPx activities were unaffected by the form of Se but were different ( P < .01) over time. However, there was no consistent trend in direction. Selenium concentrations in the skeletal muscle and liver of lambs (Table 4) did not differ with Se form.
Discussion Except for the small advantage of selenate over selenite for support of serum Se concentration in lactating dairy cattle (Table 31, the greater bio-
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Horses
Ingredient/diet
1968
PODOLL ET AL.
1982).
In laboratory studies with rats and chicks, it was found that selenate was absorbed actively by the ileal mucosa, using the same transport system as sulfate, with apparently some passive absorption in the duodenum. Selenite seemed to be absorbed by passive diffusion throughout the tract
Table 3. Serum selenium and glutathione peroxidase (GSHPxl concentrations as affected by supplements of .3 milligrams of selenium-per kilogram of dietary DM from sodium selenate or sodium selenitea Days of supplementation ~
~
~
~~
Species
Se form
0
3
7
10
Dairy cattle Sheep
Selenate Selenite Selenate Selenite Selenate Selenite
,047
,080
,050
.073 ,108 .lo3 .138 .136
,076 .073 ,117 ,109
.078 ,077 .097 ,103 .140 .167
14
28
42
49
.069 ,062 .133 .129 .164 .165
,087 ,078 ,131 ,132 ,190 ,199
,116 ,107
-
-
,131 .128 ,180
,113 .lo5
,087 ,112 .152 ,185 ,559 ,610
56
Se, pg/mL
Horses
,084 .076 ,093 ,101
,131 ,136
.079 ,075 .I14 ,122 ,157 ,165
-
-
,182
GSHPX, U/mLb
Dairy cattle Sheep Horses
Selenate Selenite Selenate Selenite Selenate Selenite
,135 ,165 ,089 ,141 ,677 .674
,074 .075 .167 ,083 .630 .970
,175 .150 ,258 ,249 ,593 ,668
,136 .124 .20 1 .181 .485 ,626
,113 ,115 ,123 ,129 .505 ,606
,104 .114 ,643 ,603
.133 ,121
-
-
,228 ,269 ,802 ,715
-
-
aFor all three species, the mean square error (MSE) for serum Se was < .002 for form and < ,0002 for time. For cattle and sheep, the MSE for GSHPx was c ,008 for form and < .004 for time. For horses, the MSE for GSHPx was ,117 for form and .017 for time. enzyme unit (U) equals 1 p o l of glutathione oxidized per minute.
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(Arduser et al., 1985; Wolffram et al., 1985; Humaloja and Mykkanen, 1986). Disposition of inorganic selenium in the diet of ruminants is obviously influenced by the complicated ruminal milieu (Hidiroglou et al., 1972; Hudman and Glenn, 1984). Based on Se concentrations in serum, skeletal muscle, and liver of lambs and feeder cattle (Ullrey, et al., 19771, supplemental Se as sodium selenite was not absorbed or retained as efficiently a s organic forms. Despite the opportunity for incorporation of selenite-Se into selenoamino acids by ruminal microorganisms, a significant portion of the selenite must have been lost in the feces by adsorption to indigestible particles or as insoluble elemental Se or selenides. This is consistent with the observations of Cousins and Cairney (1961) and others mentioned previously, and the present study provides only indirect evidence of significantly improved absorption or retention of Se from selenate in dairy cattle. Although there were minimal differences in serum Se response between selenate and selenite, serum Se concentration approximately doubled for each species from the beginning to the end of the study. If one assumes that serum Se concentrations of 2 .08 ,ug/mL indicate that dietary supplies of Se are adequate (Ullrey, 19871, then only lactating dairy cows showed initial evidence of inadequacy. Within 3 d of supplementation, mean serum Se concentrations of dairy cattle rose approximately 409'0, but 42 d of supplementation were required before serum Se values of all individuals were 2 .07 pg/mL. Based on a daily DMI of 24 kg/d and an analyzed Se concentration of .3 mg/kg of DM, 7.2 mg of Se was consumed per
availability predicted for Se in sodium selenate was not observed. Estimates of relative bioavailability may be influenced by the amount of Se fed, the species involved, and the criteria used. In rats, estimates of true absorption, using whole-body Se retention from 75Se-labeled selenate or selenite fed in small amounts, ranged from 86 to 95% or 86 to 92%, respectively (Thomson and Stewart, 1973; Mason and Weaver, 1986). For prevention of liver necrosis in rats, sodium selenate providing .1 mg of Se/kg of diet had a Se bioavailability of 122% compared with sodium selenite (Schwarz and Foltz, 1957). In chicks, sodium selenate had a bioavailability of 7 4 % for prevention of exudative diathesis compared to sodium selenite when both compounds provided .1 mg of Se/kg of diet (Cantor et al., 1975). However, when sodium selenate provided 6 mg of Se/kg of diet in a short-term study with chicks, tissue Se concentrations were 101O/O of those provided by sodium selenite (Echevarria, 1986). When the same technique was used with lambs, sodium selenate had a relative bioavailability of 133%(Henry et al., 1988). It is interesting that sodium selenate added to the diet of poults to provide .2 mg of Se/kg had a bioavailability of 134% for support of plasma Se concentration compared to sodium selenite (Cantor and Tarino,
SELENATE VS SELENITE IN LIVESTOCK DIETS
Table 4. Muscle and liver selenium concentrations [mglkg of DM) in sheep as affected by supplements of .3 milligrams of selenium per kilogram of dietary DM from sodium selenate or sodium selenitea Se form
Muscleb ~
Selenate Selenite
.33
*
Liver' ~
,011
.36 f. .016
~
1.79 f .144 2.10 ?r .487
day to produce this response. Stowe et al. (1988) have proposed that Se intakes of pregnant, lactating dairy cows should be 5 to 7 mg/d to sustain adequate Se concentrations in serum. Serum GSHPx activities (Table 31 were not helpful in assessing relative Se bioavailability under the conditions of this study. Likewise, there was no continuous increase in GSHPx activities over time with supplemental Se, suggesting that Se status a t the beginning was not limiting GSHPx synthesis. Stadtmore et al. (1982) have shown that whole-blood GSHPx activity is poorly correlated with whole-blood Se concentration in grazing beef cows and ewes consuming herbage with .09to .24 mg of Se/kg of DM, levels that would encompass dietary Se concentrations consumed by our experimental animals before this study. Although diets unsupplemented with Se were fed for 2 wk before treatments were begun, this was done only to modulate to some degree the immediate effects of prior Se supplementation on serum Se concentration. The 2-wk period would not be long enough to deplete Se reserves significantly. Selenium concentrations in skeletal muscle and liver of lambs a t the end of the study were comparable to those reported for lambs fed a complete, pelleted diet containing .29 mg of Se/kg of diet, with .20 mg of Se/kg provided by sodium selenite (Ullrey et al., 1977).
Implications Under the conditions of this study, sodium selenate was not superior to sodium selenite as a source of supplemental Se in diets for feeder lambs or idle, adult horses, and was only slightly superior (P < .05) for lactating dairy cows. Supplemental Se a t .3 mg/kg of dietary dry matter, provided by either sodium selenate or sodium selenite, supported normal serum Se concentrations and glutathione peroxidase activities in all three species.
Literature Cited Arduser, F., S. Wolffram, and E. Scharrer. 1985. Active absorption of selenate by rat ileum. J. Nutr. 115:1203. Burk, R. F. 1991. Molecular biology of selenium with implications for its metabolism. FASEB J. 5:2274. Cantor, A. H., M. L. Scott, and T. Noguchi. 1975. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exudative diathesis in chicks. J. Nutr. 10596. Cantor, A. H., and J. Z. Tarino. 1982. Comparative effects of inorganic and organic dietary sources of selenium on selenium level and selenium-dependent glutathione peroxidase activity in blood of young turkeys. J. Nutr. 112:2187. Cousins, F. B., and 1. M. Cairney. 1961. Some aspects of selenium metabolism in sheep. Aust. J. Agric. Res. 12:927. Echevarria, M. G. 1986. Effect of time and dietary selenium on selenium tissue uptake and bioavailability of selenium sources for chicks and sheep. Ph.D. Dissertation. Univ. of Florida, Gainesville. FDA. 1981a. Food additives permitted in feed and drinking water of animals: selenium. Final rule. (Friday, Aug. 28) Fed. Register 46:43415 FDA. 1981b. Food additives permitted in feed and drinking water of animals: selenium. Ducks. [Tuesday, Oct. 6) Fed. Register 46:49115 FDA. 1982. Food additives permitted in feed and drinking water of animals: selenium. Final rule. (Friday, June 4) Fed. Register 47:24292 FDA. 1987a. Food additives permitted in feed and drinking water of animals: Selenium. Final rule. (Monday, Apr. 6) Fed. Register 52:10887 FDA. 1987b. Food additives permitted in feed and drinking water of animals: selenium. Correction. (Thursday, June 4) Fed. Register 52:21001 Gill, J. L. 1978. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 1. The Iowa State University Press, Ames. Hafeman, D. G., R. A. Sunde, and W. G. Hoekstra. 1974. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104:580. Henry, P. R., M. G. Echevarria, C. B. Ammerman, and P. V. Rao. 1988. Estimation of the relative biological availability of inorganic selenium sources for ruminants using tissue uptake of selenium. J. Anim. Sci. 66:2306. Hidiroglou, M., K. J. Jenkins, and E. S. Quittkat. 1972. Le sort du radioselenium administre dans le rumen ou dans la caillette du mouton. Ann. Biol. Anim. Biochim. Biophys. 12: 599. Hudman, J. F., and A. R. Glenn. 1984. Selenite uptake and incorporation by Selenornonas ruminantiurn. Arch. Microbiol. 140:252. Humaloja, T., and H. M. Mykkanen. 1986. Intestinal absorption of 75Se-labeled sodium selenite and selenomethionine in chicks: effects of time, segment, selenium concentration and method of measurement. J. Nutr. 116:142. Lopez, P. L., R. L. Preston, and W. H. Pfander. 1969. Whole-body retention, tissue distribution and excretion of selenium-75 after oral and intravenous administration in lambs fed varying selenium intakes. J. Nutr. 97:123. Mason, A. C., and C. M. Weaver. 1986. Metabolism in rats of selenium from intrinsically and extrinsically labeled isolated soy protein. J. Nutr. 116:1883. McNeal, J. M., and L. S. Balistrieri. 1989. Geochemistry and occurrence of selenium: a n overview. In: L. W. Jacobs (Ed.] Selenium in Agriculture and the dnvironment. pp 1-14. Soil Sci. SOC.of Am, Madison, WI. NRC. 1983. Selenium in Nutrition (Rev. Ed.]. National Academy Press, Washington, DC. NRC. 1985. Nutrient Requirements of Sheep (6th Ed.). National
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aProvided in trace-mineral salt containing 60 mg of Se/kg of DM. bMean ?r SE DM was 24.8 f .30%. 'Mean f SE DM was 28.0 k .29°/b.
1969
1970
PODOLL ET AL. long-term and short-term supplementation with oral selenium and vitamin E. J. Dairy Sci. 71:1830. Thomson, C. D., and R.D.H. Stewart. 1973. Metabolic studies of 75Se-selenomethionine and 75Se-selenite in the rat. Br. J. Nutr. 30:139. Ullrey, D. E. 1980. Regulation of essential nutrient additions to animal diets (selenium-a model case). J. Anim. Sci. 51:645. Ullrey, D. E. 1987. Biochemical and physiological indicators of selenium status in animals. J. Anim. Sci. 65:1712. Ullrey, D. E., P. S . Brady, P. A. Whetter, P. K. Ku, and W. T. Magee. 1977. Selenium supplementation of diets for sheep and beef cattle. J. Anim. Sci. 45:559. Whetter, P. A., and D. E. Ullrey. 1978. Improved fluorometric method for determining selenium. J. Assoc. Off. Anal. Chem. 359:927. Wolffram, S., F. Arduser, and E. Scharrer. 1985. In vivo absorption of selenate and selenite by rats. J. Nutr. 115:454. Wright, P. L., and M. C. Bell. 1966. Comparative metabolism of selenium and tellurium in sheep and swine. Am. J. Physiol. 21 1:6.
Zhang, W. R., P. K. Ku, E. R. Miller, and D. E. Ullrey. 1986. Stability of glutathione peroxidase (GSH-Px) in swine plasma samples under various storage conditions. Can. J. Vet. Res. 50:390.
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Academy Press, Washington, DC. NRC. 1989a. Nutrient Requirements of Dairy Cattle. (6th Ed.). National Academy Press, Washington, DC. NRC. 1989b. Nutrient Requirements of Horses (5th Ed.). National Academy Press, Washington, DC. Paulson, G. D., C. A. Baumann, and A. L. Pope. 1966. Fate of a physiological dose of selenate in the lactating ewe: Effect of sulfate. J. Anim. Sci. 25:1054. Peterson, P. J., and D. J. Spedding. 1963. The excretion by sheep of 75Se incorporated into red clover the chemical nature of the excreted selenium and its uptake by three plant species. N.Z. J. Agric. Res. 6:13. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra. 1973. Selenium: Biochemical role as a component of GSH-Px. Science 179:588. Schwarz, K., and C. M. Foltz. 1957. Selenium as a n integral part of Factor 3 against dietary necrotic liver degeneration. J. Am. Chem. SOC.79:3292. Stadtmore, D. L., R. L. Reid, and G. A. Jung. 1982. Selenium status of beef cattle and sheep in West Virginia and Pennsylvania. West Virginia Univ. Agric. Forestry Exp. Sta. Bull. 6797, Morgantown. Stowe, H. D., J. W. Thomas, T. Johnson, J. V. Marteniuk, D. A. Morrow, and D. E. Ullrey. 1988. Responses of dairy cattle to
Department of Animal Science, Michigan State University, East Lansing 48824
supplementation at .3 mg/kg of dietary DM. Serum Se concentrations and glutathione peroxidase (GSHPx) activities measured initially and periodically thereafter revealed no difference between Se forms in sheep and horses and only a small ( P e ,051 advantage for selenate in supporting serum Se concentration in dairy cattle. Selenium concentrations in skeletal muscle and liver of sheep were not different between Se forms. Serum Se, but not GSHPx, increased with time, and .3 mg of supplemental Se per kilogram of dietary DM from either sodium selenate or sodium selenite supported normal serum Se concentrations in sheep, dairy cattle, and horses.
Key Words: Selenium, Bioavailability, Cattle, Sheep, Horses
J. Anim. Sci. 1992. 70:1965-1970
Introduction Selenium (Se) is an essential nutrient (Schwarz and Foltz, 1957) with partially defined metabolic functions (Rotruck et al., 1973; Burk, 1991). Although Se is widely distributed in the earth's crust, its concentrations and availability in soil are extremely variable (McNeal and Balistrieri, 1989). Thus, there are regions in the United States (such as the Great Lakes area) where Se supplements must be used to ensure that domestic animals remain healthy and productive (NRC, 19831. Dietary supplements of Se not to exceed . I mg/kg of diet were approved by the U S . Food and Drug
'This research was supported by the Michigan Agric. Exp. Sta. We thank Julie h e l l , John Shelle, Paula Fulkerson. Herbert Bucholtz, Robert Kreft, Margaret Benson, George Good, and Rita House for their assistance. 2To whom correspondence should be addressed. Received September 26, 1991. Accepted February 3, 1992.
Administration in 1974 for swine and growing chickens, and up to .2 mg/kg diet for turkeys (Ullrey, 1980). A series of amendments (FDA, 1981a,b, 1982, 1987a,b) has extended approval of dietary Se supplements up to .3 mg/kg of diet for all major food-producing animals. No mention of Se supplements for horses appears in the regulations, presumably because horses are not used for human food in the United States. From first approval in 1974, either sodium selenate or sodium selenite could be used as the supplemental Se source, although the feed industry has used selenite primarily. Apparently, it was assumed that the two forms were of equal biopotency. Indeed, Mason and Weaver (19861found that, in the rat, 86% of a 75Se-labeled oral dose of either sodium selenate or selenite was absorbed. However, when sodium selenite was given orally to functional ruminants, most of the Se was excreted in the feces (Cousins and Cairney, 1961; Peterson and Spedding, 1963; Paulson et al., 1966; Wright and Bell, 1966; Lopez et al., 1969). Presumably, microorganisms and the reducing environ-
1965
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ABSTRACT: Food and Drug Administration regulations currently permit addition of .3 mg of Se per kilogram of diet for chickens, turkeys, ducks, swine, sheep, and cattle. However, field reports indicate that this level may not be adequate for ruminants in all situations. Because sodium selenite is the most common supplemental form and is known to be readily absorbed to particles or reduced to insoluble elemental Se or selenides in acid, anaerobic environments, studies were conducted with dairy cattle, sheep, and horses fed sodium selenate to determine whether Se from this source was more bioavailable than Se from sodium selenite. A 2-wk period of no Se supplementation was followed by 49 or 56 d of Se
1966
PODOLL ET AL.
Table 1. Basal diets fed to dairy cattle, sheep, and horses
Dairy cattle
Sheep
Experimental Procedures Experiments were conducted simultaneously with three herbivorous species. Two (dairy cattle and sheep) were chosen because they were ruminants but were managed quite differently. One (horses) was chosen because most of the microbial fermentation in the digestive tract occurs beyond the principal region of Se absorption. No unsupplemented controls were used throughout the study because Se deficiency has been adequately described in these species (NRC, 19831, and we preferred not to adversely affect productivity and health of the experimental animals.
Experiment 1 Eighteen lactating Holstein cows were randomly allotted from age groups to one of two treatments. Three cows in each treatment were in their first lactation, three or four were in their second, and the others were in their third to fifth lactation. All cows were housed in individual stanchions. They had ad libitum access to water, were fed twice a day, and were milked three times a day. Average daily milk production a t the beginning of the experiment was 35 f .09 kg, and average stage of lactation was 115 f .33 d. At the end of the experiment, average daily milk production was 27 k .09 kg. The basal diet (Table 1) was mechanically mixed before feeding and contained haylage, high-moisture corn, whole cottonseed, soybean meal, animal (bypass) protein, encapsulated fat, and supplemental vitamins and minerals without added Se. This diet was formulated to meet or exceed the nutrient requirements (NRC, 1989a) of the lactating dairy cow (except for Sel and was provided for 2 wk before initial collection of blood from the ventral coccygeal vein. Serum was used to establish baseline Se and glutathione peroxidase (GSHPx) values. Selenium was then added to the diet (as sodium selenate or sodium selenite) in a ground dry corn carrier in amounts to provide .3 mg of Se/kg of dietary DM. Blood serum was collected as before on d 3, 7, 10, 14, 28,
of DM
%
Species
Horses
Ingredient Haylage High-moisture corn Cottonseed Soybean meal (489'0 CP) Animal bypass) protein Encapsulated fat Mineralhitamin mix" Total Corn (80%l-Soybean meal (44% CP) (20?/01 Alfalfa-grass hay Trace mineral saltb Total Legume-grass pasture
DMI kg/d
43 30 9 13 2
1 2 100
24
66 33 1 100
1.5
100
8
"Provided in diet DM: ,3496 Ca, .05O/0 P, .09% Mg, ,496 NaCI. . l m g of I/kg, . l mg of Co/kg, 2 m g of CU/kg, 8 m g of Zn/kg , 2 0 mg of Fe/kg, 20 m g of Mn/kg. bContained 98% NaCI, 20 m g of I/kg, 20 mg of Co/kg, 200 m g of Cu/kg, 2,000 m g of Zn/kg.
42, and 49 for Se and GSHPx assays. Concentrations of Se in the diet before and after supplementation were also determined (Table 21.
Experiment 2 Twenty crossbred wether lambs, weighing a n average of 39 f .07 kg and approximately 3 mo old, were randomly allotted to one of two treatments. Lambs were housed in groups by treatment, had ad libitum access to water, and were fed twice a day. The basal diet (Table 1) was formulated to meet or exceed nutrient requirements (NRC, 19851, except for Se, and included alfalfagrass hay and a ground corn-soybean meal mix. Trace mineral salt without Se was provided for ad libitum intake for 2 wk. Blood was collected by jugular venipuncture, and serum was used to establish baseline Se and GSHPx values. Selenium was then added as sodium selenate or sodium selenite to the trace mineral salt a t 60 mg/kg of DM. Based on prior consumption of salt (about 15 g/d), this level was predicted to add .3mg of Se/kg of total dietary DM. Subsequently, blood serum was collected on d 3, 7, 10, 14, 28, 42, and 56 for Se and GSHPx assays. At 56 d, the lambs weighed 48 k .07 kg and were killed in a USDA-inspected slaughterhouse by stunning and exsanguination. Samples of skeletal muscle (common digital extensor) and liver (central lobe) were taken for Se assay. Selenium concentrations were also determined in the hay, corn-soybean meal mix, and trace mineral salt, before and after Se additions (Table 2).
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ment within the rumen promoted conversion of the selenite to less-soluble forms, such as elemental Se or selenides (Cousins and Cairney, 1961). Because field reports suggest that legal supplemental Se levels given orally as sodium selenite to ruminants may not meet requirements in all situations, this study was conducted to determine whether .3 ppm of supplemental Se from sodium selenite would support normal Se status or whether sodium selenate (which is more stable to reduction and less likely to be adsorbed to particles and excreted) would have superior bioavailability.
1967
SELENATE VS SELENITE IN LIVESTOCK DIETS
Table 2. Dry matter and selenium concentration [by analysis) in diets and diet ingredientsa DM, Species Dairy cattle Sheep
Yo
Basal mixed diet Mixed diet + .3 m g of Se/kg DM (atel Mixed diet + .3 mg of Se/kg DM (itel Corn (8Oo:ol-Soybean meal ( 4 4 % CPI (20%) Alfalfa-grass hay Trace mineral (TMI salt (no Se) TM salt + 60 m g of Se/kg DM (ate) TM salt + 60 mg of Se/kg DM (itel Legume-grass pasture Molasses [no Selb Molasses + 157 m g of Se/kg DM (ate) Molasses + 157 m g of Se/kg DM (itel
Se, mg/kg of DM
62 58 57 88 86 100 100 100
28 75 75 75
.076 .276 ,268 .068 ,330 ,000 59 58 .034 ,069 135 147
&Seadded as sodium selenate (atel or sodium selenite (itel. bWeighed 1.527 g/mL.
Expe Y imen t 3
Statistical Analyses
Twelve adult Arabian horses, 11 mares and 1 gelding, were randomly allotted to one of two treatments. All animals were kept on a legumegrass pasture with ad libitum access to water and a Se-free trace mineral salt block (Table 11, which provided all nutrient requirements (NRC, 198913) except for Se. No supplemental Se was provided for 2 wk. At the end of this period, blood was collected by jugular venipuncture, and serum was used to establish baseline S e and GSHPx values. Oral Se supplements, as sodium selenate or sodium selenite, in a molasses carrier were given in individual daily doses to supply the equivalent of .3 mg of Se/kg of total dietary DM, assuming a DMI of 2% of BW. Blood serum was collected on d 3,7, 10, 1 4 , 2 8 , 4 2 , and 56 for Se and GSHPx assays. Selenium concentrations also were determined in pasture samples and in molasses before and after Se additions (Table 2).
The serum data from each experiment were analyzed as a split-plot design; Se treatments (selenate or selenite) constituted the primary effects and days (sampling times) the subplot effects. Treatment effects were tested against animals within treatments; days were tested against the residual mean square. Regression equations (linear, quadratic, and cubic) were fit to the data when appropriate. Sheep tissue Se concentrations were compared with a n unpaired t-test (Gill, 1978). The level of significance was set a t P 5 .05.
Selenium and Glutathione Peroxidase Analyses Anhydrous sodium selenate and anhydrous sodium selenite were purchased from a commercial vendor (Alfa Division of Morton Thiokol, Danvers, MA) for use as Se supplements. Identity was qualitatively verified by selenite’s propensity for reduction to red, insoluble elemental Se when exposed in solution to ascorbic acid. Under the same conditions, selenate remained colorless and in solution. Selenium concentrations were determined in duplicate (serum) or in triplicate (feeds) according to the procedures of Whetter and Ullrey (1978). Serum GSHPx activities were determined in triplicate according to the method of Hafeman et al. (19741 within 8 h of collection (Zhang et al., 1986).
Results The effects of treatment over time on serum Se concentration and GSHPx activity are shown in Table 3. Serum Se concentrations rose ( P < ,011 during the study period in all species. This response to supplemental Se was cubic in dairy cows and quadratic in lambs and horses, but there were no differences between selenate and selenite except in dairy cows, in which selenate produced a slightly greater ( P < .05) serum Se concentration. Serum GSHPx activities were unaffected by the form of Se but were different ( P < .01) over time. However, there was no consistent trend in direction. Selenium concentrations in the skeletal muscle and liver of lambs (Table 4) did not differ with Se form.
Discussion Except for the small advantage of selenate over selenite for support of serum Se concentration in lactating dairy cattle (Table 31, the greater bio-
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Horses
Ingredient/diet
1968
PODOLL ET AL.
1982).
In laboratory studies with rats and chicks, it was found that selenate was absorbed actively by the ileal mucosa, using the same transport system as sulfate, with apparently some passive absorption in the duodenum. Selenite seemed to be absorbed by passive diffusion throughout the tract
Table 3. Serum selenium and glutathione peroxidase (GSHPxl concentrations as affected by supplements of .3 milligrams of selenium-per kilogram of dietary DM from sodium selenate or sodium selenitea Days of supplementation ~
~
~
~~
Species
Se form
0
3
7
10
Dairy cattle Sheep
Selenate Selenite Selenate Selenite Selenate Selenite
,047
,080
,050
.073 ,108 .lo3 .138 .136
,076 .073 ,117 ,109
.078 ,077 .097 ,103 .140 .167
14
28
42
49
.069 ,062 .133 .129 .164 .165
,087 ,078 ,131 ,132 ,190 ,199
,116 ,107
-
-
,131 .128 ,180
,113 .lo5
,087 ,112 .152 ,185 ,559 ,610
56
Se, pg/mL
Horses
,084 .076 ,093 ,101
,131 ,136
.079 ,075 .I14 ,122 ,157 ,165
-
-
,182
GSHPX, U/mLb
Dairy cattle Sheep Horses
Selenate Selenite Selenate Selenite Selenate Selenite
,135 ,165 ,089 ,141 ,677 .674
,074 .075 .167 ,083 .630 .970
,175 .150 ,258 ,249 ,593 ,668
,136 .124 .20 1 .181 .485 ,626
,113 ,115 ,123 ,129 .505 ,606
,104 .114 ,643 ,603
.133 ,121
-
-
,228 ,269 ,802 ,715
-
-
aFor all three species, the mean square error (MSE) for serum Se was < .002 for form and < ,0002 for time. For cattle and sheep, the MSE for GSHPx was c ,008 for form and < .004 for time. For horses, the MSE for GSHPx was ,117 for form and .017 for time. enzyme unit (U) equals 1 p o l of glutathione oxidized per minute.
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(Arduser et al., 1985; Wolffram et al., 1985; Humaloja and Mykkanen, 1986). Disposition of inorganic selenium in the diet of ruminants is obviously influenced by the complicated ruminal milieu (Hidiroglou et al., 1972; Hudman and Glenn, 1984). Based on Se concentrations in serum, skeletal muscle, and liver of lambs and feeder cattle (Ullrey, et al., 19771, supplemental Se as sodium selenite was not absorbed or retained as efficiently a s organic forms. Despite the opportunity for incorporation of selenite-Se into selenoamino acids by ruminal microorganisms, a significant portion of the selenite must have been lost in the feces by adsorption to indigestible particles or as insoluble elemental Se or selenides. This is consistent with the observations of Cousins and Cairney (1961) and others mentioned previously, and the present study provides only indirect evidence of significantly improved absorption or retention of Se from selenate in dairy cattle. Although there were minimal differences in serum Se response between selenate and selenite, serum Se concentration approximately doubled for each species from the beginning to the end of the study. If one assumes that serum Se concentrations of 2 .08 ,ug/mL indicate that dietary supplies of Se are adequate (Ullrey, 19871, then only lactating dairy cows showed initial evidence of inadequacy. Within 3 d of supplementation, mean serum Se concentrations of dairy cattle rose approximately 409'0, but 42 d of supplementation were required before serum Se values of all individuals were 2 .07 pg/mL. Based on a daily DMI of 24 kg/d and an analyzed Se concentration of .3 mg/kg of DM, 7.2 mg of Se was consumed per
availability predicted for Se in sodium selenate was not observed. Estimates of relative bioavailability may be influenced by the amount of Se fed, the species involved, and the criteria used. In rats, estimates of true absorption, using whole-body Se retention from 75Se-labeled selenate or selenite fed in small amounts, ranged from 86 to 95% or 86 to 92%, respectively (Thomson and Stewart, 1973; Mason and Weaver, 1986). For prevention of liver necrosis in rats, sodium selenate providing .1 mg of Se/kg of diet had a Se bioavailability of 122% compared with sodium selenite (Schwarz and Foltz, 1957). In chicks, sodium selenate had a bioavailability of 7 4 % for prevention of exudative diathesis compared to sodium selenite when both compounds provided .1 mg of Se/kg of diet (Cantor et al., 1975). However, when sodium selenate provided 6 mg of Se/kg of diet in a short-term study with chicks, tissue Se concentrations were 101O/O of those provided by sodium selenite (Echevarria, 1986). When the same technique was used with lambs, sodium selenate had a relative bioavailability of 133%(Henry et al., 1988). It is interesting that sodium selenate added to the diet of poults to provide .2 mg of Se/kg had a bioavailability of 134% for support of plasma Se concentration compared to sodium selenite (Cantor and Tarino,
SELENATE VS SELENITE IN LIVESTOCK DIETS
Table 4. Muscle and liver selenium concentrations [mglkg of DM) in sheep as affected by supplements of .3 milligrams of selenium per kilogram of dietary DM from sodium selenate or sodium selenitea Se form
Muscleb ~
Selenate Selenite
.33
*
Liver' ~
,011
.36 f. .016
~
1.79 f .144 2.10 ?r .487
day to produce this response. Stowe et al. (1988) have proposed that Se intakes of pregnant, lactating dairy cows should be 5 to 7 mg/d to sustain adequate Se concentrations in serum. Serum GSHPx activities (Table 31 were not helpful in assessing relative Se bioavailability under the conditions of this study. Likewise, there was no continuous increase in GSHPx activities over time with supplemental Se, suggesting that Se status a t the beginning was not limiting GSHPx synthesis. Stadtmore et al. (1982) have shown that whole-blood GSHPx activity is poorly correlated with whole-blood Se concentration in grazing beef cows and ewes consuming herbage with .09to .24 mg of Se/kg of DM, levels that would encompass dietary Se concentrations consumed by our experimental animals before this study. Although diets unsupplemented with Se were fed for 2 wk before treatments were begun, this was done only to modulate to some degree the immediate effects of prior Se supplementation on serum Se concentration. The 2-wk period would not be long enough to deplete Se reserves significantly. Selenium concentrations in skeletal muscle and liver of lambs a t the end of the study were comparable to those reported for lambs fed a complete, pelleted diet containing .29 mg of Se/kg of diet, with .20 mg of Se/kg provided by sodium selenite (Ullrey et al., 1977).
Implications Under the conditions of this study, sodium selenate was not superior to sodium selenite as a source of supplemental Se in diets for feeder lambs or idle, adult horses, and was only slightly superior (P < .05) for lactating dairy cows. Supplemental Se a t .3 mg/kg of dietary dry matter, provided by either sodium selenate or sodium selenite, supported normal serum Se concentrations and glutathione peroxidase activities in all three species.
Literature Cited Arduser, F., S. Wolffram, and E. Scharrer. 1985. Active absorption of selenate by rat ileum. J. Nutr. 115:1203. Burk, R. F. 1991. Molecular biology of selenium with implications for its metabolism. FASEB J. 5:2274. Cantor, A. H., M. L. Scott, and T. Noguchi. 1975. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exudative diathesis in chicks. J. Nutr. 10596. Cantor, A. H., and J. Z. Tarino. 1982. Comparative effects of inorganic and organic dietary sources of selenium on selenium level and selenium-dependent glutathione peroxidase activity in blood of young turkeys. J. Nutr. 112:2187. Cousins, F. B., and 1. M. Cairney. 1961. Some aspects of selenium metabolism in sheep. Aust. J. Agric. Res. 12:927. Echevarria, M. G. 1986. Effect of time and dietary selenium on selenium tissue uptake and bioavailability of selenium sources for chicks and sheep. Ph.D. Dissertation. Univ. of Florida, Gainesville. FDA. 1981a. Food additives permitted in feed and drinking water of animals: selenium. Final rule. (Friday, Aug. 28) Fed. Register 46:43415 FDA. 1981b. Food additives permitted in feed and drinking water of animals: selenium. Ducks. [Tuesday, Oct. 6) Fed. Register 46:49115 FDA. 1982. Food additives permitted in feed and drinking water of animals: selenium. Final rule. (Friday, June 4) Fed. Register 47:24292 FDA. 1987a. Food additives permitted in feed and drinking water of animals: Selenium. Final rule. (Monday, Apr. 6) Fed. Register 52:10887 FDA. 1987b. Food additives permitted in feed and drinking water of animals: selenium. Correction. (Thursday, June 4) Fed. Register 52:21001 Gill, J. L. 1978. Design and Analysis of Experiments in the Animal and Medical Sciences. Vol. 1. The Iowa State University Press, Ames. Hafeman, D. G., R. A. Sunde, and W. G. Hoekstra. 1974. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104:580. Henry, P. R., M. G. Echevarria, C. B. Ammerman, and P. V. Rao. 1988. Estimation of the relative biological availability of inorganic selenium sources for ruminants using tissue uptake of selenium. J. Anim. Sci. 66:2306. Hidiroglou, M., K. J. Jenkins, and E. S. Quittkat. 1972. Le sort du radioselenium administre dans le rumen ou dans la caillette du mouton. Ann. Biol. Anim. Biochim. Biophys. 12: 599. Hudman, J. F., and A. R. Glenn. 1984. Selenite uptake and incorporation by Selenornonas ruminantiurn. Arch. Microbiol. 140:252. Humaloja, T., and H. M. Mykkanen. 1986. Intestinal absorption of 75Se-labeled sodium selenite and selenomethionine in chicks: effects of time, segment, selenium concentration and method of measurement. J. Nutr. 116:142. Lopez, P. L., R. L. Preston, and W. H. Pfander. 1969. Whole-body retention, tissue distribution and excretion of selenium-75 after oral and intravenous administration in lambs fed varying selenium intakes. J. Nutr. 97:123. Mason, A. C., and C. M. Weaver. 1986. Metabolism in rats of selenium from intrinsically and extrinsically labeled isolated soy protein. J. Nutr. 116:1883. McNeal, J. M., and L. S. Balistrieri. 1989. Geochemistry and occurrence of selenium: a n overview. In: L. W. Jacobs (Ed.] Selenium in Agriculture and the dnvironment. pp 1-14. Soil Sci. SOC.of Am, Madison, WI. NRC. 1983. Selenium in Nutrition (Rev. Ed.]. National Academy Press, Washington, DC. NRC. 1985. Nutrient Requirements of Sheep (6th Ed.). National
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aProvided in trace-mineral salt containing 60 mg of Se/kg of DM. bMean ?r SE DM was 24.8 f .30%. 'Mean f SE DM was 28.0 k .29°/b.
1969
1970
PODOLL ET AL. long-term and short-term supplementation with oral selenium and vitamin E. J. Dairy Sci. 71:1830. Thomson, C. D., and R.D.H. Stewart. 1973. Metabolic studies of 75Se-selenomethionine and 75Se-selenite in the rat. Br. J. Nutr. 30:139. Ullrey, D. E. 1980. Regulation of essential nutrient additions to animal diets (selenium-a model case). J. Anim. Sci. 51:645. Ullrey, D. E. 1987. Biochemical and physiological indicators of selenium status in animals. J. Anim. Sci. 65:1712. Ullrey, D. E., P. S . Brady, P. A. Whetter, P. K. Ku, and W. T. Magee. 1977. Selenium supplementation of diets for sheep and beef cattle. J. Anim. Sci. 45:559. Whetter, P. A., and D. E. Ullrey. 1978. Improved fluorometric method for determining selenium. J. Assoc. Off. Anal. Chem. 359:927. Wolffram, S., F. Arduser, and E. Scharrer. 1985. In vivo absorption of selenate and selenite by rats. J. Nutr. 115:454. Wright, P. L., and M. C. Bell. 1966. Comparative metabolism of selenium and tellurium in sheep and swine. Am. J. Physiol. 21 1:6.
Zhang, W. R., P. K. Ku, E. R. Miller, and D. E. Ullrey. 1986. Stability of glutathione peroxidase (GSH-Px) in swine plasma samples under various storage conditions. Can. J. Vet. Res. 50:390.
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Academy Press, Washington, DC. NRC. 1989a. Nutrient Requirements of Dairy Cattle. (6th Ed.). National Academy Press, Washington, DC. NRC. 1989b. Nutrient Requirements of Horses (5th Ed.). National Academy Press, Washington, DC. Paulson, G. D., C. A. Baumann, and A. L. Pope. 1966. Fate of a physiological dose of selenate in the lactating ewe: Effect of sulfate. J. Anim. Sci. 25:1054. Peterson, P. J., and D. J. Spedding. 1963. The excretion by sheep of 75Se incorporated into red clover the chemical nature of the excreted selenium and its uptake by three plant species. N.Z. J. Agric. Res. 6:13. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra. 1973. Selenium: Biochemical role as a component of GSH-Px. Science 179:588. Schwarz, K., and C. M. Foltz. 1957. Selenium as a n integral part of Factor 3 against dietary necrotic liver degeneration. J. Am. Chem. SOC.79:3292. Stadtmore, D. L., R. L. Reid, and G. A. Jung. 1982. Selenium status of beef cattle and sheep in West Virginia and Pennsylvania. West Virginia Univ. Agric. Forestry Exp. Sta. Bull. 6797, Morgantown. Stowe, H. D., J. W. Thomas, T. Johnson, J. V. Marteniuk, D. A. Morrow, and D. E. Ullrey. 1988. Responses of dairy cattle to
