THE INFLUENCE OF DIETARY SELENIUM LEVELS ON BLOOD LEVELS OF SELENIUM AND GLUTATHIONE PEROXIDASE ACTIVITY IN THE HORSE J. S. Shellow, S. G. Jackson, J. P. Baker and A. H. Cantor University of Kentucky, Lexington 40546 ABSTRACT Twenty mature geldings, averaging 535 kg, were used to determine the influence of dietary selenium (Se) on the blood levels of Se and Se-dependent glutathione peroxidase (SeGSH-Px) activity in the horse. Horses were randomly assigned within breed to four treatments consisting of five horses each and fed a basal diet containing.06 ppm of naturally occurring Se. Diets were supplemented with.05,.0 and.20 ppm Se, as sodium selenite. Blood was drawn for 2 wk before, and for 2 wk following, the inclusion of supplement Se in the diets. Whole blood and plasma Se concentrations and plasma SeGSH-Px activities were determined from all blood samples. Selenium concentrations in plasma and whole blood increased linearly from wk to wk 5 and 6, respectively, in Se-supplemented horses. After these times, no significant changes in Se concentration were observed in Se-supplemented or in unsupplemented horses throughout the remainder of the 2-wk trial. Plasma Se reached plateaus of.0 to.,.2 to.4, and.3 to.4 #g/ml in horses supplemented with.05,.0 and.20 ppm Se, respectively. Whole blood Se reached plateaus of.6 to.8,.9 to.2, and.7 to.8 #g/ml in horses supplemented with.05,.0 and.20 ppm Se, respectively. Plasma SeGSH-Px activity was not significantly affected by dietary treatment. Therefore, this enzyme was not a good indicator of dietary Se in these mature horses. (Key Words: Selenium, Glutathione Peroxidase, Geldings.) Introduction Selenium (Se) has been established as an essential nutrient in several animal species, including the horse. It is an essential component of the Se-dependent enzyme, glutathione peroxidase (SeGSH-Px) and, as such, functions as part of a cellular antioxidant defense system (Chow, 979). Substantial research indicates that dietary Se is reflected by blood Se level, as well as by SeGSH-Px activity (Omaye and Tappel, 974; Chow and Tappel, 974; Hafeman et al., 974). Stowe (967) studied serum Se levels in conventionally and experimentally fed horses and estimated the equine daily requirement to be 2.4/~g Se/kg of body weight. Based on this research, the NRC (978) suggested a Se requirement for the horse of. ppm of the diet. The investigation reported in this paper (No. 84-5-83) is in connection with a project of the Kentucky Agr. Exp. Sta. and is published with approval of the Director. Received October 2, 984. Accepted May 5, 985. Although Se supplementation in equine diets has become relatively common, the normal blood levels of Se in horses receiving a diet thought to be adequate in Se have not been clearly defined. Also, the relationship between Se and SeGSH-Px may be an important diagnostic tool in determining Se status in the horse. The objectives of this study were to determine () the influence of dietary Se on whole blood and plasma Se levels and (2) the influence of dietary Se on plasma SeGSH-Px activity. Materials and Methods Twenty mature geldings of Quarter Horse and Thoroughbred breeding, averaging 535 kg, were randomly assigned within breed to four treatment groups of five horses each. Daily intake of digestible energy was 55 kcal/kg "Ts, a level equal to that suggested by the NRC (978) for horses at maintenance. One-half of the daily feed was fed as a concentrated mixture containing.077 mg/kg of naturally occurring Se (table ); the other half was fed as timothy hay containing.043 mg/kg of naturally occurring Se, as determined by analysis. Thus, 590 JOURNAL OF ANIMAL SCIENCE, Vol. 6, No. 3, 985
SELENIUM LEVELS IN HORSES 59 TABLE. COMPOSITION OF THE BASAL CONCENTRATE MIXTURE a Ingredient Otas, grain, crimped (IFN 4-03-309) Corn, grain, cracked (IFN 4-02-985) Soybean meal (44% solvent extracted (IFN 4-05-604) Molasses, sugar cane (IFN 4-04-696) Limestone (IFN 6-02-632) Dicalcium phosphate (IFN 6-0-08) Trace mineralized salt b Total 44 35 0 8 00.00 aas-fed basis. bprovided (%): 99.000 NaC,.350 Zn,.340 Fe,.200 Mn,.033 Cu,.007 I,.005 Co. the total basal diet contained.060 mg/kg of naturally occurring Se. All horses were fed the basal diet for at least a 4-wk preliminary period. In addition, the concentrate feed was supplemented with 0,.0,.20, or.40 ppm Se, as sodium selenite (table 2). Because one-half of the daily feed was fed as a concentrate mixture, horses received supplemental Se at graded levels of 0,.05,.0, or.20 ppm Se. Final Se concentrations, basal plus supplemental, for the respective treatments were.06,.,. 6 and. 26 ppm. Water and trace mineralized salt, which did not contain Se, were provided ad libitum. Blood was drawn from each horse on the same day and time at weekly intervals via jugular venipuncture for 2 wk before, and then for 2 wk following, the inclusion of supplemental Se in the diets. Weekly plasma and whole blood samples from each horse, and a composite of the concentrate diets and hay samples, were analyzed for Se according to the fluorometric method of Olson et al. (975), as modified by Cantor and Tarino (982). Weekly plasma samples were analyzed for SeGSH-Px activity using the coupled procedure of Lawrence and Burke (976). All plasma and whole blood samples were stored at 4 C upon collection and assays were performed within 2 to 3 d after sampling. The experiment was analyzed as a repeatedmeasures design (Gill, 978). The amounts of added Se were the whole plot treatments; weeks was the repeated-measures effect. The values for the 2 pretrial wk were averaged and used as single baseline values. Animal variation was used as the error term for the whole plot treatment. If a significant treatment x week interaction was detected, one-way analyses of variance were run at each week. The least significant difference procedure was used to test differences between the mean of treatment groups at each week. All analyses were performed using the General Linear Models procedure of SAS (979). Results and Discussion The effect of dietary Se on the concentration of Se in plasma measured at weekly intervals is shown in table 3. There was an increasing linear trend (P<.0) in plasma Se concentration over time. Significant treatment differences and a linear time x treatment interaction were detected. Therefore treatment means were compared at each week of the trial. At week zero (pretrial) no significant differences were observed among treatment groups. Supplementation of the diets with Se increased (P<.05) plasma Se above that of the control Diet TABLE 2. SELENIUM ANALYSIS OF THE CONCENTRATE MIXTURES a Selenium concentration, ppm b Basal concentrate.070 -+.00 Basal concentrate +.0 ppm Se.62 -+.005 Basal concentrate +.20 ppm Se.288 -+.009 Basal concentrate +.40 ppm Se.460 +-.009 aas-fed basis. beach value is mean of five replicates -+ SE.
592 SHELLOW ET AL. TABLE 3. EFFECT OF DIETARY SELENIUM ON THE CONCENTRATION OF SELENIUM IN PLASMA Time, wk Basal diet +.05 ppm Se +.0 ppm Se +.20 ppm Se Plasma Se, pg/ml a....... Pretrial.063 +.006 b.074 +.005 b.075 +.004 b.060 -+.006 b.062 +.004 b.084 +.005 b.093 +.003 d.076 +.009 bc 2.062 +.004b.084 +.005 cd.093 +.O03 d.076.009 bc 3.060 +.002 b.095 -+.007 c.9 +.006 d.0 -+.00 cd 4.063 +.002 b.099 +.007 c.24 -+.005 d.8 +.00 cd 5.068 +.003 b.098 +.005 c,24 +.004 d.30 +.00 d 6.066 -+.002 b.03 +.004 c.26 +.o04d.28 +.009 d 7.065 +.002 b.0 +.005 c.29 +.004d.33 +.0 d 8.069 -+.003 b.05 +.005 c.40 +.006 d.39 +.0 d 9.070 -+.004 b.06 +.005 c.38 -+.005 d.42 +.00 d 0.064 +.00 b.09 +.008 c.42 +.007 d.35 -+.007 d.068 -+.00 b.5 +.004 c.48 +.006 d.45 +-.008 d 2.069 -+.003 b.0 -+.004 c.43 +.004 d.40 +.008 d aeach value is the mean of five observations -+ SE. b'c'dmeans in the same row that do not have a common superscript differ (P<.05). group by the second week of the trial, and this difference continued throughout the remainder of the experiment. By wk 5, there were differences (P<.05) in plasma Se concentration between horses in the control group, those receiving.05 ppm Se and those receiving.0 or.20 ppm supplemental Se in their diet. Plasma Se concentrations for horses receiving the two highest levels of Se were greater (P<.0) than those receiving.05 ppm supplemental Se. This difference between treatment groups observed at wk 5 continued throughout the remainder of the trial; however, during this period there were no significant differences in plasma Se concentrations between those horses receiving.0 and.20 supplemental Se. Following the initiation of dietary supplementation of Se, the maximum response in plasma Se occurred by wk 5. Little change in plasma Se concentration was observed in Se-supplemented horses after 5 wk, or in unsupplemented horses throughout the entire trial. Plasma Se reached plateaus of.0 to.,.2 to.4, and.3 to.4 k/g/ml in horses supplemented with.05,.0 and.20 ppm Se, respectively. These results are consistent with other reported values. Stowe (967) reported that serum Se levels reached plateaus of.4 to.6/.lg/ml when horses were fed.5,.0, or 2.0 ppm supplemental Se, as sodium selenite. Although Stowe (967) fed much higher levels of Se, serum concentrations reached plateaus at levels comparable with those observed in the present study. Newman et al. (98) reported that a serum Se concentration of.4/~g/ml was adequate to prevent problems associated with Se deficiency in horses. Results of this study indicate that.0 or.20 ppm supplemental dietary Se yield plasma Se concentrations above those associated with Se deficiency problems. Because there were no differences between plasma Se concentrations in horses supplemented with.0 or.20 ppm Se, there appears to be no advantage in supplementing the mature idle horse with levels of Se greater than.0 ppm. The effect of dietary Se on the concentration of Se in whole blood measured at weekly intervals after the start of dietary Se supplementation is shown in table 4. The maximum response in whole blood Se concentration occurred by wk 6, with no further significant changes throughout the remainder of the trial. Whole blood Se reached plateaus of.6 to.8,.9 to.2, and.7 to.8 /~g/ml in groups supplemented with.05,.0 and.20 ppm Se, respectively. Although the blood concentration of all three supplemented groups was significantly higher than that of the basal group, dietary supplementation above.05 ppm Se did not result in further increases. These results indicate that whole blood was not as sensitive
SELENIUM LEVELS IN HORSES 593 TABLE 4. EFFECT OF DIETARY SELENIUM ON THE CONCENTRATION OF SELENIUM IN WHOLE BLOOD Time, wk Basal diet +.05 pprn Se +.0 ppm Se +.20 ppm Se Whole blood Se,/~g/ml a Pretrial.02 -+.006 bc.24 cd.36.002 d.099.02 b.098.004 b.28.009 cd.40.007 d.08 -+.0 bc 2.099.004 b.20 b.47 c.9.04 b 3.096.005 b.4 c.62 c.37 c 4.099 +-.006 b.40.02 c.64.02 c.34.04 c 5.6.006 b.60 -+.03 c.89 c.66.022 c 6.2.005 b.73 c.80 c.6 c 7.03.005 b.52 c.77 d.60 cd 8.099 -+.004 b.52.00 c.80.006 cd.66 +-.04 c 9.7.003 b.7 -+.0 c.24.003 d.92.04 cd 0.3.004 b.89.008 c.220.005 d.90.02 c.08 -+.005 b.73 c.207 -+.005 d.88 c 2.04.005 b.76.02 c.208.003 d.83.02 cd aeach value is the mean of five observations +- SE. b'c'dmeans in the same row that do not have a common superscript differ (P<.05). to changes in dietary Se as was plasma. Cantor and Tarino (982) also have reported plasma Se concentration to reflect changes in dietary Se more closely than that of whole blood in turkeys; they suggested that this difference between plasma and whole blood could have been due in part to the relatively small turnover of red blood ceils during the comparatively short experimental period. Cornelius et al. (960) reported that two Thoroughbreds had red blood cell life spans of 40 and 60 d when 4C-glycine was used as a label. Valli et al. (975) reported the life span of red blood cells in Standardbred horses to be 55 (+0) d when radioactive selenomethionine was used as a label. Because the length of the treatment in the present study was only 40 d, the lack of response of whole blood to dietary Se could have been due to the incomplete turnover of red blood cells. The effect of dietary Se on SeGSH-Px measured at weekly intervals is shown in table 5. Plasma SeGSH-Px activity was not significantly affected by dietary treatment, although an increasing trend in activity over time was observed. The lack of a dietary treatment effect suggests that the endogenous Se, and(or) the naturally occurring Se in the basal diet, was sufficient for maximizing the activity of this enzyme in the mature horse at maintenance, and supplemental Se did not significantly increase the enzyme activity. The studies of Noguchi et al. (973), Tappel (974) and Cantor and Tarino (982) were conducted with young growing animals that were initially depleted of Se. Therefore, the dietary Se had a much greater influence than did endogenous Se on plasma SeGSH-Px was probably at adequate levels in all treatment groups and was not as responsive to changes in dietary Se in these mature horses as in the studies with the young of other species. The lack of dietary treatment effect on plasma SeGSH-Px activity, and therefore the lack of correlation (R =.28, NS) between plasma Se concentration and enzyme activity, could have been due in part to the level of blood Se needed to maintain maximum enzyme activity. The results of this study suggest that these horses had sufficient blood Se levels at the beginning of the trial for maintenance of SeGSH-Px activity throughout the trial. This may have been due to a relationship similar to that reported by Blackmore et al. (982). These researchers found that erythrocyte GSH-Px activity of Thoroughbred horses reached a plateau when Se levels of.2 #g/ml of serum or.6 /ag/ml of whole blood were attained. Although plasma and whole blood Se during the present pretrial period were lower than the
594 SHELLOW ET AL. TABLE 5. EFFECT OF DIETARY SELENIUM ON PLASMA GLUTATHIONE PEROXIDASE ACTIVITY Time, wk Basal diet +.05 ppm Se +.0 ppm Se +.20 ppm Se Plasma SeGSH-Px, units/ml a' Pretrial.665 +.073.652 +.056.679.085.536.046.630.05.687.052.698.072.532.038 2.6.5.040.70.056.72.073.537 +.024 3.623 -+.037.660 +.048.679.074.580 +.026 4.657.053.73.052.68.075.60 +.025 5.69.028.666 +.04.677.079.604.027 6.652.037.739 +.O47.722.085.67 +.055 7.635.040.68.053.674.070.650.057 8.623.032.706.042-.648.075.628.050 9.665.043.77.050.662.055.646.044 0.650.046.743.045.639.058.626.038.674.020.744.039.736.055.72.025 2.690.027.743.O4.736.056.742.07 9 aeach value is the mean of five observations + SE; one unit, /Jmol NADPH oxidized/rain. levels reported by Blackmore et al. (982), a plateau in enzyme activity may already have been reached at the beginning of this trial, and supplemental Se did not further enhance enzyme activity. L iteratuce Cited Blackmore, D. J., C. Campbell, C. Dant, J. E. Holden and J. E. Kent. 982. Selenium status of Thoroughbreds in the United Kingdom. Equine Vet. J. 4:39. Cantor, A. H. and J. Z. Tarino. 982. Comparative effects of inorganic and organic dietary sources of selenium on selenium levels and seleniumdependent glutathione peroxidase activity in the blood of young turkeys. J. Nutr. 2:287. Chow, C. K. 979. Nutritional influence on cellular antioxidant defense systems. Amer. J. Clin. Nutr. 32:66. Chow, C. K. and A. L. Tappel. 974. Response of glutathione peroxidase to dietary selenium in rats. J. Nutr. 04:444. Cornelius, C. E., J. J. Kaneko, D. C. Benson and J. D. Wheat. 960. Erythrocyte survival studies in the horse, using glycine-2-c t4. Amer. J. Vet. Res. 2:23. Gill, J. L. 978. Design and Analysis of Experiments, Vol. 2. Iowa State Univ. Press, Ames. Hafeman, D. G., R. A. Sunde and W. G. Holkstra. 974. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 04:580. Lawrence, R. R. and R. F. Burke. 976. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Commun. 7:952. Newman, L. E., P. R. Weaver and J. T. Bryans. 98. Selenium responsive problems and serum selenium levels in horses in Kentucky. Proc. 24th Annu. Amer. Assoc. Vet. Lab. Diagnostitions. p 76. Noguchi, T., A. H. Cantor and M. L. Scott. 973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 03:502. NRC. 978. Nutrient Requirements of Domestic Animals, No. 6. Nutrient Requirements of Horses. Fourth Revised Ed. National Academy of Sciences - National Research Council, Washington, DC. Olson, O. E., I. S. Palmer and E. E. Cary. 975. Modification of the official method for selenium in plants. J. Assoc. Official Anal. Chem. 58:7. Omaye, S. T. and A. L. Tappel. 974. Effect of dietary selenium on glutathione peroxidase in the chick. J. Nutr. 04:747. SAS. 979. SAS User's Guide. Statistical Analysis System Institute, Inc., Cary, NC. Stowe, H. D. 967. Serum selenium and related parameters of naturally and experimentally fed horses. J. Nutr. 93:630. Tappel, A. L. 974. Selenium-glutathione peroxidase and vitamin E. Amer. J. Clin. Nutr. 27:960. Vatli, V. E., J. H. Lumsden, E. I, Carter and B. J. McSherry. 975. The kinetics of hematopoiesis in the normal light horse. Proc. st International Symposium on Equine Hematology. Amer. Assoc. of Equine Practitioners, Michigan State Univ., East Lansing.