Influence of Dietary Levels of Vitamin E and Selenium on Tissue and Blood Parameters in Pigs L. G. Young, J. H. Lumsden, A. Lun, J. Claxton and D. E. Edmeades*

ABSTRACT Eighteen barrows approximately three weeks of age were used in a 3 x 3 factorial arrangement to investigate the effect of level of supplemental vitamin E and selenium on tissue and blood parameters. Tissue selenium concentrations increased in a quadratic manner with increased selenium intake with kidney tissue containing considerably greater concentrations than liver, heart or muscle. Supplementation of the diet caused a threefold increase in serum selenium within the first week with a slight tendency to further increases in subsequent weeks. Serum vitamin E of unsupplemented pigs declined by fifty percent during the experiment, whereas supplemental vitamin E resulted in increased serum vitamin E. There was a considerable variation in percent peroxide hemolysis. Correlations of -0.63 between percent peroxide hemolysis and vitamin E intake and -0.85 between percent peroxide hemolysis and serum vitamin E were observed.

RESUME Les auteurs ont utilise 18 porcelets castres et 'ages d'environ trois semaines, dans une experience a arrangement factoriel de 3 x 3. Cette etude visait 'a determiner l'effet de l'ad-

*Department of Animal and Poultry Science, Ontario Agricultural College (Young, Lun and Edmeades) and Department of Pathology, Ontario Veterinary College (Lumaden and Claxton), University of Guelph, Guelph, Ontario NlG 2W1. Submitted June 24, 1975.

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dition 'a la diete de differentes quantites de vitamine E et de selenium, sur les parametres tissulaires et sanguins. La teneur des tissus en selenium augmenta de faqon quadratique 'a mesure qu'augmentait l'ingestion de cet element; les reins en contenaient aussi beaucoup plus que le foie, le coeur on les muscles. L'incorporation de selenium a la diJete tripla la concentration serique de cet element au cours de la premiere semaine et provoqua aussi une legere tendance a sa hausse, au cours des semaines ulterieures. La teneur en vitamine E du serum des porcelets dont la diete n'en etait pas enrichie diminua de 50% au cours de l'experience; par ailleurs, l'enrichissement de la diete avec cette vitamine en augmenta la quantite dans le serum. On nota une variation considerable du pourcentage de l'hemolyse par le peroxide d'hydrogene. Le rapport entre ce pourcentage et l'ingestion de vitamine E s'1tablissait 'a -0.63; il etait par ailleurs de -0.85 relativement 'a la teneur du serum en vitamine E.

INTRODUCTION Jenkins and Hidiroglou (10) have recently reviewed selenium and vitamin E responsive problems in livestock. A high incidence of vitamin E-selenium related deficiency diseases in pigs fed soybean meal-high moisture corn diets has been reported (19). This experiment was initiated to investigate the influence of supplemental selenium and/or vitamin E in a soybean meal-high moisture corn diet on various blood and tissue parameters in pigs

Can. J. comp. Med.

as well as to evaluate hydrogen peroxide hemolysis as a diagnostic technique for the

deficiency disease.

MATERIALS AND METHODS Eighteen barrows approximately three weeks of age and 7.0 kg initial weight were used in an experiment with a 3 x 3 factorial arrangement. The factors were three levels of supplemental vitamin E (0, 60, 120 ppm) and three levels of supplemental selenium (0.0, 0.60, 1.20 ppm) added to a protein supplement-high moisture corn diet (Table I). Supplemental vitamin E was provided as a-tocopherol acetate and selenium as sodium selenate (41% Se). The barrows were obtained from sows which had been fed a corn-soybean meal diet unsupplemented with either vitamin E or selenium commencing approximately three weeks prior to farrowing and were maintained on the diet until the pigs were weaned. After weaning the pigs were placed in individual stainless steel cages in an air conditioned room and were fed the same diet as fed to their dams for three days in and then offered their experimental diets. The pigs were self-fed their respective diets and water was available in separate trays. The pigs were bled and weighed at weekly intervals for nine weeks with the TABLE I. Protein Supplement

Supplement Soybean meal (49%) ................... 84.0 Salt, cobalt, iodized .2.0 Calcium phosphate (18.5% Ca, 20.5%P) 5.0 Linestone ............................ 4.6 Trace mineral premixb .................. 0.4 Vitamin premixe ....................... 2.0 Corn ........................... 2.0 aComplete diets prepared by mixing the supplement and high moisture corn in a ratio of 1:4 bProvided the following in ppm of the supplement: Mn 240, Fe 280, Cu 40, Zn 400 *Provided the following per kg of supplement: riboflavin 17.6 mg, pantothenic acid 35.2 mg, niacin 79.6 mg, choline chloride 441.2 mg, vitamin B,2 79.6 ag, vitamin A 13, 236 IU, vitamin D2 1, 323.6 IU

Volume 40 - January, 1976

exception of the eighth week when no blood satmples or weights were obtained. The pigs were electrocuted at the termination of the experiment then necropsied. Samples of heart, liver, kidney and semitendenosis muscle tissue were obtained for chemical analyses and heart, liver and semitendenosis muscle were taken for histological examination. Diet vitamin E and all selenium values were determined according to procedures previously described (18). The serum vitamin E values were obtained by the method described by Fabianek et al (4) except the extraction was done using n-hexane instead of o-xylene. The hydrogen peroxide hemolysis tests were peformed according to the method of Gordon, Nitowsky and Cornblath (5). The osmotic fragility tests were performed as described by Davidsohn (2). Serum iron and iron binding capacity were determined by the methods of Ramsay (13,14). The data were analysed by analysis of variance technique (22). Stepwise regression equations and correlation coefficients were calculated for certain parameters.

RESULTS The diets containing the intermediate level of vitamin E assayed lower than anticipated as did some of the diets supplemented with selenium (Table II). All pigs survived and gained approximately 0.32 kg per day during the trial. The rate of gain was not influenced by supplemental selenium or vitamin E (Table III). However, the weekly gain of pigs which did not receive supplemental vitamin E or selenium appeared lower during the last week of the experiment as compared with pigs supplemented with vitamin E or selenium (5.52, 6.98, 6.62 kg). One pig fed the basal diet had focal cellular infiltration in the cardiac muscle. No other gross or microscopic pathological conditions were noted which could be related to the dietary treatments. Tissue selenium concentrations increased with an increase in dietary selenium (Table III). Kidney tissue contained considerably greater concentration of selenium than other tissues.

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The blood serum obtained at the initiation of the experiment contained approximately 0.05 ,ug of selenium per milliliter (Table IV). The level varied slightly in pigs which were not supplemented with selenium and may have decreased slightly toward the end of the experiment. Supplementation with selenium caused a threefold increase in the serum selenium levels during the first week with a slight tendency to further increases in subsequent weeks of the experiment. Supplemental selenium did not influence the level of serum vitamin E. (Table IV). There was a decline in the level of serum vitamin E in pigs which did not receive supplemental vitamin E from an initial level of 1.08 ,ug/ml to 0.56 Jug/ml at the end of the experiment. Supplemental vitamin E resulted in an increase in serum vitamin E. However, the increase was less rapid than that observed for selenium. The percentage of hydrogen peroxide hemolysis varied considerably both within treatment groups and from week to week (Table V). A correlation of -0.63 between the percent peroxide hemolysis and vitamin E intake was observed and a correlation of -0.85 between percent peroxide hemoly-

sis and serum vitamin E (Table VI). Selenium did not influence the hydrogen peroxide hemolysis. No significant changes in erythrocyte osmotic fragility were observed among treatment groups. Correlations among various other parameters were calculated (Table VI). High correlations (0.85 to 0.93) were observed between selenium intake and the selenium level in various tissues. Correlations among various blood parameters obtained from samples of blood taken at weekly intervals were calculated. The magnitude of the correlations for each criteria within each week were similar from week .to week and only the correlations for the blood samples obtained just prior to electrocution are reported. There appeared to be a relationship between serum iron and selenium intake (r = 0.70). Other correlations were of a lower magnitude. Regression equations were calculated relating dietary treatment to tissue selenium concentration (Table VII). Selenium intake was the major factor influencing tissue selenium with the linear term accounting for most of the RS value (7090%). The addition of the quadratic term to the regression equation for kidney sele-

TABLE II. Chemical Analyses of the Experimental Dietsa Diet Number 3 4

8 6 7 5 60 0 120 60 60 120 0 0.0 0.60 1.20 1.20 0.0 0.60 0.60 Chemical Analyses 77.27 77.54 77.53 77.51 76.99 77.08 77.09 76.73 Dry Matter % ............ 0.24 38.00 104.10 0.45 36.00 138.80 0.24 49.70 Vitamin E ppm as fedb 0.01 0.01 0.01 0.45 0.48 0.44 0.91 0.89 Selenium ppm as fede 'Complete diets prepared by mixing the supplement and high moisture corn in a ratio of 1:4 bOne analysis per diet eAverage of five analyses per diet 1

Level of Vitamin E ppm Level of Selenium ppm

.....

2

9

120 1.20

0 0.0

.....

.......

77.09 122.20 0.97

TABLE III. Influence of Supplemental Vitamin E and Selenium on Rate of Gain and Tissue .Selenium Average Daily Gain

Av. Dietary Selenium ppm 0.01 0.46 0.92 Av. Dietary Vitamin E ppm 0.31 42.0

kg

Kidney

0.30 0.34 0.33

6.80b 7.12b

Tissue Selenium PPM Dry Basis Heart Muscle Liver

2.54'

5.11 0.33 5.69 0.33 122. 5.65 0.31 "nmeans within columns with different superscripts are significantly

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0.26&

2.06b

2.87'

0.29' 0.93b 1.29o

0.88 1.57 0.82 1.96 0.80 1.66 different at P = 0.05

0.20' 0.56e

0.47b

0.38 0.40 0.45

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nium increased the R2 value by 15 per- observed until a frank deficiency occurred. All of the pigs survived until the termincentage units and to a lesser extent for the ation of the experiment. In another experiother equations. The linear and quadratic effects of vita- ment conducted at the same time using min E intake on percent hemolysis consti- similar diets and pigs selected from the tuted 40 and 66 percentage units of the R' same farrowing and fed under more pracvalue. Selenium intake contributed a small tical conditions a high incidence of death and lesions attributed to deficiencies of amount of the R' value. Vitamin E intake was the main factor vitamin E-selenium occurred. The main influencing serum vitamin E with the difference between the two experiments linear and quadratic terms contributing appeared to be the environmental condi64 and 20.5 percentage units to the RI tions including temperature variation and floor space per pig. Thus stress and exervalue. cise may hasten the development of vitamin E-selenium deficiencies. The increase in tissue selenium concentrations with increased dietary selenium levels has been reported previously (3, 7, DISCUSSION 16). There appeared to be a slight increase in kidney selenium concentration with increased vitamin E supplementation (Table Others (18, 19) have observed the lack III). This tendency has been reported preof effect of vitamin E or selenium intake viously (3, 6, 7, 17). However, examination on rate of gain as was observed in the of the regression equation for kidney sepresent experiment. The tendency for de- lenium indicates a very minor effect of vicreased rate of gain of the unsupplemented tamin E on kidney selenium. pigs observed during the last week of the The levels of serum vitamin E at the experiment may indicate that the pigs were start of the experiment of 1.08 to 1.28 ,ug/ approaching a deficiency state of either or ml were slightly lower than 1.79 and 1.45 both of these nutrients and that the effect ,ug/g reported for plasma tocopherol in of these nutrients on gain would not be normal pigs (11). However, at the end of TABLEIIV. Influence of Supplemental Vitamin E and Selenium on Serum Vitamin E and Selenium

Period Initial Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week

Av. Dietary Selenium ppm 0.01 0.46 0.92

9j

0.13b 0.17b

Serum Se &g/ml 0.03' 0.05' 0.14b 0.15b 0.16b 0.20' 0.21b 0.24b

0.03a 0.16b

0.16b

0.22.

0.19b 0.24.

0.05 0.12 0.04 0.12 0.06 0.12

0.11 0.10 0.13

0.12 0.12 0.12

0.14 0.13 0.14

0.14 0.13 0.14

0.16 0.14 0.15

1.28 1.34(5)d 1.18 1.78(4)d 1.13 1.47(6)d

1.77 1.80 1.75

2.38 2.58 2.25

2.43 2.50 2.35

2.57 2.58 2.45

0.05 0.04& 0.05 0.14b 0.05 0.18b

0.04'

0.02'

0.23'

0.03'

0.02'

Av Dietary

Vitamin E ppm 0.31 42.00 122.00

Av. Dietary Selenium ppm 0.01 0.46 0.92

0.14 0.12 0.13

0.16 0.15 0.14

Serum vitamin E pg/ml 1.87 2.02 1.93

2.28 2.42 2.25

2.28 2.37 2.20

Av. Dietary Vitamin E ppm 1.08 0.85(4)d 0.88& 0.31 0.88& 0.73a 0.73. 0.68' 0.63' 1.24 1.26(6)d 42.00 1.95b 2.10b 2.83b 2.62b 2.88b 2.88b 1.28 2.35(5)d 2.48. 122.00 2.83' 3.38, 3.50e 3.65. 3.77' '-b.means within columns with different superscripts are significantly different at P = 0.05 'number in brackets indicates the number of samples

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0.56'

3.05b 3.98.

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TABLE V. Influence of Supplemental Vitamin E and Selenium on Percent Red Blood Cell Hemolysis Hemolysis (%) Initial Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 9 Dietary Selenium ppm 0.01 0.46 0.92 Dietary Vitamin E ppm 0.31 42.00 122.00

31.5 25.3 29.3

53.5 53.5 54.5

61.0 45.8 50.2

48.5 43.8 50.9

42.5 54.5 60.0

13.3 25.0 27.7

39.7 47.3 39.7

26.7 36.3 37.8

24.3 30.5 33.2

38.2 26.5 21.5

72.3 50.0 39.2

94.8 38.0 24.2

73.3 30.8 39.1

86.0 43.3 27.7

54.7 8.8 2.5

71.3 33.5 21.8

66.7 17.3 16.8

56.5 15.7 15.8

TABLE VI. Correlations Among Various Parameters Used to Assess Vitamin E-Selenium Status of Pigs

Kidney selenium Liver selenium Heart selenium Muscle selenium Serum vitamin E 9th week % Hemolysis H202 9th week Serum selenium 9th week 9th week Serum iron 9th week TTBC

Vit. E Se Intake Intake 0.86 0.19 0.12 0.93 0.95 0.01 0.25 0.85 0.86 -0.01 0.14 -0.63 0.10 0.91 0.70 0.16 0.54 0.48

TIBC

Serum Iron

0.30

0.17

0.55

0.32

%

Serum Se

Hemolysis

-0.85

-0.08 0.25

TABLE VII. Prediction Equations Relating Dietary Vitamin E and Selenium Intake to Tissue Selenium, Serum Vitamin E and Red Blood Cell Hemolysis R2

Kidney selenium ppm Liver selenium ppm Heart selenium ppm Muscle selenium ppm Serum selenium ppm Hemolysis (%)a

0.3224 Se - 0.00588 Se2 + 0.0000007 (E x Se)2 0.1237 Se - 0.00185 Se2 + 0.0000006 (E x Se)2 0.0412 Se - 0.00043 Se2 - 0.0000301 (E x Se)2 0.0183 Se - 0.00026 Se2

2.394 + 0.1950 + 0.3221 + 0.1872 + 0.0172 + 0.0111 Se - 0.00014 Se2 43.13 - 0.025 E + 0.000004E2 - 0.0217 Se2 - 0.000849 Se x E + 0.0000169E Se2 + 1.39169 Se Serum Vit E p.g/mla = 0.6433 + 0.0015E - 0.000002E2 Serum obtained during the 9th week of the experiment = = = = = =

the experiment the unsupplemented pigs had levels of vitamin E about one-half the initial level, whereas the levels in supplemented pigs were two to three times higher than initial levels. The in vitro hydrogen peroxide hemolysis test has been considered a useful index of tocopherol deficiency in rats (15) and men (12) when vitamin E assays were not readily available for diagnostic purposes. Lack of reproducibility of the test has been noted and several factors influencing peroxide hemolysis have been investigated (1, 8, 9, 20, 23). There was greater within group variation for the pig erythrocytes

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93.25 96.70 94.32 81.39 90.68 81.62 94.50

from week to week than occurred for the normal human- erythrocytes used as a control for the technique. Examination of the data indicated that less experienced technicians contributed to the variability within groups when they were required to assist with the test. Seventy-two percent of the variability in peroxide hemolysis results could be accounted for in the serum vitamin E and peroxide hemolysis correlation (-0.85). Measurement of the secondary formation of malonyldialdehyde is said to be a more specific and sensitive measure of lipid antoxidation (21, 23) and warrants further investigation in swine.

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comp.

Med.

ACKNOWLEDGMENTS This work was supported through financial assistance provided by the Canada Department of Agriculture, (C.D.A. Grant 9091) and the Ontario Ministry of Agriculture and Food. REFERENCES 1. ALFIN-SLATER, R. B., H. HANSEN, R. S. MORRiB and D. MELNICK. Dietary fat composition and tocopherol requirements: L. Lack of correlation between nutritional indices and results of in vitro peroxide hemolysis tests. J. Am. Oil Chemists Soc. 46: 563-567. 1969. 2. DAVIDSOHN, C. Clinical Diagnosis by Laboratory Methods. 13th Ed. Davidson and Wells. Philadelphia, London: Saunders. 1962. 3. EWAN, R. C. Effect of vitamin E and selenium on tissue composition of young pigs. J. Anim. Sci. 32: 883-887. 1971. 4. FABIANEK, J., J. DeFILIPPI, T. RICKARDS and H. HERP. Micromethod for tocopherol determination in blood serum. Clin. Chem. 14: 456462. 1968. S. GORDON, H. H., H. M. NITOWSKY and M. CORNBLATH. Studies of tocopherol deficiency in infants and children. I. Hemolysis of erythrocytes in hydrogen peroxide. Am. J. Dis. Child. 90: 669-681. 1955. 6. GROCE, A. W., E. R. MILLER, J. P. HITCHCOCK, D. E. ULLREY and W. T. MAGEE. Selenium balance in the pig as affected by selenium source and vitamin E. J. Anim. Sci. 37: 942-947. 1978. 7. GROCE, A. W., B. R. MKLLER, D. E. ULLREY,

P. si. KU, K. IL KEAHEY and D. J. ELLIS. Selenium requiremets In corn-o diets fo growx ing-finishing swine. J. Anim. Si. 37: 948-956. 1973. 8. HORWITT, M. K., C. C. HARVEY. G. 0. DUNCAN and W. C. WILSON. Effects of limited tocopherol intake in man with relationships to erythrocyte hemolysis and lipid oxidations. Am. J. Clin. Nutr. 4: 408419. 1966. 9. JACOB, H. S. and S. E. LUXL Degradation of membrane phospholipids and thiols in peroxide hemolysis: studies in vitamin B deficiency. Blood

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32: 549468. 1968. 10. JENKINS, K. J. and IL HEDIROGLOU. A review of selenium/vitamin B responsive problems in livestockl: A case for selenium as a feed additive in Canada. Can. J. Anim. Sci. 52: 591-620. 1972. 11. LINDBERG, PAUL. Plasma tocopherol in pigs. Symposium, vitamin E in Animal Nutrition at Hindsgavl Castle, Denmark. September 8-11, 197) 12. NITOWSKY, H. M, M. CORNBLATH and H. H. GORDON. Studies of tocopherol deficiency in infants and c.hildren. IL Plasma tocopherol and erythrocyte hemolysis in hydrogen peroxide. Am. J. Dis. Child. 92: 164-174. 1956. 13. RAMSAY, W. N. M. The determination of iron in blood plasma or serum. Clinica chim. Acta 2: 214-220. 1957. 14. RAMSAY, W. N. M. The determination of the total iron-binding capacity of serum. Clinica chim. Acta 2: 221-226. 1957. 16. ROSE, C. S. and P. GYORGY. Specificity of hemolytic reaction in vitamin E-deficient erythrocytes. Am. J. Physiol. 168: 414418. 1952. 16. SHARP, B. A., A. A. van DREUMEL and L. G. YOUNG. Vitamin E, selenium and methionine supplementation of dystrophogenic diets for pigs. Can. J. comp. Med. 36: 898-402. 1972. 17. SHARP, B. A., L. G. YOUNG and A. A. van DREUMEL. Vitamin E and selenium responsive diseases in swine: Nutritional aspects. Nutr. Conf. for Feed Manufacturers. Univ. of Guelph. p. 9. 1970. 18. SHARP, B. A., L. G. YOUNG and A. A. van DREUMEL. Dietary induction of mulberry heart disease and hepatosis dietetica in pigs I. Nutritional aspects. Can. J. comp. Med. 36: 871-376. 1972. 19. SHARP, B. A, L. G. YOUNG and A. A. va DREUMEL. Effect of supplemental vitamin B and selenium in high moisture corn diets on the incidence of mulberry heart disease and hepatosis dietetica in pigs. Can. J. comp. Med. 36: 893-897. 1972. 20. SHOHET, S. B. Hemolysis and changes in erythrocyte membrane lipids. New Engl. J. Med. 286: 577483. 1972. 21. SINNHUBBER, R. 0. and T. C. YU. 2-thiobarbituric acid method for the measurement of rancidity In fishery products. II. The quantitative determination of malonaldehyde. Fd Technol. 12: 9-12. 1958. 22. STEEL, R. G. D. and J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw-Hill Book Co. 1960. 23. STOCKS, J. and T. L. DORMANDY. The autoxidation of human red cell lipids Induced by hydrogen peroxide. Br. J. Haemat. 20: 95-111. 1971.

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