EFFECTS OF SELENIFEROUS GRAINS AND INORGANIC SELENIUM ON TISSUE AND BLOOD COMPOSITION AND GROWTH PERFORMANCE OF RATS AND SWINE 1

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1 EFFECTS OF SELENIFEROUS GRAINS AND INORGANIC SELENIUM ON TISSUE AND BLOOD COMPOSITION AND GROWTH PERFORMANCE OF RATS AND SWINE 1 T. B. Goehring 2, I. S. Palmer 3, O. E. Olson 3, G. W. Libal 2 and R. C. Wahlstrom 2 South Dakota State University, Brookings Summary Two experiments were conducted to determine the effect of varying dietary selenium (Se) levels and Se source on growing swine. In Exp. 1, seleniferous wheat and oats were used to formulate diets containing.47, 2.58, 5.60 or 8.40 ~tg/g organic Se. Dietary Se level had no effect on pig performance during the 6-wk experiment as measured by daily gain, daily feed intake or feed/gain. Blood composition and enzyme activity were not affected by dietary treatment. Selenium concentrations of blood, hair, liver, kidney, heart, spleen and diaphragm muscle were increased linearly (P<.01) as dietary. Se increased. In addition, liver weight as a percentage of body weight was increased linearly (P<.01) as dietary Se level increased. No signs of chronic Se poisoning were observed. Dietary treatments in Exp. 2 were similar to Exp. 1 with the exception that sodium selenite was utilized as the Se source and the diets were fed for 17 wk. Inorganic Se levels of.54, 2.63, 5.69 or 8.33 /ag/g had no effect on pig performance as measured by daily gain, daily feed intake or feed/gain. Selenium concentrations of blood, hair, liver, kidney, spleen and diaphragm muscle were significantly increased as dietary Se level increased. Liver weight as a percentage of body weight was increased at the two highest dietary Se levels. Blood glutathione peroxidase activity was significantly increased by dietary treatment, while other blood variables were not affected. 1Published with the approval of the Director of the South Dakota Agr. Exp. Sta. as Pub. No. "1967 of the Journal Series. 2 Dept. of Anita. and Range Sci. 3 Dept. of Chem. Received December 16, Accepted March 14, No signs of chronic Se poisoning were observed. These results suggest that the toxic level of Se for growing pigs fed diets of this type is greater than 8.33/~g/g regardless of the Se source. The data for rats, while generally indicating the same degree of effects as the data for swine, suggest that rats are more susceptible to chronic poisoning by selenium. (Key Words: Selenium Toxicity, Pig Growth, Blood Selenium, Body Composition, Blood Enzymes.) Introduction Selenium is established both as an essential micronutrient and as a natural toxicant for domestic livestock. The Food and Drug Administration has approved sodium selenite and sodium selenate as supplements to pig starter diets up to a level of.3 ~tg/g of Se (U.S. Dept. Health and Human Services, 1982). Consequently, the potential exists for Se poisoning to occur through accidental contamination, incorrect mixing or incorrect formulation of the diet. Reports of Se poisoning in swine are limited and not well documented. Selenium poisoning has been experimentally produced (Miller and Schoening, 1938; Wahlstrom et al., 1955, 1956; Wahlstrom and Olson, 1959; Moxon and Mahan, 1981) but seldom was the experimental objective to establish the maximum level of dietary Se that could be tolerated without affecting pig performance. The level at which Se becomes toxic to swine is thought to be about 4 to 5 /ag/g, depending on diet composition and other factors (NRC, 1983). This value was derived primarily from early Se research with considerable extrapolation of data from other species and needs verification if accidental cases of poisoning are to be properly diagnosed. This research was conducted to determine the effect of dietary Se level and source on pig 725 JOURNAL OF ANIMAL SCIENCE, Vol. 59, No. 3, 1984

2 726 GOEHRING ET AL. TABLE 1. COMPOSITION OF DIETS FOR EXPERIMENT 1 Item Diet: Selenium, #g/g Calculated: Analyzed: Seleniferous wheat, 20 ~tg Se/g Wheat (IFN ) Seliniferous oats, 7 #g Se/g Oats (IFN ) Corn (IFN ) Soybean meal (IFN ) Dicalcium phosphate (IFN ) Calcium carbonate (IFN ) Trace mineralized salt a Vitamin-antlbiotic premix b Crude protein by analysis, %c % asupplied the following per kg of diet: Zn, 30 mg; Mn, 30 mg; Fe, 24 mg; S, 6.6 mg; Cu, 2.4 mg; I,.6 mg and Co, 75 ~g. bsupplied the following per kg of diet: vitamin A, 4,000 IU; vitamin D, 400 IU; vitamin E, 8.8 IU; vitamin K, 3.5 mg; riboflavin, 4.4 mg; pantothenic acid, 17.6 rag; niacin, 28.2 mg; choline, 176 mg; vitamin B12, 17.6 ~g; selenium,.158 mg; sulfamethazine, 110 mg; chlortetracycline, 110 mg and penicillin, 55 mg. CAs-fed basis. performance, blood composition, tissue Se concentration, blood enzyme activity and liver weight as a percentage of body weight. In addition, the diets were fed to rats to determine whether effects of Se were similar for the two species. Materials and Methods Pig Studies. Two experiments were conducted utilizing 24 crossbred pigs of predominantly Yorkshire, Hampshire and Duroc breeding in each experiment. Six pigs were assigned randomly to each treatment from outcome groups based on ancestry, sex and weight in Exp. 1 and on ancestry and weight in Exp. 2. Average initial weight of pigs was 8.0 and 8.4 kg in Exp. 1 and 2, respectively. Pigs were maintained in individual pens in an environmentally controlled nursery having plastic or plastic-coated expanded metal floors for the 6-wk duration of Exp. 1 and for the initial 6 wk of Exp. 2. From 6 to 17 wk, pigs in Exp. 2 were housed in an enclosed building with concrete-floored pens bedded with wood shavings. Feed and water were provided ad 4Sasco, Inc., Omaha, NE. libitum. Weight gains and feed intakes were measured weekly. The experimental diets used in Exp. 1 (table 1) were formulated to contain 0, 3, 6 or 9/~g/g of naturally occurring Se. Wheat and oats were replaced with seleniferous wheat and oats to obtain the desired dietary Se levels. The seleniferous grains were normal varieties grown in South Dakota on soils naturally high in selenium. The basal diet in Exp. 2 was the same as the nonseleniferous control diet in Exp. 1 (diet 1) except chlortetracycline was used as the antibiotic. Sodium sele-nite in a ball milled premix with a soybean meal carrier was added at the expense of soybean meal to provide Se levels similar to those in diets 2, 3 and 4 of Exp. 1. Selenium contents by analysis for diets 1 to 4, respectively, were.47, 2.58, 5.60 and 8.40/ag/g for Exp. 1 compared with.54, 2.63, 5.69 and 8.33/ag/g for Exp. 2. Rat Studies. Two 4-wk experiments were conducted with white, male, Sprague-Dawley rats 4 averaging about 60 g and fed the same diets used in the two swine experiments. Seven rats assigned randomly to each diet were housed in individual stainless steel cages at 25 to 28 C with a light period of 12 h/d. They were allowed feed and deionized water ad libitum.

3 EFFECTS OF SELENIUM SOURCE ON RATS AND SWINE 727 Blood, Hair and Tissue Analyses. Blood samples were obtained at the termination of each pig and rat experiment and also at 6 wk in the second pig experiment. Pigs were bled from the anterior vena cava into heparinized tubes and rats were anesthetized with diethyl ether and bled by heart puncture into heparinized tubes. Packed cell volume (PCV) was determined with a microhematocrit centrifuge and expressed as percentage volume of packed red cells in whole blood. Total hemoglobin concentration was determined colorimetrically (Sigma Chemical Co., 1980b) and total plasma bilirubin was determined by the method of Sigma Chemical Co. (1981). Selenium was determined fluorometrically (Olson et al., 1975). Blood plasma glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activities were determined by the method of Sigma Chemical Co. (1980a) and expressed as Sigma-Frankel (S-F) units/ml, The indirect method of Lawrence and Burk (1976) was used to determine plasma and cellular glutathione peroxidase (GSH-Px). The GSH-Px values are expressed as enzyme units (EU)/mg protein and EU/mg hemoglobin, with EU representing nmol NADPH oxidized/rain. Hair samples were obtained from all pigs 1 d before slaughter. At the termination of each experiment, all pigs and rats were killed. Heart, liver, kidney and spleen were removed and weighed. Samples of each organ and of diaphragm muscle in pigs and leg muscle in rats were removed and frozen for Se analysis. Rat livers were scored on a scale of 0 (no liver damage) to 5 (severe necrosis). Statistical Analysis. Each experiment was analyzed as a randomized, complete block design using a least-squares procedure (SAS, 1979). Single degree of freedom orthogonal polynomial regression analysis was used to TABLE 2. EFFECT OF NATURALLY OCCURRING SELENIUM IN DIETS ON PERFORMANCE, SELENIUM IN TISSUES AND BLOOD COMPOSITION OF SWINE FED FOR 6 WEEKS (EXP. 1) a Item Avg daily gain, kg Avg daily feed, kg Feed/gain Liver wt, % body wt b Liver, ~g/g wet tissueb Kidney, ~g/g wet tissue b Heart, #g/g wet tissue b Spleen,/~g/g wet tissue b Muscle, tzg/g wet tissue b Hair, #g/g air-dry hair b Selenium, ~g/ml b Packed cell volume, % Hemoglobin, g/100 ml GSH-Px, EU/mg protein (cells) GSH-Px, EU/mg hemoglobin (cells) GSH-Px, EU/mg protein (plasma) GOT, S--F units/ml (plasma) GPT, S-F units/ml (plasma) Diet and selenium content by analysis,/~g/g as fed SE Performance Tissue selenium Blood composition asix individually fed pigs per treatment, avg initial wt, 8.0 kg. blinear effect (P<.01).

4 728 GOEHRING ET AL. examine the linear, quadratic and cubic effects of dietary Se treatment. The Waller-Duncan test (Steel and Torrie, 1960) was used to identify selenium levels at which the variables might suggest toxicosis. Results Exp. 1. The effects of naturally occurring dietary Se at various concentrations are presented in table 2. No significant differences in daily gain, daily feed intake or feed efficiency were observed among treatments. However, pigs fed the highest Se level had the lowest daily gain and feed intake. This response suggests that the Se concentration in this diet approached a toxic level. Liver weight as a percentage of body weight increased linearly (P<.01) with increasing Se level (table 2). Selenium concentrations in liver, kidney, heart, spleen, diaphragm muscle and hair all increased linearly (P<.01) with increasing level of dietary Se. Kidney Se concentration was similar to that of liver for pigs fed diets 2, 3 or 4. Selenium levels of the blood increased (P<.01) with increasing dietary Se level. Increasing dietary levels of Se had no effect on PCV. The level of cellular or plasma GSH-Px was not significantly influenced by dietary Se level. In addition, dietary Se level had no effect on plasma GOT or plasma GPT activity. TABLE 3. EFFECT OF CONCENTRATION OF SELENIUM AS SELENITE IN DIETS ON PERFORMANCE, SELENIUM IN TISSUES AND BLOOD COMPOSITION OF SWINE FED FOR 17 WEEKS (EXP. 2) a Diet and selenium content by analysis, ~g/g as fed Item SE Avg daily gain, kg Avg daily feed, kg Feed/gain Liver wt, % body wt b Liver, ~zg/g wet tissue cd Kidney, #g/g wet tissuec Heart, ~g/g wet tissue c Spleen, ~g/g wet tissue ce Muscle, ~g/g wet tissue c Hair,/ag/g air-dry hair c Selenium, ~g/ml c Packed cell volume, % Hemoglobin, g/100 ml GSH-Px, EU/mg protein (cells) d GSH-Px, EU/mg hemoglobin (ceils) d GSH-Px, EU/mg protein (plasma)fg GOT, S-F units/ml (plasma) GPT, S~F units/ml (plasma) Performance Tissue selenium Blood composition asix individually fed pigs per treatment, avg initial wt, 8.4 kg. bcubic effect (P<.05). CLinear effect (P<.01). dquadratic effect (P<.01). ecubic effect (P<.01). fquadratic effect (P<.05). glinear effect (P<.05).

5 EFFECTS OF SELENIUM SOURCE ON RATS AND SWINE 729 Exp. 2. Pig performa, nce did not differ (P>.05) when the basal diet was supplemented with sodium selenite (table 3). Liver weight as a percentage of body weight was affected cubically (P<.05) by dietary treatment, increasing at the two higher Se concentrations. Kidney, heart, diaphragm muscle and hair Se concentrations each increased linearly (P<.01) as dietary Se level increased. Selenium concentration in spleen and liver increased cubically (P<.01) and quadratically (P<.01), respectively, as supplemental Se level increased. Liver Se concentrations were again similar to those of kidney at all levels of Se supplementation. Increasing levels of supplemental Se resulted in a linear increase (P<.01) in blood Se concentration. Packed cell volume and hemoglobin levels of the blood were not affected by dietary treatments. Cellular and plasma GSH-Px levels increased quadratically (P<.05) as the level of supplemental Se increased. The lowest levels of sodium selenite supplementation (diets 2 and 3) produced the maximum increase in both cellular and plasma GSH-Px. A slight reversal of this trend occurred with the feeding of the highest level (diet 4) of sodium selenite. Dietary treatment had no influence on GOT and GPT activities. Rat Studies. Data obtained from feeding rats the swine diets containing various levels of Se from seleniferous grains or sodium selenite are presented in table 4. There were significant linear, quadratic or cubic responses to dietary Se for all variables measured except for blood GOT activity from rats fed sodium selenite and for GPT activity of rats fed seleniferous grains as the Se source. Daily gains, PCV and hemoglobin levels of rats fed diets 3 and 4 containing selenite were less (P<.01) than those of rats fed diet 1. Similar differences were observed for rats fed seleniferous grain except for PCV. The data from these three variables indicate that the two higher dietary levels of Se were toxic to rats. Because the two rat studies were not conducted simultaneoulsy, the degree of toxicity of the two selenium sources was not compared. Discussion The pig performance data indicate that the level of dietary inorganic or organic Se required to produce a reduction in growth rate and feed intake is greater than 8.4/~g/g when pigs are fed diets containing 35% wheat, 20% oats, 17% corn and 25% soybean meal. In contrast Wahlstrom et al. (1955) and Wahlstrom and Olson (1959) reported a reduction in growth rate and feed intake in pigs fed a 15% protein cornsoybean meal diet supplemented with sodium selenite at levels of 7 or 10 /ag/g Se. Later research (Moxon and Mahan, 1981) found sodium selenite supplementation of a 20% protein corn-soybean meal diet at a level of 5 /~g/g Se caused a reduction in growth rate and feed intake when fed to weanling swine. In view of the observed greater toxicity of selenium for rats on corn-based diets as compared with wheat-based diets (Palmer et al., 1983), the lesser toxicity of selenium in the pig experiments reported herein may have been because of the inclusion of wheat in the diets. Organ and tissue Se concentrations were found to be directly proportional to the dietary Se concentration. This finding is in agreement with results of studies in which pigs were fed adequate but not excessive Se (Ewan, 1971; Ku et al., 1972, 1973 ; Young et al., 1976; Meyer et al., 1981). In comparisons across experiments, levels of Se in organs and tissues of pigs fed the control diet for 17 wk (Exp. 2) were similar to levels found in those pigs that received this diet for only 6 wk (Exp. 1). However, the Se content of organs and tissues of pigs fed diets supplemented with sodium selenite was considerably less than that of pigs fed similar levels of Se derived from seleniferous grains. Similar results have been reported by Jenkins and Winter (1973) and Ku et al. (1973). A high percentage of the naturally occurring Se in many plants is in the form of selenomethionine (Allaway et al., 1967). Chavez (1979b) theorized that Se in the form of Se-methionine can be incorporated nonspecifically and perhaps undistinguishably from the sulfur-amino acids into tissue proteins. Chavez also stated that when ingested in an inorganic form the pathway of Se into tissue proteins is more under control mechanisms than when organic forms, readily incorporated into protein, are ingested. Thus, the difference in Se levels of organs and tissues between pigs fed inorganic or organic Se may be accounted for by the difference in metabolism of the two forms of Se. In both experiments, liver and kidney were found to have higher Se concentrations than heart, spleen and diaphragm muscle at each.dietary Se level. This observation has previously been made with pigs fed excess Se (Dudley,

6 730 GOEHRING ET AL. TABLE 4. EFFECT OF SELENIUM IN SWINE DIETS FED TO RATS FOR 4 WEEKS a Diet number and/~g Se/g Se Grain: Variable measured source Selenite: SE Avg daily gain, g Grain b Selenite bc Liver wt, % body wt Grain b Selenite c Liver scoresg Grain bc Selenite b Liver Se,/ag/g wet tissue Grain b Selenite b Kidney Se, ~tg/g wet tissue Grain b Selenite bc Heart Se, gg/g wet tissue Grain b Selenite b Spleen Se, ~tg/g wet tissue Grain bc Seleniteb Muscle Se, #g/g wet tissue Grain b Selenite b Blood Se, gg/ml Grain bde Selenite b Packed cell volume, % Grainbd Selenite b Hemoglobin, g/100 ml Grain b Seleniteb GSH-Px, EU/mg protein (ceils) Grain bee Seleniteb GSH-Px, EU/mg hemoglobin (cells) Grain bce Selenite b GSH-Px, EU/mg protein (plasma) Grain f Selenite bd GOT, S-F units/ml (plasma) Grainb Selenite GPT, S--F units/ml (plasma) Grain Selenite df aseven rats per treatment. Avg initial wt, 61.4 and 60.0 selenite, respectively. blinear effect (P<.01). CQuadratic effect (P<.01). dquadratic effect (P<.05). ecubic effect (P<.05). flinear effect (P<.05). go = no damage; 5 = severe necrosis. g for rats fed diets containing selenized grain or 1935) and adequate levels of Se (Ku et al., 1973; Young et al., 1977; Meyer et al., 1981). Regardless of the Se source or dietary level, it is apparent that liver and kidney are major organs involved in accumulation and storage of Se. Pigs fed 8.40 ~tg/g of naturally occurring Se were found to have about 7 ~tg/g Se in liver and kidney. Pigs fed a similar level of inorganic Se were found to have less than 4 ~tg/g Se in liver and kidney. Herigstad (1972) found, in general, pigs with acute selenosis had greater concentrations of Se in liver than in kidney and those not developing selenosis had greater concentrations of Se in kidney than in liver. In the experiments reported herein, liver and kidney Se concentrations were similar at all dietary Se levels above 3/ag/g. The concentration of Se in these organs was considerably less than

7 EFFECTS OF SELENIUM SOURCE ON RATS AND SWINE 731 the 20 #g/g in liver or kidney that identified acute selenium poisoning in pigs (Herigstad, 1972). The accumulation of appreciable amounts of Se in the hair of pigs in these experiments is consistent with observations of Wahlstrom et al. (1984) who reported hair Se levels of 3.7 to 12.2 #g/g in pigs fed diets containing 8/ag/g of Se. Also, they reported hair Se level was lowest in red hair and highest in black hair. Wahlstrom et al. (1955, 1956) reported that the liver becomes necrotic and degenerates due to chronic selenosis. Liver weight as a percentage of body weight increased with increasing dietary Se level in the experiments presented herein. Obermeyer et al. (1971) made a similar observation in rats fed excess trimethylselenonium chloride. This enlargement may be an indication of an attempt by the animal to adapt to a foreign compound and may not necessarily be a sign of liver damage due to selenosis. Blood Se has been shown to be a sensitive indicator of Se intake when feeding swine diets low or adequate in Se content (Young et al., 1976; Chavez, 1979a,b; Meyer et al., 1981). Data reported here indicate blood Se to be equally sensitive in response to excessive dietary Se intake. Pigs fed approximately 6 or 9 #gig se were found to have blood Se concentrations over 2 #g/g. This finding is consistent with results reported by Moxon and Mahan (1981), who reported blood Se to be over 2 /ag/g when pigs were fed diets containing 10/~g/g Se. Diagnostic indicators of anemia include low PCV (hematocrit), low hemoglobin level or increased serum bilirubin. A decrease in hemoglobin level due to chronic selenosis has been observed in rats (Franke and Potter, 1934; Halverson et al., 1966) and dogs (Moxon, 1937). Serum bilirubin can be used in differential diagnosis of hemolytic anemia and liver dysfunction in domestic livestock (Smith et al., 1972). Hatverson et al. (1966) found serum bilirubin increased in anemic rats suffering from chronic organic selenosis. Packed cell volume and hemoglobin levels of pigs in these experiments were within normal ranges for these hematological variables (Kaneko, 1973). Plasma bilirubin levels were determined but were too low for accurate analysis. In these experiments, GSH-Px activity was not a sensitive index of excessive dietary Se intake. This finding is in contrast to results of studies where adequate but not excess Se was fed (Sivertsen et al., 1977; Chavez, 1979a,b). Blood glutathione peroxidase activity was not influenced by dietary Se level when pigs were fed diets containing naturally occurring Se (Exp. 1). However, the enzyme increased quadratically (P<.05) as supplemental sodium selenite increased in Exp. 2. The difference in GSH-Px response when pigs were fed diets containing inorganic or organic Se may be explained by the difference in bioavailability of the two forms of Se. Cantor and Scott (1974) found the biological availability of selenomethionine to be only 37% of that of sodium selenite for prevention of exudative diathesis in chicks. These authors also observed sodium selenite produced higher GSH-Px levels than seleno-dl-methionine at the same dietary Se level. Plasma GOT and GPT levels found in both experiments approximate normal levels for these enzymes (Kaneko, 1973). In the trials reported here, rats fed the swine diet without added Se gained similarly to rats that were fed wheat or corn-based diets without added Se in experiments of Palmer et al. (1983). Daily gains of rats were reduced by the two higher levels of Se (diets 3 and 4) in contrast to the response of pigs fed these diets. Palmer et al. (1983) reported reduced gains in rats fed Se levels of 8/~g/g from corn, wheat or selenite. Weight gains were similar to those reported herein for rats fed a similar level of Se. In general, blood and tissue data for rats were similar to those for swine. As with swine, Se contents of tissues were higher for rats fed seleniferous grains. Liver and kidney had the highest concentration of Se of all tissues in both species. In contrast to the pig data, Se concentrations were higher in rat kidney than in liver at all levels of Se. The liver scores indicated slight liver damage for rats fed diets 3 and 4. However, liver weights as percentage of body weight do not rerlect the degree of visible liver damage. The data indicate that a lower level of Se will cause chronic selenosis in rats compared with pigs. However, this difference might be the result of differences in biological age rather than species. Literature Cited Allaway, W. H., E. E. Cary and C. F. Ehlig The cycling of low levels of selenium in soils, plants and animals. In: O. H. Muth (Ed.) Symposium: Selenium in Biomedicine. pp The AVI Publishing Co., Inc., Westport, CT. Cantor, A. H. and M. L. Scott Biological activity of selenium compounds in chickens.

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Moxon Value of dietary selenium and vitamin E for weanling swine as measured by performance and tissue selenium and glutathione peroxidase activities. J. Anim. Sci. 52 : 302. Miller, W. T. and H. W. Schoening Toxicity of selenium fed to swine in the form of sodium selenite. J. Agr. Res. 56:831. Moxon, A. L The effects of diets containing selenium in the form of seleniferous grains and inorganic salts on the blood picture of the dog and the selenium content of the tissues and organs of these animals. M.S. Thesis. South Dakota State College, Brookings. Moxon, A. L. and D. C. Mahan Toxic dietary selenium levels for weaning swine. In: J. M. Howell, J. M. Gawthorne and C. L. White (Ed.) Trace Element Metabolism in Man and Animals. pp Springer-Verlag, New York. NRC Selenium in Nutrition (Revised). National Academy of Sciences-National Research Council, Washington, DC. pp Obermeyer, B. D., I. S. Palmer, O. E. Olson and A. W. 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