Journal of Agricultural Science in Finland Maataloustieteellinen Aikakauskirja Vol 63:1—74 METABOLISM OF SELENITE, SELENOMETHIONINE AND FEED-INCORPORATED SELENIUM IN LACTATING GOATS AND DAIRY COWS Selostus: Seleeniaineenvaihdunta maitoa tuottavalla vuohella ja lehmällä PENTTI ASPILA Department of Animal Husbandry, University of Helsinki SF-00710 Helsinki, Finland Academic Dissertation to be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium xii, Unioninkatu 34 Helsinki, on May nth 1991, at io O'clock. SUOMEN MAATALOUSTIETEELLINEN SEURA • HELSINKI https://www.c-info.fi/en/info/?token=QTL3-2z7oIwWZtTD.DKTUerQuV4QlEmrF1ZaMiQ.0TvFDyChWexichU-wXUq5sz6SpZBTVnCn9YwKexDsh4pZ1jVSJxKLXWXXszXwmT7qxMXO9E_lFHg9TpOyyw1HY-YoYLRQ2TPrld2QL9_2crjrkG5wFbeInahr_6IfWyGtcQzva8rHKoPiWUrR4KncwyYADlwvp2U3PUc 3 ACKNOWLEDGEMENTS The present study was carried out in the Department of Animal Husbandry, University of Helsinki in collaborationwith the Department of Food Chemistry and Technology and the Instrument Center of the Faculty of Agriculture and Forestry, University of Helsinki. I want to express my sincere gratitude to prof. Liisa Syrjälä-Qvist, who proposed the subject for my dissertation and has supported it in all stages. I also want to express my warm gratitude to prof. Esko Poutiainen for his en- couragement to continue my studies after graduation. My special gratitude I express to Antti Uusi-Rauva for his cooperation and ad- vice in conducting experiments with radioisotopes. I am very grateful to Dr. Marja Mutanen and Dr. Ruth Blauwiekel, the referees of this work, who have given invaluable advice and criticism in finalizing this thesis. My sincere thanks as well to my colleagues especially to Mikko Tuori, associate prof. Matti Näsi and Dr. Pekka Huhtanen for discussions and interest during the work. I also want express my gratitude to prof. Pekka Koivistoinen for his interest and support in the study. I am indebted to Jari Lehto and Jukka Loimaranta for carrying out selenium analysis, to Dr. Satu Sankari for blood analysis and to Dr. Vieno Piironen for tocopherol analysis. I am indebted to Raija Toropainen, Jukka Niemi and Timo Laitinen for their technical assistance in carrying out the experiments. I also want to express my best thanks to Seppo Karttunen, Jorma Tossavainen and technical staff in Suitia experimental farm for their cooperation in carrying out the experiment on cows. This study was initially supported by Foundation for Promotion of Food Pro- duction. Funds were also awarded by Ministry of Agriculture and Forestry, The Tiura Foundation, The Finnish Cultural Foundation, Finnish Academy, University of Hel- sinki, Kemira ltd. and Valio ltd. 5 Metabolism of selenite, selenomethionine and feed-incorporated selenium in lactating goats and dairy cows Contents ACKNOWLEDGEMENTS 3 LIST OF ABBREVIATIONS 7 ABSTRACT 9 1. INTRODUCTION 11 2. REVIEW OF THE LITERATURE 12 2.1. Selenium metabolism in the rumen 12 2.2. Absorption of selenium 13 2.2.1. Site of selenium absorption 13 2.2.2. Mechanism of selenium absorption 14 2.2.3. Factors affecting selenium absorption 14 2.2.4. Resecretion of Se into intestine 15 2.3. Metabolism and chemical forms of selenium in tissues 15 2.3.1. Selenium in erythrocytes and plasma 16 2.3.2. Selenium metabolism in the liver 18 2.3.2.1. Enzymatic synthesis of selenocysteine in liver 18 2.3.2.2. Selenoprotein synthesis in liver 18 2.3.2.3. Synthesis of methylated selenocompounds 19 2.3.3. Selenium metabolism in kidney 19 2.3.4. Selenium metabolism in the mammary gland 21 3. OBJECTIVES OF THE STUDY 23 4. MATERIALS AND METHODS 24 4.1. Experiment 1 (goats fed Se depleted and supplemented diets) 24 4.1.1. Experimental design 24 4.1.2. Feeds, feeding and milking 24 4.1.3. Preparation of labeled doses 25 4.1.4. Introduction of doses 25 4.1.5. Sampling 25 4.1.6. Laboratory analyses 25 4.1.7. Calculations and statistical analyses 26 4.2. Experiment 2 (goats fed diets supplemented with sodium selenite or selenited barley) 27 4.2.1. Experimental design 27 4.2.2. Feeds, feeding and milking 27 4.2.3. Preparation of labeled doses 28 4.2.4. Introduction of doses 28 4.2.5. Sampling 28 4.2.6. Laboratory analyses, calculations and statistical analyses 28 6 4.3. Experiment 3 (dairy cows fed diets supplemented with sodium selenite or selenited silage) 29 4.3.1. Experimental design 29 4.3.2. Preparation of selenited feeds 29 4.3.3. Feeding, dietary composition and production 29 4.3.4. Sampling 31 4.3.5. Analytical methods 31 4.3.6. Calculations and statistical analyses 31 S.RESULTS 33 5.1. Experiment 1 33 5.1.1. 75Se given orally in selenited grass 33 5.1.1.1. Excretion of "Se 33 5.1.1.2. Rate constants 35 5.1.1.3. 75Se in plasma and erythrocytes 35 5.1.2. 75Se given intraruminally as sodium selenite 35 5.1.2.1. Excretion of "Se 35 5.1.2.2. Rate constants 38 5.1.2.3. 75 Se in plasma and erythrocytes 38 5.1.3. 75 Se given intravenously as selenomethionine 38 5.1.3.1. Excretion of 75 Se 38 5.1.3.2. Rate constants 41 5.1.3.3. 7! Se in plasma and erythrocytes 41 5.2. Experiment 2 41 5.2.1. 7!Se given orally and intraruminally 41 5.1.2.1. Excretion of 75 Se 41 5.1.2.2. Rate constants 42 5.1.2.3. 75 Se in plasma, erythrocytes and hair 44 5.2.2. 75Se given intravenously as sodium selenite and selenomethionine 44 5.2.2.1. Excretion of "Se 44 5.2.2.2. Rate constants 45 5.2.2.3. 75Se in plasma, erythrocytes and hair 45 5.3. Experiment 3 48 5.3.1. Blood parameters 48 5.3.2. Plasma selenium 48 5.3.3. Erythrocyte selenium 49 5.3.4. Erythrocyte and plasma GSH-Px 50 5.3.5. Milk selenium 51 6. DISCUSSION 52 6.1. Absorption of selenium 53 6.2. Selenium in blood and hair 54 6.3. Excretion of selenium in urine 55 6.4. Excretion of selenium in milk 57 7. GENERAL CONCLUSIONS 60 8. REFERENCES 61 SELOSTUS 69 APPENDIX 70 7 List of abbreviations AP alkaline phosphatase ASAT aspartate amino transferase Bq Bequerelle, disintegration per second CK creatine kinase cpm counts per minute DM dry matter FSe selenium in other than mineral feeds (energy and protein feeds) y-GT Y-glu'amyl transferase GSH reduced glutathione GSH-Px glutathione peroxidase GSSeSG selenodiglutathione k rate constant kat katal, mmol of substrate oxidized per second I.R. intraruminal I.V. intravenous 10 inorganic selenium OR organic selenium MSe selenium in mineral feeds TMRT total mean retention time TMSe trimethylselenonium ion TT transit time TV, half-life JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen A ikakauskirja Vol. 63: 9—74, 1991 Metabolism of selenite, selenomethionine and feed-incorporated selenium in lactating goats and dairy cows PENTTI ASPILA Department of Animal Husbandry, University of Helsinki SF-00710 Helsinki, Finland Abstract. The objective of this study was to investigate the metabolism of inorganic and organic Se sources at different dietary Se levels in lactating goats and dairy cows. The study consists of two experiments on goats dosed singly, either orally with grass sprayed with Na 2 75 5e0 3 one week before cutting, intraruminally (1.R.) with Na 275 5e0 3 , or intravenously (1.V.) with Na2 7! Se0 3 or 75 Se-selenomethionine. Follow-up periods were from 15 to 28 d long. Dietary Se levels were 0.05, 0.22 and 0.34 mg/kg DM. Values for 75 Se absorption, excretion in milk, urine and faeces, 75 Se activity in plasma, erythrocytes and hair are presented. In an- other experiment lasting 539 d, 48 dairy cows were fed either Na 2 Se03 or grass silage sprayed with Na 2Se0 3 one week before cutting. Dietary Se levels were from 0.03 to 1.8 mg/kg DM. Se content in milk, plasma and erythrocytes, and GSH-Px activity in erythrocytes and plasma are given. True absorption of 75 Se was 63 % and 65 °7o, and excretion of 75 Se in milk 4 % and 7 % in the goats dosed I.R. with Na 2 75 5e0 3 and orally with 7 'Se-labeled grass. The effect of dietary Se content was non-significant. After I.V. dose, 3.6 % and 33 %of 75 Se was excreted in milk in goats dosed with Na 2 7! Se03 and 75Se-selenomethionine, respectively, Na 2 75 5e03 being eliminated mainly via urine. In cows receiving selenium as Na 2 Se0 3, milk contained 0.011, 0.011, 0.016 and 0.020 mg Se/1 at dietary Se levels 0.11, 0.17, 0.42 and 0.68 mg/kg DM, respectively. In cows receiving Se-sprayed silage, milk Se content was 0.023, 0.020, 0.029 and 0.040 mg/1 when the diet con- tained 0.09, 0.20, 0.45 and 1.20 mg Se/kg DM. Se incorporated into silage was more efficient (p<0.001) in raising milk Se content than selenite. When the animals were fed Se depleted diets, milk Se content of the cows supplemented with selenite decreased more rapidly (p< 0.001) than that of the cows supplemented with selenited silage. Index words: Selenium, feed selenium, selenite, selenomethionine, metabolism, glutathione peroxidase, milk, blood hair, dairy cow, goat 9 1. INTRODUCTION The selenium content of feed and food grown in Finland has been well below the ac- cepted dietary requirements (Sippola 1979, Varo & Koivistoinen 1981). There has also been evidence linking dietary selenium content to some diseases (e.g. cardiovascular diseases, Salonen et al. 1982). These assumptions led in the early 1980's to the need to investigate possibilities to increase dietary selenium intake in the Finnish population. Even though since 1969 commercial mineral mixtures had been fortified with inorganic selenium this had not had any distinct impact on selenium content in milk or meat. There was also evidence from previous studies that organic forms of selenium would be trans- ferred into milk and retained in tissues more efficiently than inorganic Se (e.g. Jacobsen et al. 1965, Conrad and Moxon 1979, Maus et al. 1980). Thus fortifying selenium in fer- tilizers was anticipated to be the best means of supplying the Finnish population and also domestic animals with adequate selenium. In- vestigations of plant uptake of various sele- no compounds were succesfully carried out by Dr. Toivo Yläranta. Based on his results, it was concluded that sodium selenate would be the most suitable form of selenium to be added to fertilizers given prevailing conditions in Finland (Yläranta 1984a). This research programme was established to investigate appropriate feed selenium con- tent for achieving suitable selenium content in milk and meat products. Prof. Liisa Syrjälä- Qvist was the chairman of the group and Mr. Pentti Aspila was requested to work as researcher. The programme was implemented in 1982 and its objective was to develop recommendations for raising the selenium content in cow milk and in beef to an ap- propriate level. The chemical form of Se em- ployed in this study was sodium selenite, a reduced product of sodium selenate, because it is the best documented seleno compound and in foliar applications it has been similar to sodium selenate (Yläranta 1984b). In this doctoral thesis the results from two experi- ments on lactating goats and the results from one experiment on lactating dairy cows are presented. Since 1984 Finnish fertilizers have been sup- plemented with sodium selenate, which has in- creased the selenium content of feeds from the level of 0.02 mg/kg DM to the level of 0.2 mg/kg DM. As a result the Se content of ani- mal products has also increased and at the present, the intake of Se from the average Finnish diet meets standard recommenda- tions. Meat products contribute 40 °/o and milk products 20 % of the total selenium in- take (Ekholm et al. 1991). 11 2. REVIEW OF THE LITERATURE 2.1. Selenium metabolism in the rumen Investigations on the chemical nature of seleno-compounds of plant origin have dem- onstrated the presence of several seleno com- pounds, e.g. selenomethionine, selenocys- teine, selenite, selenocysteic acid, selenocys- tathione, Se-methylselenomethionine, Se- methylselenocysteine and others. (Peterson and Butler 1962, Jenkins and Hidiroglou 1967, Shrift 1969, Olson et al. 1970, Nigam and McConnell 1976, Burk 1976, Ulrey 1981, Gissel-Nielsen 1987). Of these the main selenium compounds ingested by rumi- nants are isologs of methionine and cystine. In soya proteins selenium is probably in the form of selenocysteine (Mason and Weaver 1988). About 70 % of selenium is protein- bound in lucerne (Peterson and Spedding 1963, Jones and Godwin 1963) and about 60 % in brome grass (Jenkins and Hidiro- glou 1967). Gissel-Nielsen (1976) suggested that normally 80 °/o of selenite-Se given to plants is incorporated into either protein or free amino acids. Fortifying sulphur and nitrogen might have some influence on the dis- tribution and chemical form of selenium in plants (Gissel-Nielsen 1982). In the rumen, plant proteins undergo exten- sive bacterial proteolysis resulting in the liber- ation of a considerable proportion of the ami- no acids and reutilization for microbial pro- tein synthesis. Compared to sulphur, selenium has been shown to be incorporated in vitro more rapidly into bacterial protein; the incor- poration of selenite is faster than that for selenate or selenomethionine(Paulson et al. 1968). A small amount of inorganic selenium could also be incorporated into seleno-amino acids by rumen microbes. The compounds identified are mainly selenomethionine, sele- nomethionine selenoxide, selenocysteine and compounds resembling taurine and homocys- teine (Hidiroglou et al. 1968, Hudman and Glenn 1984). Selenite and selenate, however, were not incorporated into selenomethionine even though under the same conditions sul- phate-sulphur was utilized in methionine syn- thesis with subsequent incorporation into microbial proteins (Paulson et al. 1968). At low sulphur intake (Pope et al. 1979) or at high selenium intake (Rosenfeld 1962) incor- poration of inorganic selenium into microbial protein tends to increase. Incorporation of selenite is inhibited by sulphite and nitrite (Hudman and Glenn 1984). Some selenomethionine can be metabolized by the rumen bacteria to form selenocysteine in a such way that both compounds are incor- porated into bacterial protein (Whanger et al. 1967, Hidiroglou et al. 1974). This con- version, however, has not been found in all studies (Paulson et al. 1968). The reason for this might be the instability of selenocysteine (Butler and Peterson 1967). Incorporation of seleno-amino acids into rumen bacteria is a rapid process resulting in about half of the dosed selenomethionine being associated with rumen bacteria within the first 6 hrs. There- after the proportion of selenium associated with rumen bacteria is slowly decreased with a coinciding increase in proportion of ingesta + protozoa associated Se. It is assumed that only a minor proportion of this selenium is bound to the protozoa. The proportion of protein-bound selenium is found to be about 12 80 % in ingesta +protozoal fraction, but only about 65 % in bacterial protein (Hidiroglou et al. 1974). The average turnover rate for protein-bound selenium has been only 70 % of that observed for sulphur (Hidiroglou et al. 1968). Supplementing selenium to sheep has been shown to increase the number of rumen bac- teria and also to have some effect on rumen bacterial composition (Hidiroglou et al. 1968). This might result in improved microbial protein synthesis. The effect of sulphur on selenium metabolism may arise from the fact that increasing sulphur in the diet has in- creased the population of Desulphovibrio bac- teria in the rumen (Shrift 1973) and thus more selenium is enzymatically reduced to H 2Se (Pope et al. 1979). H2 Se is an unstable compound, which might be reduced further to elemental selenium or to highly insoluble metal selenides (Hidiroglou and Jenkins 1973). Some of H 2Se can also be eliminated from the rumen via eructation (Lopez et al. 1968). Handerek and Godwin (1970) found one percent of dosed selenium in expired air. However, Paulson et al. (1968) could not find any volatile selenium even under the same conditions in which some volatile sulphur was formed. High selenium intake (Lopez et al. 1968) and also in some cases high dietary pro- tein level (Ganther et al. 1966) may increase expired selenium. Part of the selenium given to animals in or- ganic form or as sodium selenite or selenate is metabolized to elemental selenium, which is utilized to some extent by rumen microbes (Handerek and Godwin 1970, Hudman and Glenn 1984), or further to highly insoluble selenides which can be utilized neither by mi- crobes nor by the host (Pope et al. 1979). 2.2. Absorption of selenium 2.2.1. Site of Se absorption There is no net absorption of selenite from the rumen (Wright and Bell 1966) although a small amount of selenomethionine may be absorbed (Hidiroglou and Jenkins 1973). Absorption of selenomethionine from the ru- men is a rapid process, but accounts for only about 2 % of the total absorption of seleno- methionine. The absorption is similar to that of methionine (Venkov 1969). Cysteine is ab- sorbed from the rumen to a much lesser ex- tent than methionine (Lazarov and Ivanov 1970), but there are no studies of the ruminal absorption of selenocysteine. Some selenium is, however, recycled back into the rumen, mostly via saliva (Dejneka et al. 1979), resulting in almost zero net absorption from the rumen (Hidiroglou and Jenkins 1974). In the omasum some selenomethionine is absorbed, but some work suggests that more selenium is secreted back to the omasum leading to the zero or even negative net ab- sorption of selenium from the omasum (Hidiroglou and Jenkins 1973). However, Langlands et al. (1986) found a slight ten- dency for decreased outflow of Se from the omasum when compared to outflow from the rumen. Selenite may be absorbed in the abomasum to some extent in sheep (Wright and Bell 1966), but not in the rat (Whanger et al. 1976) or in swine (Wright and Bell 1966). In contrast to selenite, some selenomethionineis absorbed in mice (Hanson and Jacobsen 1966), rats (Whanger et al. 1976) and sheep (Langlands et al. 1986). Absorption is greater from the pyloric and fundic portions of the abomasum than from the mucosal epithelial segment (Hanson and Jacobsen 1966). The main site for selenium absorption varies from species to species, but is always located in a small intestine. In rats (Whanger et al. 1976) and in chicks (Pesti and Combs 1976, Humaloja and Mykkänen 1986) selenite and selenomethionine are absorbed from the duodenum more efficiently than from the jejunum or ileum, but in sheep absorption is most efficient the from midjejunum (Hidi- roglou and Jenkins 1974). Selenate-Se is ab- sorbed in rats most efficiently from the ileum followed in descending order by the proximal 13 jejunum and large intestine (caecum and co- lon) (Wolframm et al. 1985). In swine and sheep net absorption is greatest in the distal 4/5 of the small intestine (Wright and Bell 1966), probably due to extensive secretion of Se into the duodenum. 2.2.2. Mechanism of Se absorption Most investigations on the absorption mechanism of Se are carried out with labora- tory animals and results in ruminants are limited. Ruminant nutritionists therefor are compelled to extrapolate from data obtained from laboratory animals. Selenomethionine is transferred actively across the intestinal wall in the golden hamster (McConnell and Cho 1967), the chick (Humaloja and Mykkänen 1986) and the rat (Thomson and Sterwart 1973). Transport is dependent upon the ATP- dependant sodium pump (McConnell and Cho 1967). The mechanism is probably the same as that for methionine. Also selenate transport may be active in rat ileum (Arduser et al. 1985, Wolframm et al. 1985). Absorption of selenate is more rapid than that of selenite (Turner et al. 1990). Selenate ab- sorption probably involves a carrier-mediated mechanism, which is relatively unspecific for selenate (Wolframm et al. 1985), is depen- dant on a Na + gradient across the intestinal membrane (Arduser et al 1985), and may de- rive energy from Na + K + -ATPase (Turner et al. 1990). In contrast to selenomethionine, simple diffusion probably dominates in the absorp- tion of selenite (McConnell and Cho 1965, Wolframm et al. 1985) and selenocysteine (McConnell and Cho 1965). Humaloja and Mykkänen (1986) have proposed, however, that a carrier system transports selenite in the chick intestine. Furthermore, there is evidence that Se-dicysteine could be formed extracel- lularly from selenite and cysteine in pig (Wolframm et al. 1988) and sheep jejunum (Wolframm et al. 1987). Se-dicysteine would thenbe transported across the intestinal brush border by the active Na + -dependant trans- port system existing for neutral amino acids (Preston et al. 1974, Wolframm et al. 1989). L-cysteine stimulates absorption of Se from selenite probably by generation of selenodi- cysteine and cysteine selenopersulfide. In- tracellular formation of Se-dicysteine may contribute to the stimulatory effect of L-cys- teine on the uptake of Se from selenite by maintaining the concentration gradient for passive uptake of selenite (Wurmli et al. 1989). Reduced glutathione (GSH) and y-gluta- myltransferase (y-GT) may play some role in the absorption of selenite in the rat intestine (Anundi et al. 1984). A large amount of GSH is excreted in the bile in rats (Sies et al. 1979, Eberle et al. 1981) probably resulting in the formation of oxidized selenodiglutathione (GSSeSG). This would act as a substrate for y-GT and result in the formation of amino acids and dipeptides, for which active trans- port mechanisms exist in intestinal cells. Se- lenium bound to these products might thus be- come available for active transport (Anundi et al. 1984). 2.2.3. Factors affecting Se absorption The efficiency of selenium absorption ap- parently is not regulated by dietary selenium level (Lopez et al. 1968, Cary et al. 1973, Kiker and Burk 1974), although Humaloja and Mykkänen (1986) reported that at a toxic Se dietary level selenium absorption tended to increase. Supplementing methionine to ham- sters (McConnel and Cho 1967) and sulphur to sheep (Pope et al. 1979) or rats (Arduser et al. 1985) tended to decrease the rate of sele- nium absorption although the results with sul- phur are not consistent (e.g. Paulson et al. 1966, White and Somers 1977). Some ca- tions, especially bivalent cations like silver (Rahim et al. 1986) and lead (Mykkänen and Humaloja 1984, Neathery et al. 1987), may decrease selenium absorption when ad- ministered together with Se. However, neither Cu (Rahim et al 1986, Koenig et al. 1989), Fe, Cd, Mo, Mn (Rahim et al. 1986) nor Co 14 (van Ryssen et al. 1987) affected selenite ab- sorption. Absorption of selenium may be decreased on low or high calcium intake (Alfaro et al. 1987), with the optimal dietary calcium level being 8 g/kg DM for selenium absorption in the dairy cow (Harrison and Conrad 1984b). Vitamin A (Combs 1976) and ascorbic acid (Combs and Pesti 1976, Combs and Scott 1974) in chicks and ascorbic acid in rats (Rahim 1985) increase selenium absorption, suggesting that factors which inhibit the oxi- dation of dietary selenium promote its absorp- tion. In contrast to this Mykkänen and Mutanen (1983) reported that moderate doses of ascorbic acid can inhibit the intestinal absorption of selenite in chicks when ad- ministered together intraduodenally. In their later study in non-fasted chicks (Mykkänen and Mutanen 1986) concluded that orally ad- ministered ascorbic acid had no effect on selenium absorption, but there might be some differences between the Se sources. Lowered absorption due to ascorbic acid might be due to reduction of selenite to elemental Se (Hill 1979). Ascorbic acid status of chicks has no effect on selenium absorption (Mykkänen and Mutanen 1983). The ionophores narasin and monensin have enhanced selenite absorp- tion in steers (Costa et al. 1985). In rat selenium absorption is enhanced with increasing age (Raghib et al. 1986), but the opposite has been found in sheep (Grace and Watkinson 1988). 2.2.4. Resecretion of Se into intestine A significant amount of absorbed selenium is excreted back to the intestine in rat (Im- bach and Sternberg 1967, Gregus and Klaassen 1986), sheep (Hidiroglou and Jenkins 1974,Langlands et al. 1986) and bo- vine (Symonds et al. 1981 a and b). Two to three times the amount of ingested Se enters the proximal portion of the small intestine in sheep (Langlands et al. 1986). Secretion is highest in the first fifth of the small intestine (Wright and Bell 1966) or in the midjeju- num (Hidiroglou and Jenkins 1974) in sheep and in the first fifth of the small intestine in swine (Wright and Bell 1966). Resecreted Se enters the gastrointestinal tract mainly via sali- va and bile. In sheep saliva Se concentration is reported to be 0.3 ng/1 and in bile is nearly three times higher (Langlands et al. 1986). There was no correlation between Se concen- tration in blood and that of saliva or bile. Dejneka et al. (1979) reported about 2 %, but Langlands et al. (1986) as much as 28 % of a Se dose to be excreted via bile in sheep. The corresponding figures for the rat are 4 % (Levander and Baumann 1966) to about 6 % (Imbach and Sternberg 1967) and for cattle about 2 °?o with selenite and about 1 % with selenate (Symonds et al. 1981b). Biliary excre- tion does not increase proportionally with dos- age suggesting that the hepatobiliary transport of selenium is saturable (Gregus and Klaas- sen 1986). The total secretion of selenite into the intestine in cattle is about 9 % of the to- tal dosage of selenite and about 14—17 % of selenate (Symonds et al. 1981b), suggesting that digestive juice plays a major role. In rats 11 % of selenite was secreted in digestive juice (Imbach and Sternberg 1967). The major selenocompound secreted in bile is probably selenotaurocholic acid (Rosenfeld 1962). In cats 1.4 °/o of intravenously injected 75Se-se- lenomethionine was excreted in pancreatic juice, mainly in protein-bound form (Hanson and Blau 1963). 2.3. Metabolism and chemical forms of selenium in tissues Although the chemistry of selenium resem- bles that of sulphur in several respects, these elements have important biochemical differ- ences and are therefore not completely inter- changeable in animals. The quadrivalent Se in selenite tends to undergo reduction, while the quadrivalent S in sulfite tends to undergo oxidation. Thus Se compounds tend to be metabolized in animals to more reduced states, while S compounds tend to be oxidized (Combs and Combs 1984). Although the 15 analogous oxyacids of Se and S have com- parable strengths, the hydride H2Se is a much stronger acid than H2S. This difference is reflected in the dissociation constants of the selenohydryl group of selenocystine (pKa 5.24) and the sulphydryl group of cystine (pKa 8.25) (Huber and Criddle 1967). Whereas thiols such as cystine are mainly protonated at physiological pH, the selenohydryl groups of selenols such as selenocystine are largely dissociated. This behavior appears to be im- portant in the catalytic role of selenium in selenoenzymes. 2.3.1. Selenium in erythrocytes and plasma Studies concerning selenium metabolism in erythrocytes are abundant and have been car- ried out in rats (Burk 1973, Butler et al. 1985), mice (Sandholm, 1973a, Sandholm 1974), chicks (Jenkins et al. 1969), humans (Burk et al. 1967, Lee et al. 1969, Burk 1974, Mas et al. 1988), sheep (Wright and Bell 1966) and cattle (Sandholm 1973b, Jenkins and Hidiroglou 1988). Glutathione peroxi- dase (GSH-Px) is the best documented seleno- compound in animals. The molecular weight of this enzyme in cattle erythrocytes has been reported to be 84 000 to 88 000 U (Oh et al. 1974, Jenkins and Hidiroglou 1988). The role of Se in GSH-Px was first demonstrated by Rotruck et al. (1973) and Flohe et al. (1973) and the structure was described by Ladenstein (1979). Plasma proteins do not bind selenium as selenite unless erythrocytes are present, sug- gesting that a transformation of selenite oc- curs within the erythrocytes (Mas et al. 1988). Exchange of selenite between plasma and the erythrocyte is a rapid process. With- in one minute 50 80 % of selenite selenium was accumulated inside erythrocytes in hu- mans (Lee et al. 1969), in mice (Sandholm 1973a) and in rats (Gasiewicz and Smith 1978). The uptake of selenite by bovine, chick and ovine erythrocytes is also rapid, but not as rapid as that of human or rat erythrocytes (Jenkins 1968, Jenkins and Hidiroglou 1972, Sandholm 1973b, McMurray and Davidson 1979). In a cow at low plasma selenium con- centration (<0.2 mg/1) selenite was taken up in one to two minutes by erythrocytes and thereafter the ejection exceeded the uptake. At higher selenium concentrations (>2 mg/1) the bulk of selenium remained associated with erythrocytes. At a very high selenium concen- tration (200 mg/1) the uptake of Se by the red blood cells was restricted (Sandholm 1973b). Some of selenite may stay unaltered inside the erythrocytes and extract as selenite. This hap- pens whenever a relatively small number of erythrocytes is present (McMurray and Davidson 1979). The uptake of selenium by erythrocytes is dependant upon the availability of reduced glutathione (GSH) (Jenkins and Hidiroglou 1972, Sandholm 1973b, Gasiewicz and Smith 1978) or sulphydryl groups (Porter et al. 1979) and the amount of selenium in blood (Sandholm 1973b). However, increased sele- nite concentration tends to deplete erythro- cyte GSH (Gasiewicz and Smith 1978). Hae- moglobin is reported to be the binding site for newly-reduced Se and thus haemoglobin would play a role in the erythrocyte uptake of selenite (Mas et al. 1988). Jenkins and Hidiroglou (1988) reported that in calves fed selenium at a dietary level of 5 ppm 35 to 40 % of erythrocyte Se was in GSH-Px, 50 % in haemoglobin and 5 % in a selenite plus sele- nopolypeptide fraction 72 hrs post dosing. In pigs Se has been reported to be distributed equally between GSH-Px and haemoglobin (Xia et al. 1985). In rhesus monkeys fed selenite 68 % of erythrocyte Se was associated with GSH-Px while in animals fed seleno- methionine only 34 % of erythrocyte Se was in GSH-Px. In contrast, more Se was asso- ciated with haemoglobin in animals fed selenomethionine than selenite (Butler et al. 1990). In haemoglobin Se is retained in the globin portion (Beilstein and Whanger 1986a). Subsequent to selenite's appearance in the erythrocyte it reacts with GSH to form sele- nodiglutathione (GSSeSG) (Ganther 1968, 16 Sandholm and Sipponen 1973). GSSeSG is further reduced by Nadph and glutathione reductase to form selenopersulphide (GSSeSH) and further to H 2 Se (Gasiewicz and Smith 1978). H2Se or a similar reduced product of GSSeSG is the final product of selenite me- tabolism in the rat erythrocyte. McMurray and Davidson (1979) called this product com- pound X. However, release of selenium from rat erythrocytes occurs through a different mechanism when compared to that observed for diglutathione (GSSG) (Gasiewicz and Smith 1978). Selenite metabolism in animals leading to the formation of methylated sele- nides is believed to occur via H2Se (Ganther and Hsieh 1974, Hsieh and Ganther 1977). H2Se is an unstable compound, which may undergo oxidation to elemental selenium (Ganther 1971) and further, combine with tissue macromolecules such as albumin (Hsieh and Ganther 1975) or other seleno- compounds. However, when receptor sites on plasma proteins exist, compound X is excreted from the erythrocyte to be bound to this ac- ceptor protein. If plasma proteins are satu- rated compound X is retained by the erythro- cyte and becomes attached to proteins within the erythrocyte. These processes are reversi- ble. Binding of selenium within the erythro- cyte is an alternative process and formation of protein-bound selenium in plasma is usually favored (McMurray and Davidson 1979). In the rat 85 °/o (Gasiewicz and Smith 1978) and in the sheep 90 % (McMurray and Davidson 1979) of the selenium released into plasma is found to be protein-bound. The majority of this selenium is incorporated into selenocys- tines in polypeptides (Cummins and Martin 1967, Jenkins 1968). Incorporation of seleni- um into serum proteins need not be enzymatic. In addition to proteins, biologically significant sulphydryl reducing agents such as CoASH, cysteine and GSH have also been reported to form selenotrisulphides upon reaction with selenous acids (Ganther 1968, Sandholm and Sipponen 1973). The reaction of selenite with the thiol groups of these compounds is an important pathway through which inor- ganic selenium is initially incorporated into living systems (Kice 1981). In contrast to selenite and selenomethio- nine, selenate is probably taken into the erythrocyte only by diffusion (Jenkins and Hidiroglou 1972). Even though selenite as such does not bind to plasma proteins, H2Se and GSSeSG are readily incorporated to plas- ma proteins even in the absence of erythro- cytes (Gasiewicz and Smith 1978). Albumin (Sandholm 1973a, Mas et al 1988) and an- other protein having a molecular weight of greater than 200 000 U (Mas et al. 1988) are the initial acceptors for the selenium com- pound released from erythrocytes. Increasing the selenium dose has generally increased the total selenium binding to albumin (Jenkins and Hidiroglou 1988), but decreased the binding to the a-globulins in rats and dogs (Hirooka and Galombos 1966). Jenkins et al. (1969) postulated that albuminbinds selenium only when supraphysiological levels of selenite are employed. This was confirmed by Her- man and McConnell (1974). Also McMur- ray and Davidson (1979) found that some proteins other than albumin play an impor- tant role in the binding of selenitemetabolized by erythrocytes. P-lipoprotein is suggested to be an important site for selenium attachment (Sandholm 1975). Only a small percentage of the total plasma selenium is identified as selenium-dependant GSH-Px (Behne and Wolters 1979). In erythrocytes, however, GSH-Px represents most of the total selenium, being 75 % in sheep (Oh et al. 1974) and 100 °7o in rat (Behne and Wolters 1979). The proportion of erythrocyte Se associated with GSH-Px is dependant on the Se source, or- ganic forms of selenium being incorporated more efficiently into other proteins than into GSH-Px (Beilstein and Whanger 1986a, 1986b, Butler et al. 1990). Plasma proteins are important carriers for the incorporation of selenium into lympho- cytes. Uptake of protein-bound selenium by lymphocytes is three times as high as uptake of selenite. There are neither energy nor pro- tein synthesis requirements for the uptake of 17 18 seleno proteins or selenite by lymphocytes, but there is need for sulphydryl groups for uptake of both protein bound selenium and selenite (Porter et al. 1979). Protein synthesis is also not needed in leucocytes for uptake of selenite (Cavalieri et al. 1967). 2.3.2. Selenium metabolism in the liver The liver plays an essential role as the sele- nium processing center of the body. In the liver selenium is converted to compounds which are available for other organs and through which selenium is excreted from the body (Behne and Höfer-Bosse 1984). In several studies the plasma concentration of intravenously given selenium has decreased sharply reaching the minimum 15 to 40 min- utes after the injection (Sandholm 1973b, Al- len and Miller 1981a, Symonds et al. 1981b). During the first 30 minutes bovine liver takes up 40 °/o of the selenium removed from the plasma (Symonds et al. 1981a, 1981b). After reaching the minimum level, the plasma selenium begins to increase due to the release of selenium from the liver back to plas- ma, a- and Y-globulins bind the selenium metabolites released from the liver (Symonds et al. 1981b). Motsenbocker and Tappel (1982a, 1982c) found in rat and monkey liver and in plasma a selenocysteine-containing pro- tein, which had a short half-life. They called it selenoprotein P. Other studies have con- firmed the hypothesis that this protein is serv- ing as a selenium transportation protein from the liver to other tissues (Motsenbocker and Tappel 1984, Beilstein et al. 1984, Gometz and Tappel 1989). A similar selenium trans- port protein has been identified in swine plas- ma (XIA et al. 1985). At a high intake of selenite Se deposition occurs mainly in the cytosol fraction of the liver, differing from muscle, kidney and testes where an excess of Se is deposited in the nuclear fraction (Dea- gen and Whanger 1985). 2.3.2.1 Enzymatic synthesis of selenocysteine in liver Synthesis of selenocysteine plays a very im- portant role in selenium metabolism. More than 80 °?o of the selenium in selenite fed rats is found to be in the form of selenocysteine (Hawkes et al. 1983, Butler et al. 1985). The number of proteins containing selenocysteine has been estimated to be 9 to 19 in rats (Hawkes et al. 1983), GSH-Px being the most important compound containing selenocys- teine (Deagen and Whanger 1985). Se dosed as selenomethionine is first de- posited in liver as selenomethionine, but then is slowly converted to selenocysteine (Butler et al. 1985) probably through the same path- way as cysteine from methionine: selenome- thionine —* Se-adenosylselenomethionine —* Se-adenosylhomocysteine —» seleno- homocysteine —» selenocystathione —* selenocysteine. These reactions are catalyzed by cystathione 0-synthetase and cystathione Y-lyase (Esaki et al. 1981, Soda et al. 1981). Selenocysteine is synthesized also from selenite (Hawkes and Tappel 1983). Conversion of selenite to selenocysteine to be used for the GSH-Px synthesis occurs faster than conver- sion to selenomethionine (Butler et al. 1985). However, the mechanism of seleno- cysteine synthesis from selenite still remains unclear. One possible pathway is the reverse of the selenocysteine lyase reaction, which would contribute selenocysteine formation from alanine and H 2 Se (Esaki et al. 1985). However, no selenocysteine has been found to be synthesized from H 2Se and serine. In rat liver selenocysteine, but not cysteine, is catabolized rapidly by selenocysteine lyase into alanine and H 2Se (Esaki et al. 1985). Selenocysteine is synthesized and catabolized continuously and thus the selenium retention in various tissues is in constant flux. 2.3.2.2. Selenoprotein synthesis in liver GSH-Px and selenoprotein P are synthe- sized in rat liver (Burk and Gregory 1982, Motsenbocker and Tappel 1982c). In ruminants, liver GSH-Px synthesis does not play a major role in selenium metabolism. Only 10 °7o of hepatic selenium was incorpo- rated into GSH-Px in ruminants whilst 75 % of hepatic selenium in rat liver was found in the form of GSH-Px (Sunde et al. 1978). There exist two possible mechanisms for in- corporation of selenium into GSH-Px. Sunde and Hoekstra (1980 and 1981) proposed post-translational incorporation, in which or- ganic selenium has to be converted into the inorganic form before its attachment to an amino acid residue of the GSH-Px polypep- tide chain. However, this theory was criticized by Hawkes et al. (1982), who introduced translational incorporation involving seleno- cysteine attaching to the polypeptide chain of GSH-Px during the protein synthesis. They also found selenium-specific selenocysteyl- tßNA in rat liver. Selenocysteyl-tRNA was later confirmed to be synthesized from selenite in rat liver (Hawkes and Tappel 1983) and in- corporating its selenocysteine moiety to the polypeptide chain of GSH-Px during protein synthesis. Selenoprotein P is synthesized both in vitro and in vivo in the rat hepatocyte. The mecha- nism by which selenocysteine attaches into selenoprotein P is probably the same as pro- posed for GSH-Px (Motsenbocker and Tap- pel 1982c). Hepatic synthesis of selenopro- tein P is a rapid process and the highest con- centration in plasma has been reached three hours after injection of selenium. Selenopro- tein P is also removed rapidly from plasma to other tissues (Motsenbocker and Tappel 1982c). Selenium deficient rats are shown to retain twice as much selenium in selenoprotein P when compared to selenium sufficient rats (Motsenbocker and Tappel 1982c). Obvious- ly the liver prioritizes the synthesis of selenium transportation protein above that of other selenoproteins (e.g. GSH-Px). This enables an animal to maintain a sufficient level of avail- able selenium for tissues where selenium is most needed (Burk and Gregory 1982). In this way the liver redistributes selenium be- tween different tissues and Se compounds in selenium deficient animals (Motsenbocker and Tappel 1982c). 2.3.2.3. Synthesis of methylated selenocompounds Selenomethionine, selenocysteine and H 2Se are toxic for animals and are converted to harmless dimethyl selenide and trimethyl- selenonium ions (Martin 1973). Methylated selenocompounds are mainly produced in the liver, but the kidney also is able to synthesize dimethylselenide (Ganther and Hsieh 1974, Hsieh and Ganther 1977), a direct precursor of trimethylselenonium ion (TMSe). Toxicity of TMSe is only one-tenth that of selenome- thionine and selenite (Obermeyer et al. 1971). The biological role of TMSe is negligible and it is excreted readily in urine (Tsay et al. 1970, Foster et al. 1986b). Methylation of selenomethionine probably arises from the synthesis of selenocysteine, which is further catalyzed to H2Se and alanine via selenocys- teine lyase (Esaki et al. 1981, 1982). H 2Se then is metabolized further to TMSe. Selenocysteine probably is not first catabo- lized to H2Se when synthesizing TMSe (Fos- ter and Ganther 1984). Precursors for di- methylselenide are H 2Se or other interme- diate products from the reduction of selenite. In this reaction the hydrogen of the precur- sors is substituted enzymatically by methyl groups (Ganther and Hsieh 1974). There exist at least two different types of methyl- transferases, one existing in hepatic micro- somes and the other in the cytoplasm (Hsieh and Ganther 1977). If the methylation ca- pacity is exceeded the excess of dimethylsele- nide is excreted via the lungs. TMSe is not an inert compound and may undergo demethyla- tion followed by remethylation (Foster et al. 1986a). 2.3.3 Selenium metabolism in kidney More than 99 % of kidney selenium is found in the cortex and only a small fraction in the medulla (Lopez et al. 1968). Most of the cellular selenium of goat kidney is found in the nuclear fraction (Allen and Miller 1981b). In rats about half of the kidney sele- nium is distributed in the cell organelles with the highest concentration found in the lyso- 19 somes (Motsenbocker and Tappel 1982b). Only a small fraction of this lysosomal Se was identified chemically. Xia et al. (1985) re- ported most ofkidney Se to be associated with GSH-Px in pigs. Motsenbocker and Tappel (1982b) speculated that selenium in plasma selenoprotein P is made available for the tis- sues by catabolizing selenoprotein P in the lysosomes of the cell. Thus high lysosomal selenium content might represent intermediate products of selenoprotein P catabolism. In rat kidney, specific activity of 75 Se was highest one hour after injection of 75Se-la- beled selenite or selenomethionine (Millar et al. 1973). In the kidney selenium is rapidly in- corporated into proteins and within a few hours after injection of selenite, selenate or selenomethionine most of selenium has been found in the protein fraction (Millar 1972, Millar et al. 1973). At least four different selenoproteins have been discovered in the kidney. Only 10 °/o of the selenium in the rat kidney is in the form of GSH-Px (Behne and Wolters 1983), with most of GSH-Px in the mitochondrial frac- tion (Motsenbocker and Tappel 1982b). In addition to GSH-Px, selenoprotein-Pl and two smaller selenoproteins have been found in the kidney (Motsenbocker and Tappel 1982b). These two smaller proteins may have a specific function in the kidney, because these selenoproteins were found neither in the liver nor testis. It seems likely that there exists some specific function for selenium in the kidney, because in the selenium-deficient sheep (Lo- pez et al. 1968) and rat (Burk et al. 1973, Behne and Höfer-Bosse 1984) a high propor- tion of selenium is retained in kidney. Boz- kurt and Smith (1981) speculated that selenoproteins might have some effect against the toxicity of cadmium. It is more likely, however, that cadmium reacts with selenite, but not with selenoproteins (Motsenbocker and Tappel 1982b). After absorption organic Se is incorporated immediately into tissue proteins (Vokal- Borek 1980) and is not filtered by nephrons. Free selenomethioninecould be filtered in the glomerulus, but is readily reabsorbed in the proximal nephron as is methionine (Robinson et al. 1985). Excretion of selenium into urine with in- creasing dietary selenium intake is due to elevated production of organic selenium metabolites, like TMSe. In addition to TMSe, Kiker and Burk (1974) were able to demon- strate three other urinary selenium-containing products, which they called U-2, U-3 and U-4. These products were not formed in vitro from selenite. The methodology used by Kiker and Burk (1974) to identify these compounds was, however, later criticized by Nahapetian et al. (1983), even though they also found at low Se intake that non-TMSe contributes most of the Se excreted in urine. TMSe was the primary selenocompound in rat urine when selenate, selenomethionine, selenocysteine, Se-methylselenocysteine or wheat selenium were fed (Palmer et al. 1970, Nahapetian et al. 1983). U-2 also was detected after all these selenium sources were fed. Some volatile selenocompounds have been detected in urine (Palmer et al. 1970), especially when organ- ic selenium has been involved. Kiker and Burk (1974) demonstrated in vitro synthesis of some urinary compounds, which they re- ferred to as inorganic compounds. Nahape- tian et al. (1983) postulated, however, that little or no selenite is excreted in urine in the rat. The first selenocompounds occurring in urine after selenite injection to rats are inor- ganic, but later the proportion of inorganic metabolites of selenium in urine plateaus at the level of 20 °7o of total urinary Se in sele- nium sufficient rats and at 50 °7o in selenium deficient ones. The proportion of TMSe in urine rises dra- matically with increasing selenium dose. The change was from 10 % to 69 % (Nahapetian et al. 1983) or from 2 % to 70 % (Morris and Levander 1986) when the Se dose was increased by a factor of 100. Thus very little TMSe is excreted with selenium deficient diets, but it is the primary excretion metabolite when the intake exceeds the requirement (Kiker and Burk 1974, Nahapetian et al. 1983). 20 This probably illustrates detoxification of in- gested Se (Nahapetian et al. 1983). Selenium level has effected also the excretion of U-2 (Kiker and Burk 1974). Thus selenium excre- tion in urine is regulated through the produc- tion of TMSe and U-2. Proportion of sele- nium excreted as TMSe has been smaller af- ter dosing selenomethioninethan after dosing selenate (Nahapetian et al. 1983, Morris and Levander 1986). TMSe was found to be about 10 times less toxic than inorganic (selenite or selenate) and organic (selenocysteine or selenomethionine) selenium and furthermore to be an inactive compound that was almost completely reco- vered from urine when it was fed to Se-de- ficient rats (Tsay et al. 1970). In some studies, however, TMSe is found to be the ma- jor urinary metabolite of Se even at low levels of selenite administration in rats (Palmer et al. 1969, Kiker and Burk 1974, Burk 1976) indicating that TMSe is a normal product of selenite metabolism. These discrepancies may, however, reflect different routes of dosing (e.g. intravenous vs. oral). Thus a slower rate of absorbed Se entering the blood stream, as compared to that after injection, might result in differences in the extent and rate of forma- tion of TMSe in the liver and kidney. Na- hapetian et al. (1983) suggested that in urine there is no free selenite, but it could be in a bound form together with other urinary metabolites, which were either neutral or negatively charged compounds. In addition to the chemical form of Se and dietary Se level several other factors have been reported to affect urinary Se excretion. High intakes of vitamin E (He and Zhu 1988) and vitamin B 2 (Parson et al. 1985) have been shown to decrease urinary Se excretion while the opposite trend has been reported with Mn (Meng et al. 1987), sulphur containing ami- no acids (Greger and Marcus 1981) and sul- phate (Ganther and Baumann 1962, Pope et al. 1979). The effect of sulphate was not, how- ever, confirmed by Paulson et al. (1966) us- ing reasonably high dietary S content. Dietary protein content has been shown to be corre- lated positively with urinary excretion of selenite in rats (Amiot et al. 1982), but nega- tively in humans (Greger and Marcus 1981). There also exist small differences in the amounts of Se compounds excreted in urine between young and adult animals. In adults the main compound has been TMSe, while in young animals Se-glutathione, selenotrisul- phide and an unidentified neutral substance are predominant. However, the overall com- ponents were the same (Ostadolova et al. 1988). 2.3.4. Selenium metabolism in the mammary gland Transfer of selenite into the mammary gland involves production of a reduced sele- no compound in the liver followed by diffu- sion of this compound into mammary gland (Allen and Miller 1981a). The theory of passive diffusion is supported by the fact that after injection of 75Se-selenite plasma 75Se concentration remains higher than milk 75 Se (Fuss and Godwin 1975, Allen and Miller 1981b). Maus et al. (1980) also concluded that selenite Se transfer from plasma into milk is not actively regulated by the mammary gland. However, some active transfer to the epithelial cells has been speculated to occur (Allen and Miller 1981a). Secretion into milk was highest two hours after the liver started to secrete its selenium metabolite into blood (Allen and Miller 1981a, 1981b), and is not related to the release of the erythrocyte metabolite of Se. However, small amounts of selenite and erythrocyte metabolite are ex- creted in milk. Selenite ion does not bind in vitro to cow or goat milk proteins, but in mammary secre- tory cells a system may exist which is able to reduce selenite to a form which could be bound to proteins. Reduced H 2Se binds easily to milk proteins and binding correlates positively to milk casein content (Allen and Miller 1981a). Selenium is bound to milk proteins between cysteine residues as seleno- trisulphide (Jenkins and Hidiroglou 1971). Sulphur in cysteine is able to form bonds both 21 internally and between polypeptide chains. The selenium atom may be bound between sulphur atoms either before the formation of sulphur bonds or after breakage of sulphur bridges in re-formation (Jenkins 1968). Some selenite may also be incorporated to seleno amino acids. In sheep milk more than 3 % of intraruminally dosed selenite-Se (Godwin et al. 1971) and also a small fraction of in- travenously dosed selenite-Se (Fuss and God- win 1975) were reported to be in the form of selenomethionine. Research concerning selenomethionine me- tabolism in the mammary gland is more scarce than that of selenite. There are differences be- tween these two seleno compounds, however. Much less selenium is excreted into milk af- ter the dose of selenite than after a dose of selenomethionine (Jacobsen et al. 1965,Fuss and Godwin 1975, Khirwar and Arora 1977) or feeding natural selenium containing feeds (Condrad and Moxon 1979, Maus et al. 1980). Some authors contest these results (Jenkins and Hidiroglou 1971). The reason for high excretion of selenomethionine into milk may arise from the fact that in the course of milk protein synthesis selenomethionine substitutes for methionine (Jenkins and Hidiroglou 1971). This is supported by the finding that selenomethionine is able to at- tach the same transfer-RNA as methionine (McConnell and Hoffman 1972). Selenomethionineis transferred from blood into mammary gland epithelial cells probably through some selective mechanism. In the first hours after injection of selenomethionine in sheep, selenium concentration is significant- ly higher in milk than in plasma. Peak con- centration in milk has been measured at two hours after dosing. After 75Se-selenomethio- nine injection relatively high 75Se activity was found in a methionine-rich casein fraction (Jenkins and Hidiroglou 1971). However, methionine-poor milk proteins ((3-lactoglobu- lin, a-lactalbumin and albumin) also retained high specific activity of 75Se after selenome- thionine injection. This was supposed to be due to the ability of selenomethionine to sub- stitute both cysteine and methionine in milk proteins (Jenkins and Hidiroglou 1971). Allen and Miller (1981b) reported spe- cific activity of 75Se to be higher shortly af- ter injection of 75Se-selenite in whey proteins than in casein. Later 75 Se activity in casein exceeded that in whey proteins. This may be due to a different and faster pathway of Se incorporation into whey proteins than into secretory vacuoles (Keenan and Dylewski 1985). Selenium in milk is mainly in the pro- tein fraction, and thus it could be assumed that the secretory mechanism from epithelial cells to alveoli is same as for proteins gener- ally (Allen and Miller 1981a). Casein poly- peptide chains are built in the ribosomes of therough endoplasmic reticulum, before they are transported inside the endoplasmic re- ticulum. From there casein micelles are trans- ported to the Golgi apparatus for phospho- rylation. Whey proteins (3-lactoglobulin and a-lactalbumin) probably follow the same pathway (Keenan and Dylewski 1985). In the Golgi apparatus proteins and thus selenium containing vacuoles are formed. These vacu- oles migrate towards the apical membrane and finally fuse with the membrane and release their contents into the alveolar lumen (Allen and Miller 1981a, Keenan and Dylewski 1985). Fat droplets are enveloped by cell mem- brane and thus take part of the membrane into milk. Membrane from protein secretory vacu- oles replaces the cell membrane which is lost in fat secretion (Keenan and Dylewski 1985). Selenium in fat droplets may thus be bound to the proteins in the membranes covering these droplets (Allen and Miller 1981a). Debski et ai. (1987) were able to identify B—l 28—12 selenoproteins in human milk. They also estimated about 30 °Io of milk selenium to be associated with GSH-Px, with the proportion being relatively higher in goat milk than in cow milk. The proportion of casein associated Se has been reported to be 29 % (Debski et al. 1987), 40 °7o (Yoshida et al. (1981) or 60 % Matthias et al. (1967) in cow milk and about 60 "Vo in goats (Debski et al. 1987). 22 3. OBJECTIVES OF THE STUDY Although results concerning selenium me- tabolism in laboratory animals are abundant, investigations of seleniummetabolism in lac- tating ruminants are relatively scarce. Re- search is mainly concentrated on inorganic selenium sources and there are only a few studies which illustrate metabolism of feed selenium. The aim of this study was to investigate selenium metabolism in lactating cows and goats fed either sodium selenite or feed incor- porated selenium. Sodium selenite was the in- organic seleno compound used in the study. It either was sprayed on growing stands and hence incorporated into plants in organic forms, mainly protein bound compounds, or it was fed or injected directly into animals. In addition selenomethionine was dosed intra- venously into goats to obtain more detailed information on the metabolism of organic seleno compounds in lactating animals. Various dietary selenium levels were em- ployed in the study to obtain information on the effects of selenium status of the animal on the metabolism of seleno compounds either from inorganic or organic sources. The study was intended to include dietary selenium lev- els over the range of deficient up to toxic with particular emphasis on levels having the op- timal selenium content. The present study addresses the metabolism of selenium in tissues closely related to sele- nium absorption and excretion in lactating animals. 23 4. MATERIALS AND METHODS 4.1. Experiment 1 (goats fed Se depleted and supplemented diets) 4.1.1. Experimental design The experiment was carried out with four goats having average milk production of 1.4±0.1 kg/day (xiSE), and body weight of 38.9±4.4 kg. At the initiation of the ex- periment the goats were an average of 1.8 years old and they were in their sixth or seventh lactation month. The goats were kept in individual metabolism cages to enable quantitative collection of urine and faeces throughout the whole experiment. Goats 1 and 4 were fed selenium depleted (0.05 mg Se/kg DM) and goats 2 and 3 with selenited barley (see exp. 3, p. 29) supple- mented rations (0.22 mg Se/kg DM). This supplementation level was chosen because our previous unpublished research had shown the optimal dietary Se content to be 0.2 to 0.4 mg Se/kg DM. Experimental rations were fed during the two months preceding the experi- ment. The experiment consisted of three consecu- tive phases lasting 16, 18 and 18 days, respec- tively. In the beginning of the first phase all animals were given orally 75Se labeled timo- thy grass (Phleum pratense). In the beginning of thephase II they were dosed intraruminal- ly with 75Se labeled sodium selenite and in the beginning of the phase 111 intravenously 75Se labeled selenomethionine (Figure 1). This order for dosing was chosen to avoid high background activity remaining after the intra- venous dose. 4.1.2. Feeds, feeding and milking The animals were fed twice daily at 8.00 and 15.00 hrs. The rations were formulated to meet energy and protein as well as mineral standards presented in Finnish feed tables (Salo et ai. 1982). The average intakes of goats 1 and 4 were 0.49 kg fresh grass (fro- zen) 0.06 kg hay and 0.62 kg barley-oats- mixture (50:50) on a dry matter basis, and in goats 2 and 3 0.58 kg fresh grass (frozen), 0.08 kg hay, 0.43 kg barley-oats-mixture (50:50) and 0.17 kg selenited barley (see exp. 3, p. 29) on a dry matter basis. Twenty grams of min- eral mixture (Se content 0.26 mg/kg) was given once daily mixed with concentrate to each goat. The goats were milked twice daily and dur- ing the first days after Se doses more often as indicated in connection with sampling. At Selenium depleted (Goats 1 and 4) Selenium supplemented (Goats 2 and 3) Phase I, 16 days Oral dose of 7S Se labeled grass Phase 11, 18 days Intraruminal dose of Na 2 "Se03 Intravenous dose of 75 Se-selenomethioninePhase 111, 18 days Figure 1. Experimental design in experiment 1. 24 each milking all milk was removed and weighed. 4.1.3. Preparation of labeled doses The oral dose was produced by spraying 67.34 MBq of Na2 75 5e03 (Amersham SCSI, A =20.65 MBq/ml) on growing timothy grass (Phleum pratense) six days before cutting. 75Se-label was diluted in 20 ml distilled water and surface tension reducer (5 ul Citowett) was added to improve selenium uptake. About 33 °7o of the label was retained in the grass. The intraruminal dose of 75Se-selenite was prepared by adding 0.40 ml (8.00 MBq) of 75 Se-labeled Na 2Se03 (Amersham SCSI, A = 20.65 MBq/ml) to 50 ml distilled water. The intravenous dose was prepared by adding 0.56 ml (12.80 MBq) of 75Se-labeled sele- nomethionine (Amersham SCI2, A = 22.92 MBq/ml) to 25 ml of 0.9 % NaCl (final con- centration 0.51 MBq/ml). 4.1.4. Introduction of doses The oral dose of timothy grass was offered in the morning before feeding animals, refusals were weighed and their 75Se activity was determined. Dose was 2.87, 3.16, 3.23 and 2.77 MBq for goats 1,2, 3 and 4, respective- ly. The intraruminal dose of 75Se-selenite (1.60 MBq) was injected through the rumen wall and the syringes were flushed twice with distilled water to ensure that the entire dose of label was introduced into the rumen. For the intravenous dose the goats were catheterized in Vena jugularis (Cavafix 1.4 mm catheter). 75Se-selenomethionine at a dose of 2.56 MBq was introduced through the catheters and to ensure proper flushing of the catheter syringes were filled twice with blood drawn from the catheter and then reemptied into the vein. 4.1.5. Sampling During phases I and II blood samples were taken from Vena jugularis in 10 ml hepa- rinized Vacutainer tubes and put immediate- ly in ice. All milk, urine and faeces were col- lected and weighed throughout the whole ex- periment. The goats were milked twice daily, and urine and faeces were collected twice daily or at times indicated and sampled represen- tatively. During phase I blood, milk, urine and faecal samples were taken at 0, 2 (only blood), 4,8, 14, 21, 28, 36, 45, 52, 69, 77 (only milk), 92, 116 (only milk, urine and faeces), 140, 188, 236, 332 and 380 hrs post dosing. During phase II blood samples were taken at 0,4, 8, 14, 22, 30, 54, 94, 142, 190, 262, 334 and 430 hrs post dosing. Milk urine and faecal samples were taken at 0,4, 8, 14, 22, 30, 46, 54, 70, 77 (only milk), 94, 118, 142, 166, 190, 214, 262, 334 and 430 hrs post dosing. Blood samples during the first two days of phase 111 were taken through the catheters. The first 2 ml of blood was discarded and thereafter 10 ml of blood was taken with a heparinized syringe. Finally about 2 ml of heparinized (5 IU heparin/ml, final concen- tration) sterile physiological saline was in- jected through the catheter to avoid clotting. After removing the catheters blood samples were taken in vacutainer tubes as during phases I and 11. During phase 111 blood sam- ples were taken at 0, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2,4, 7, 10, 20, 34, 46, 77, 101, 166, 214, 262 and 334 hrs post dosing. Milk, urine and faecal samples were taken at 0, 1.5 (only milk and urine), 4,7, 10, 20, 28, 34, 46, 53, 70, 77, 94, 101 (only milk), 118, 142, 166, 214, 262 and 334 hrs post dosing. Hair samples were shaved from rear and back at the initiation of the experiment and thereafter at the end of each phase and 29 days after the last dose. 4.1.6. Laboratory analyses Radioactive samples were counted with a 1280 Ultro-Gamma (LKB-Wallac Ltd., Fin- land) automatic gamma counter with 3 " X 3 " NaJ(TI) hollow crystal for 10 minutes or for 10 000 counts. 25 Blood samples were centrifuged (550 Xg, 10 min) to separate plasma and erythrocytes. Hematocrit in whole blood was determined by centrifuging samples at 13 000xg 5.5 for minutes. In the case of inadequate volume blood was diluted with 0.9 % NaCl. Radioac- tive assays were done in singlet for erythro- cytes, duplicate for plasma and triplicate for milk and urine in the volume of 2.5 ml. For the radioactive determination of faeces, 70 g of faeces and 130 g of distilled water were homogenized in Stomacher 400 homogenizer (2 min mixing). For the determinations 2.5 g of homogenate was weighed in triplicate. Fae- cal samples were corrected for different geom- etry in counting. Hair samples were ashed in vacuum at 50°C in oxygen flow for four hours in a Tracerlab LTA-600 asher. Ash was dissolved in 4-N HCI and diluted with distilled water for assay. Timothy grass was dried at 60° C and ashed in the same way as hair samples. Feed selenium content was determined by hydride method (Saari and Paaso 1980). Electrothermal atomic absorption spectromet- ric method was used for milk and erythrocyte assays (Kumpulainen et ai. 1983) and for plasma assays (Alftan and Kumpulainen 1982). Feed chemical composition was deter- mined by standard methods, and milk fat and protein content by standard NIR-method. 4.1.7. Calculations and statistical analyses Values for 75Se-activity for milk, blood, urine and faeces were interpolated for days when no samples were taken. All the values were corrected for decay. Values during the phases II and 111 were corrected for back- ground from the preceding doses by extra- polating excretion curves of the preceding phase over the following phase and subtract- ing background values from the determined values. For calculating blood volume the value of 0.07 1/kg live weight was assumed (Kolb 1967). For calculating true absorption of 75Se en- dogenous excretion in faeces during the phase 111 was used. True absorption = (100 -F)/(100-Fe); F = proportion of 75Se actually excreted in faeces, Fe = proportion of endogenous 75 Se from ab- sorbed 75 Se (based on results during the phase III) Figures were produced using Harvard Graphics programme with curve linear fitting. Standard errors were calculated based on ac- tual values resulting in some inaccuracy in ex- pressing them as deviations from the curve; they are therefore given as deviations from the actual values. Rate constants (k-values) were calculated by using a graphic curve analysis technique ("curve peeling", Shipley and Clark, 1972). Values on the linear declining portion of the line were determined by the linear regression equation (y =a —bt, where t = time: linear, and y = 75Se-activity: In). Transit time (TT) expressing time for the first appearance of 75Se in the samples (Grovum and Williams, 1973) was calculated accord- ing to equation (1), time for increasing 75 Se activity from 0 to a maximum (T) according to equation (2) and total mean retention time (TMRT) according to Grovum and Phillips (1973) by using equation (3). m T ln(A2 )-ln(A|) k2 -k, where A, and A 2 are intercept values for 75Se activity in the samples , and k, and k 2 are rate constants associated with the kinetics of 75Se in the body. ln(k2 )-ln(k,) k 2 -k, (3) TMRT =TT+l/k, + l/k2 +l/k 3 Thus time for maximum excretion was de- fined as TT + T. Half-lives (T,,) are ex- pressed based on values for k 3 (or the slowest 26 component within scope of determination) and were calculated as ln2/k3 (Shipley and Clark, 1972). Differences in rate constants between the selenium levels were tested by analysis of var- iance, differences in excretion of 75Se-label were analyzed by split-plot analyses of vari- ance (Snedecor and Cochran 1980) using the following model: Yijk i =\i+ L( + e i; +Tk + (LT) jk + eijkl , where L and T are selenium level and time effects, e;; the main plot error and eijkl the sub- plot error. Differences between the selenium levels in overall excretion of 75Se-label within the path and differences between regression coeffi- cients on slopes of excretion curves were tested by t-test (Sokal and Rohlf 1981). 4.2. Experiment 2 (goats fed diets supplemented with sodium selenite or selenited barley) 4.2.1. Experimental design The details of animal housing, sampling and analyses during experiment 2 were simi- lar to experiment 1. Four goats housed in in- dividual metabolism cages with an average milk production of 1.0± 0.1 kg/d and an aver- age body weight of 35 kg were involved the experiment. The goats were in their first lac- tation and were five months postpartum at the initiation of the experiment. The source of selenium for goats 1 and 3 was a sodium selenite preparation (see exp. 3, p. 29) given with concentrate (0.38 mg Se/kg DM), and for goats 2 and 4 was selenitedbar- ley (see exp. 3, p. 29) at level of 0.34 mg Se/kg DM. The goats were fed the experimental ra- tions for two months preceding the experi- ment. The experiment consisted of two phases lasting 28 days each. At the beginning of phase I the animals were given a 75Se-labeled dose intraruminally (goats 1 and 3) as Na2 75 5e0 3 or orally (goats 2 and 4) as 75Se-labeled grass. In the beginning of phase II the goats were dosed intravenously with 75Se-labeled sodium selenite (goats 1 and 3) or 75Se-labeled sele- nomethionine (goats 2 and 4) (Figure 2). This order for dosing was chosen to avoid high background remaining after the intravenous dose. This also made possible the introduction of a dose containing high 75Se activity, be- cause milk collected after the I.V. dose was used in other research. 4.2.2. Feeds, feeding and milking Feeding of the animals was similar to ex- periment 1 and was as follows: fresh grass (frozen) 0.2 kg, hay 0.4 kg, oats 0.3 kg and barley 0.2 kg on a dry matter basis. Mineral mixture (Se content 0.26 mg/kg) was offered twice daily. Average daily consumption of mineral mixture for goats 1,2, 3 and 4 was 8.8 g, 13.7 g, 7,7 g and 10.7 g, respectively. The goats were milked as in experiment 1. To obtain a reliable excretion pattern in milk dur- ing the first four hours of phase II oxytocin was injected via Vena jugularis prior to milk- ing. Dietary Se source Inorganic (goats 1 and 3) Organic (goats 2 and 4) Phase I, 28 days Intraruminal dose Oral dose 75Se-labeled grassNa 2 "SeOj Phase 11, 28 days Intravenous dose Intravenous dose "Se-selenomethionineNa, 7! SeO, Figure 2. Experimental design in the experiment 2. 27 4.2.3. Preparation of labeled doses The intraruminal 75Se-dose for the goats 1 and 3 was prepared by adding 0.37 ml (7.41 MBq) of 75Se-labeled Na 2Se0 3 (Amersham SCSI, A = 20.65 MBq/ml) and 0.5 ml of 0.01 M Na2Se03 to 19.13 ml distilled water. The oral dose for the goats 2 and 4 was produced by spraying 24.16 MBq of 75Se- labeled Na 2Se03 on growing Italian ryegrass (Lolium multiflorum) one week before the third cutting. 75Se-label was diluted in 20 ml distilled water and surface tension reducer (5 |xl Citowett) was added to improve selenium uptake. About 65 % of the label was retained in the grass. Italian ryegrass was grown in a green house in a 0.2 m 2 pot. The intravenous dose was prepared for the goats 1 and 3 by adding 0.30 ml (6.20 MBq) of 75Se-labeled Na 2Se03 to 12 ml of 0.9 % NaCl (final concentration 0.51 MBq/ml), and for the goats 2 and 4 by adding 0.37 ml 75Se- labeled L-selenomethionine(Amersham SCI2, A =22.92 MBq/ml) to 16 ml of 0.9 % NaCl (final concentration 0.52 MBq/ml). 4.2.4. Introduction of doses The intraruminal dose of 75Se-selenite (3.52 MBq) for goats 1 and 3 was injected through the rumen wall. The oral dose for goats 2 (3.65 MBq) and 4 (3.62 MBq) was offered in the morning before feeding. Introduction of the intraruminal dose for goat 1 failed as the dose was probably introduced into the rumen or abdominal wall. The results from this goat were thus excluded from further analysis. For introducing the intravenous dose the goats -were catheterized in Vena jugularis (Cavafix 1.4 mm catheter) 20 hrs before dosing. The dose for goats 1 and 3 was 2.52 MBq, and for goats 2 and 4 was 2.59 MBq. After dosing the syringes were filled twice with blood drawn through the catheter and reemp- tied into the vein to ensure proper flushing of the catheter. 4.2.5. Sampling Blood, milk, urine and faecal samples were taken as described in connection with experi- ment 1. During phase I blood samples were taken at 1,3, 6, 10, 20, 27, 34, 46, 144, 216, 312, 504 and 672 hrs post dosing, and milk, urine and faecal samples at 1 (only urine), 3 (only milk and urine), 6, 10, 20, 27, 34, 34, 46, 54, 70, 78, 96, 120, 144, 168,216, 312, 384, 512 and 672 hrs post dosing. During phase II blood samples were taken at 0,1, 2,4, 8, 15, 30, 45, 60, 90, and 120 min, and at 3,4, 6, 10, 21, 27, 33, 46, 54, 72, 144, 240, 408, and 672 hrs. Milk samples were taken at 0,1, 2,3, 4,6, 10, 21, 27, 33, 46, 54, 72, 96, 120, 192, 240, 336, 384, 512, and 672 hrs. Urine and faecal samples taken at 0, 3, 6 and 10hrs and thereafterat the same time as milk samples. Hair samples were shaved from rear and back at the initiation of the experiment, at days 13 and 27 during phase I, and at days 7 and 14 during phase 11. The hair shaved on respective days was weighed and the shaved area measured and quantity of hair cm-2 de- termined. 4.2.6. Laboratory analyses, calculations and statistical analyses Analyses were carried out as described in experiment 1 with the exceptions of cen- trifuging of blood samples 10 minutes for 1200xg, and analyzing hematocrit in addition to whole blood from the erythrocyte suspen- sion. The results were calculated as described in experiment 1. Erythrocyte 75Se-counts were corrected for plasma contamination using erythrocyte hematocrit values and plasma 75Se-counts. 75 Se retained in hair was calculated as- suming equal hair growth rate over the whole body and surface area of the body (m-2 ) : A = 0.12xW 0- 66 , W =live weight (kg) (Lloyd et al. 1978). Statistical analyses were carried out as described in connection with experiment 1 (see chapter 4.1.7., p. 26). 28 4.3. Experiment 3 (dairy cows fed diets supplemented with sodium selenite or selenited silage) 4.3.1. Experimental design The experiment involved all dairy cows at the Suitia experimental farm. Cows were housed in a free-stall barn. The experimental design was a 2 x 2 factorial, with sodium sele- nite and selenited silage as main effects (dur- ing period V, selenited barley, Table 1). There were thus four experimental groups: Group 1: No sodium selenite J> Control silage Group 2: Sodium selenite added^^ Group 3: No sodium selenite Selenited silage Group 4: Sodium selenite added -"^ Only cows having records from at least four periods were included analyses. Total number of cows included in the analyses was 48 of which 45 were Friesians and 3 were Ayrshires. The cows were distributed in the groups as fol- lows: group 1: 14, group 2: 11, group 3: 11 and group 4: 12 animals. The whole experiment consisted of six pe- riods with different levels of selenium intake. During the two months preceding the experi- ment the cows were fed to meet requirements. The periods are shown in Table 1. Table 1. Experimental periods and the selenium content of the diet in experiment 3: Dietary selenium (mg/kg DM) Group period Days 1' 2 2 3 3 4" I—l4 0.200.31 0.210.21 15—70 0.070.17 0.200.29 71—147 0.080.42 0.450.78 148—308 0.060.68 1.201.81 309—420 0.030.11 0.090.17 421—539 0.040.04 0.040.04 I' II 111 IV" V VI 1 control group, no added Se, (n= 14) 2 sodium selenite as source of Se, (n= 11) 3 selenited silage (or selenited barley during the period V) as source of Se, (n= 11) 4 sodium selenite and selenited silage (selenited barley) as source of Se, (n= 12) 5 standardization period, feeding was adjusted to meet requirements 6 days 260—308 are excluded from further analysis, be- cause of unequal quality of roughage among groups. 4.3.2. Preparation of selenited feeds Selenited silage was produced by spraying sodium selenite on growing grass one week be- fore cutting. Selenium used per hectare was 5 g, 3 g and 4 g for the Ist, 2nd and 3rd cut- ting, respectively. Silage was slightly pre- wilted (DM content between 23 and 30 %). A more detailed description of the silage preparation is presented elsewhere. Control si- lage was made on the same days with the same method as selenited silage, only without spray- ing selenite. Selenited barley was produced by spraying 100 g Se/ha as sodium selenite on growing barley when coming into axe. Sodium selenite was fed as a preparation manufactured by Kemira Ltd. The prepara- tion was produced by mixing sodium selenite with feed grade lime and granulated using ben- tonite. The final content of selenium in the preparation was predicted to be 100 mg/kg (measured content 116 mg Se/kg). 4.3.3. Feeding, dietary composition and production The feeding was based on wilted silage. Both silage and hay were given ad libitum and the cows had free access to them during the whole day. Hay consumption was, however, less than one kg per day. Concentrate : rough- age ratio was on average 35:65 on a dry mat- ter basis. Half of the cows were given concen- trates 0.4 kg/kg FCM exceeding 5 kg milk production and protein according to standards (Salo et. al. 1982). The other half of the animals in each group were fed according to the flat rate system (see Tuori and Poutiai- nen, 1982) i.e. fixed concentrate level at a maximum of 6 kg/d including 1.5 kg/d pro- tein supplement during the first six lactation weeks and thereafter 0.5 kg/d until 100 days after calving. After that the feeding pattern was changed to standard feeding (periods I—- IV) or concentrates were reduced to 3 kg/d (periods V —VI). An average concentrate mix- ture contained 46.5 % oats, 46.5 % barley, 3.7 % molassed beet pulp, 0.5 % urea and 29 2.8 °/o mineral mixture (Ca : P-ratio 2.5 :1). In addition the cows were given daily 200 g of mineralmixture per cow on the top of the silage. Roughage was given separately for each group. Refusals were removed and weighed daily. Concentrate, protein supplement, selenited barley (during period V), and sele- nium preparation were given individually twice daily. Concentrate refusals were weighed at each feeding time and selenium preparation refusals were estimated. Dietary selenium originating from mineral feeds (MSe) including the selenium prepara- tion and the commercial mineral mixture is presented in Table 2. This selenium is con- sidered inorganic selenium in the later discus- sion, even though no chemical analyses of the selenium forms in the feeds were done. Sele- nium in energy and protein feeds is called "feed selenium" (FSe) and regarded as or- ganic selenium. The content of selenium and of other minerals in feeds used in the experi- ment 3 are presented in Appendix 2. The highest dietary Se content fed to the cows was 2.4 mg/kg DM and was thus close to the toxic level (Underwood 1981), but no symptoms of toxicity were observed. The average dietary mineral and trace ele- ment content are presented in Table 3. Higher Ca content in the groups 2 and 4 was mainly Table 3. Average dietary mineral and trace element con- tent in cows fed various Se diets (for group explanation see Table 1, p. 29). Group 12 3 4 8.8 9.4 8.3 9.0 4.5 4.6 4.8 4.7 2.2 2.2 2.2 2.2 1.8 1.8 2.0 2.0 680 650 550 580 Ca, g/kg DM P, g/kg DM Mg, g/kg DM S, g/kg DM Fe, mg/kg DM Zn, mg/kg DM 89 95 90 86 due to the high content of Ca in the selenium preparation (370 g/kg DM). During phase IV, when the allotment of the selenium prepara- tion was greatest, groups 2 and 4 received 18 % of their Ca intake in this supplement. Variation in mineral and trace element con- tent between the periods was great (see Ap- pendix 3). Milk production was recorded twice daily on five days in a week with an accuracy of 0.1 kg. Cows were weighed at four week intervals before the afternoon feeding and one day af- ter calving. Average feed intake was 16.6, 17.4, 16.8 and 16.4kg DM/d, live weight 585, 583, 564 and 563 kg, and milk production 20.2, 22.2, 21.0 and 19.7 kg/d in the groups 1,2, 3 and 4, respectively. More detailed information will be published later. Table 2. Mineral selenium (MSe) and feed selenium (FSe) intake (mg/kg DM) 1 in cows fed various Se diets (for group explanation see Table 1, p. 29). Period Group 12 3 4 MSe FSe MSe FSe MSe FSe MSe FSe II 0.005 0.069 0.103 0.083 0.005 0.219 0.102 0.183 111 0.005 0.064 0.349 0.098 0.005 0.449 0,381 0.503 IV 0.006 0.056 0.701 0.074 0.005 1.168 0.615 1.242 V 0.005 0.024 0,108 0.033 0.007 0.116 0.096 0.099 VI 0.006 0.035 0.005 0.034 0,005 0.036 0,007 0.031 Mineral selenium includes Se from commercial mineral mixture and selenium preparation, and feed selenium Se from the rest of the feeds. Proportion of inorganic and organic Se during period I are not given because of the relatively high proportion of Se originating from protein concentrate, which contained an inorganic Se component in addition to natural feed selenium. 30 4.3.4. Sampling During the first four periods quality and en- siling losses of silage were monitored by the buried bag method described by Ettala et. al. (1972). Total number of sacks was 53. Dur- ing the fifth and sixth periods silage was sam- pled weekly. Barley and oats were sampled be- fore grinding and hay was sampled approxi- mately every two months. Protein supple- ment, mineral mixture and molassed beet pulp were sampled once for each set of these feeds purchased. Selenited barley and selenite preparation were sampled once. Milk was sampled at four week intervals for fat and protein content and somatic cell count. Milk samples for selenium determinations and blood samples were taken on days 7, 14, 42, 70, 147, 259, 420 and 539. Blood samples were taken before feeding from Vena jugularis. Samples for haemoglobin, hematocrit, whole blood GSH-Px and selenium were taken in 5 ml EDTA tubes and samples for the rest of determinations into heparinized 20 ml tubes. 4.3.5. Analytical methods Dry matter content of feeds was determined by oven drying at 103°C for 24 h. Samples for analysis were dried in vacuum at 50°C for two to three days and milled through a 1 mm screen. Feed analyses were made according to standard procedures. Silage DM content was corrected for volatile losses by adding 1.4 per- cent units to the measured DM content (Hui- da et al. 1986). Selenium in feeds was deter- mined by hydride method, calcium, magne- sium, iron and zinc by atomic absorption and phosphorus by vanadium molybdate method as described by Saari and Paaso (1980). Sul- phur was determined by LECO-132 sulphur analyzer. In earlier studies these minerals and trace elements are speculated to interfere with Se metabolism (Rodvien et al. 1974, Pope et al. 1979, Harrison and Conrad 1984b, Rah im et al. 1986). In vitro digestibility (Tilley and Terry 1963) was measured in all rough- age samples. Net energy in fattening feed units and digestible crude protein were calculated according to chemical composition and digest- ibility coefficients taken from Finnish feed ta- bles (Salo et. al 1982). Milk fat and protein content and somatic cell count were determined with standard NIR-technique. Milk and whole blood sele- nium was determined by hydride method as described by Tykkyläinen et al. (1985) and plasma selenium by electrothermal atomic ab- sorption spectrometric method (Alftan and Kumpulainen, 1982). GSH-Px in whole blood and plasma (on days 7, 70 and 147) were determined by modi- fied Gunzler (1974) method as described by Sankari (1985), hemoglobin by cyanomethe- moglobin method (van Kampen and Zijlstra 1961), and hematocrit as described in experi- ment 1. Aspartate aminotransferase (ASAT), alkaline phosphatase (AP), creatine kinase (CK) (Anon. 1974), and y-glutamyl transfer- ase (y-GT) (Anon. 1976) were determined ac- cording to the recommendations of the Com- mittee on Enzymes of the Scandinavian So- ciety. Plasma glucose was determined by o- toluidine method (Hyvärinen and Nikkilä 1962), total protein by biuret reaction (Hen- ry et al. 1974), albumin by bromcresol green method (Bartholomew and Delaney 1964), cholesterol by enzymatic colorimetric method (Talke and Schubert 1965) and plasma Ca, P and Mg with Gilford 3500 Auto Analyzer. Plasma tocopherols on days 14 and 259 (four animals in each group) were determined by a high performance liquid chromatographic method (Piironen et al. 1985). 4.3.6. Calculations and statistical analyses Erythrocyte Se content and GSH-Px-ac- tivity were calculated using hematocrit value, and whole blood and plasma Se/GSH-Px values. For calculating blood volume 0.077 1/kg live weight (Kolb 1967) and for esti- mating mammary uptake of Se from plasma 500 1 blood circulating through the mammary gland per one liter milk produced (Rook and Thomas 1983) were assumed. 31 Differences in blood parameters and milk Se content were analyzed by split-plot analyses of variance using the following model: Yijkl =n+D; + e y +Pk + (DP)jk + eijk„ where D and P are diet and period effects, ey the main plot error and eijk) the sub- plot error. Significance levels given in the tables were obtained by analysis of variance separately within each period, and differences between the groups using Q-test (Snedecor and Cochran 1980). 32 2 5. RESULTS 5.1. Experiment 1 5.1.1. 7SSe given orally in selenited grass 5.1.1.1. Excretion of 75Se During the 16 day follow-up period (phase I) after the oral 75Se dose, depleted goats ex- creted 6.7 ±l.O (x ±SE), 5.7 ±1.6 and 37.5 ± 4.2 % of the dose in milk, urine and faeces, respectively. Selenium supplemented goats ex- creted during the same time interval 8.2 ± 1.1, 15.2±2.6 and 38.5±0.3, respectively. Total excretion was thus 49.9±6.8 for depleted and 61.9±l.l for selenium supplemented goats. Only excretion in urine was significantly dif- ferent (p<0.05) in Se depleted and sup- plemented groups. Maximum excretion in milk occurred at 25.710.1 and 23.3 ±0.2 hrs after dosing in selenium depleted and supplemented goats, respectively. The difference in maximum values was significant (p<0.05, Figure 3a). Variation in 75Se excretion in urine was great, mostly due to uneven volume of urine (ml/h) voided during the first two days when sam- pling interval was frequent. However, maxi- mum 75Se activity was reached in less than one day (Figure 3b) and rate of excretion of 75Se tended to be lower for depleted goats than for supplemented ones (1.9 vs. 3.6 °/o of dose/d). Maximum faecal 75Se losses oc- curred at 31.2 ± 1.4hrs and 25.9± 1.2 hrs af- ter dosing (Figure 3c) and total 75Se losses at 28.1 ± 1.2 hrs and 22.8 ±0.2 hrs after dosing in depleted and supplemented goats (p > 0.05), respectively. The time when maximum 75Se excretion occurred was different (p<0.05) in milk and in faeces. During the first 20 hours urine was the pri- mary route for 75 Se excretion, but later 75 Se excretion in faeces exceeded that in urine. On day two the proportion of 75Se excreted in faeces was highest with an average of 88 % for depleted and 80 °/o for supplemented goats (p>0.05). Subsequently there was a steady de- crease in the proportion of 75Se excreted in faeces and an increase in the proportion ex- creted in urine. In supplemented goats 75 Se excretion in urine exceeded that of faeces at day four after the dose, but in depleted goats faeces remained the primary route for 75 Se excretion over the 16 day follow-up period. In depleted goats 75Se excretion in milk resembled that in urine after 24 hrs, but in supplemented goats milk 75Se was only one- third of that excreted in urine between 6 and 16 days after the dose (Figure 4). During the first 36 hrs there was no difference in propor- tional excretion between depleted and sup- plemented goats. During the time interval be- tween 36 hrs and 5 days proportionally more (p<0.01) of 75Se was excreted in urine in supplemented goats and less in faeces (p< 0.05) than in depleted ones. After five days proportional excretion of 75Se was lower in urine and higher in faeces in depleted goats (p<0.05). A slightly higher proportion of 75Se was excreted via milk in depleted goats than in supplemented ones (26.4 % vs. 15.9 %, p = 0.09). Interaction between dietary sele- nium level and timewas significant (p < 0.001) only in faeces and in total excretion indicating more rapid excretion of selenium in selenium supplemented goats than in depleted ones. 33 34 Figure S. Excretion of 7! Se after oral dose a) in milk b) in urine c) in faeces in experiment 1, (n = 2, x±SE). 5.1.1.2. Rate constants There were no differences between the two selenium treatments in rate constants calcu- lated based on 75Se excreted in milk, urine or faeces (see Appendix 1). Only k,-values based on total excretion of 75Se differed be- tween the selenium levels (p<0.05). Urine k,- values are rough estimates due to a great vari- ation in the volume of urine voided. The only differences found were in k 2-values between excretion paths (p< 0.001) and in interaction between the path and selenium supplementa- tion (p<0.05). 5.1.1.3. 75Se in plasma and erythrocytes Plasma 75Se values reached their maximum two days after the dose. The maximum value for depleted goats was 7.9 ±0.6 and for sup- plemented ones, 4.6 ±O.l % of dose (p< 0.05). After the maximum values there was a steady decrease in plasma 75Se-activity, but the regression analysis of the slope revealed no difference between two selenium treat- ments (Figure 5). Erythrocyte 75Se-activity tended to be slightly higher (1.0 vs. 0.5 % of dose) in de- pleted goats than in supplemented ones. There was great variation in the time when erythro- cyte 75Se-activity reached maximum. How- ever, at 1.5 days after the dose erythrocyte 75Se values were close to maximum and they remained at this level over the 16 day follow- up period. 5.1.2. 75Se given intraruminally as sodium selenite 5.1.2.1. Excretion of 75Se During the 18 day follow-up period (phase II) after the intraruminal 75Se-dose total ex- cretion was 56.7 ±3.2 and 64.810.1 <7o of the dose in depleted and supplemented goats, re- spectively. During the same time interval in depleted goats 4.0 ± 0.7, 10.2 ± 2.9 and 42.5 ± 0.9 % of the dose was excreted in milk, urine and faeces, respectively, and in selenium sup- plemented goats 4.3±0.6, 22.1 ±2.2 and 38.4 ± 1.7 %, respectively. The difference be- tween the selenium treatments was significant (p<0.05) only in urine. In depleted goats there was no actual peak in 75Se excretion in milk; 75Se activity slowly Figure 4. Proportional excretion of "Se after oral dose in experiment 1, (n =2). 35 increased reaching its highest activity at 3.1 d post dosing. Maximal 7!Se excretion in milk differed between the two selenium treatments (p0.05). Time after the dose had a significant effect (p< 0.001) in all cases. Interaction between the selenium level and path for excretion was sig- nificant (p < 0.05), and there was a strong ten- dency for selenium level to affect excretion path (p = 0.07). Total mean retention time (TMRT) and half-life (T, XI) based on 75Se ex- creted in urine were different between the selenium levels (p<0.05). Dietary level signifi- cantly affected the excretion paths (p<0.001). The interaction between the path and the selenium level significantly affected both TMRT and T l/2 (p<0.01). During the first 20 hrs urine was the pri- mary route for 75Se excretion followed by milk. Thereafter 75Se excreted in faeces ex- ceeded that of urine and milk. The propor- tion of 75 Se excreted in faeces was highest at day two after the dose at 92 % of total daily excretion for depleted and 78 % for sup- plemented goats (p>0.05). At the same time the proportion of 75Se excreted in milk and urine reached their minimums. Thereafter a steady increase in proportion of 75Se excreted in milk and urine resulted in the proportion of 75Se excreted in the urine exceeding that in faeces at day five in supplemented goats and at day nine in depleted goats. The proportion of 75 Se excreted in milk exceeded that in fae- ces on day 11 in both depleted and supple- mented goats (Figure 7). The proportion of 75 Se excreted in urine and faeces differed sig- nificantly in the two groups (p < 0.05), but was similar in milk between groups. In milk the proportion of excreted 75Se was greater (p < 0.05) during the time interval between 5 and 18 days than during the first five days. In urine the proportion was lowest (p<0.05) during the time interval between two and five days Figure 5. "Se in plasma and erythrocytes after oral dose in experiment 1, (n =2, x ± SE) 36 37 Figure 6. Excretion of 75 Se after I.R. dose a) in milk b) in urine c) in faeces in experiment 1, (n =2, x±SE). after the dose, during which time the faecal proportion was highest (p< 0.001). 5.1.2.2. Rate constants Each of excretion curves consisted of three components, except the curve describing the 75Se excretion in milk in depleted goats, which had only two components. There were no differences in rate constants either between the selenium levels or the excretion paths (Ap- pendix 1). However, in k 3 the interaction be- tween the selenium level and excretion path was significant (p<0.01). 5.1.2.3. 75 Se in plasma and erythrocytes The maximum values of 75Se activity in plasma were reached two days after the dose and they were higher in depleted goats than in supplemented ones (13.9 % vs. 7.5 % of the dose, p<0.01). Thereafter there was a steady decrease in plasma 75Se values, al- though the slope of the regression was not af- fected by selenium level. However, at the end of 18 day follow-up period plasma 75Se ac- tivities were higher (p < 0.05) in depleted goats than in supplemented ones (Figure 8). 75 Se-activity in the first samples of erythro- cytes had a high proportion of background re- maining from the first phase and they were thus omitted from the analysis. Maximum values in supplemented goats were reached at day two after the dose and thereafter values steadily decreased. In depleted goats, how- ever, erythrocyte 75Se values began to in- crease again four days after the dose. At day 14 the values exceeded those found at day two and the maximum values were reached at the end of the 18 day follow-up period. Regres- sion analysis indicates that the slope of 75 Se activity in erythrocytes (p<0.05) as well as 75Se activity in erythrocytes at the end of the follow-up period (p<0.01) were different be- tween the selenium treatments. 5.7.5. 75Se given intravenously as selenomethionine 5.1.3.1. Excretion of 75Se During the 18 day follow-up period (phase Figure 7. Proportional excretion of "Se after I.R. dose in experiment 1, (n =2). 38 ili) after the intravenous 75Se-selenomethio- nine dose total excretion was 43.514.2 and 67.915.4 % of the dose in depleted and sup- plemented goats, respectively. During the same time interval 33.213.8, 4.810.4 and 5.410.01, and 35.811.8, 25.717.6 and 6.310.4 % of the dose was excreted in milk, urine and faeces in depleted and supplemented goats, respectively (Figure 9). The differences between the selenium treatment groups were non-significant even though there was a trend (p = 0.09) for different excretion rates in uri- nary 75Se when corrected to 100 % excretion. Excretion of 75Se in urine and milk was a very rapid process and maximum excretion in urine occurred in the first samples 1.5 hrs post dosing. In milk, excretion (% of dose/h) was equal during the time intervals 0—1.5 hrs and 1.5—3 hrs, indicating that maximum excretion probably occurs before 1.5 hrs post dosing. In faeces peak excretion occurred at 20 hrs post dosing, but in spite of relative frequent sampling, it was not possible to determinate transit time. At the end of the follow-up pe- riod total 75Se excretion (p<0.05) and excre- tion in urine (p<0.01) were higher in sup- plemented than in depleted goats. In depleted goats milk was the primary route for 75 Se excretion with the percentage exceeding 50 % throughout the 18 day follow- up period. In supplemented goats most of 75Se was excreted via milk during the first two days, but then excretion in urine was com- parable to that in milk and after seven days it exceeded 75Se excretion in milk. Urinary excretion of 75Se was close to 60 % at day 18 post dosing. In depleted goats excretion in urine played only a minor role with an over- all percentage of less than 20 %. In depleted goats proportion of 75Se excreted in faeces exceeded that of urine at eight hours post dosing and remained above that of urine over the follow-up period. Proportion of 75Se ex- creted in faeces plateaued at 25 % five days post dosing. In supplemented goats the pro- portion of 75Se excreted in faeces was highest at day seven post dosing and thereafterslightly decreased and plateaued at 13 % of excretion. Figure 8. "Se in plasma and erythrocytes after I.R. dose in experiment 1, (n =2, x±SE) 39 40 Figure 9. Excretion of 7S after I.V. dose a) in milk b) in urine c) in faeces in experiment 1, (n =2, x±SE). At the end of the follow-up period differences in proportional excretion between the sele- nium treatments were significant (p<0.05) in milk, urine and faeces (Figure 10). 5.1.3.2. Rate constants Each of excretion curves consisted of three components, except curves based on faecal 75Se excretion, which had only two compo- nents. Differences between the selenium levels were insignificant, but differences between the excretion paths were significant (p<0.05) in k, and k 3, and also in T, A . 5.1.3.3. 75 Se in plasma and erythrocytes 75Se retained in plasma was reduced with- in 10 minutes to 11.4 % and 8.6 % of the dose in depleted and supplemented goats, respec- tively. Diminution of 75 Se activity in plasma continued until 30 minutes after the dose whereupon 75 Se levels rose for four hours, when maximum 75Se activities in plasma were reached both in depleted and supplemented goats (17.5 vs. 13.0 % of the dose, p = 0.08). Thereafter 75Se activity decreased until the end of follow-up period, but there were no differences (p<0.05) between either the final selenium values or in the slopes of the regres- sion (Figure 11). 75Se activity of erythrocytes showed two minimums at the time points of 30 minutes, and at day two in depleted and at day four in supplemented goats. 75Se activity in erythrocytes continued to increase towards the end of follow-up period when depleted goats had maximum activity. In supplemented goats the maximum valuereached at two hours post dosing was not exceeded during the follow-up period. The difference between the selenium treatment groups was insignificant even though erythrocyte 75Se activity in depleted goats was on the average twice that of supplemented goats. 5.2. Experiment 2 5.2.1. 75Se given orally and intraruminally 5.2.1.1. Excretion of 7S Se The intraruminal dose of 75 Se-selenite failed in one of the goats, thus results from Figure 10. Proportional excretion of 75Se after I.V. dose in experiment 1, (n =2). 41 this animal were excluded from further anal- ysis. Therefore no significances between the selenium sources are given. The other goat which was dosed intraruminally with inor- ganic 75Se (IO) excreted 2.0, 21.3 and 68.8 % of the dose, and the goats given orally 75Se labeled grass (OR) excreted 7.910.76, 15.5 ± 0.2 and 59.8 ± 9.0 o.os)in IO and OR goats, respectively, with a similar type of excretion in both groups (Figure 15c). Regression analysis showed that 75Se in milk declined more rapidly in OR goats while 75 Se Figure 13. Proportional excretion of 75Se after oral and I.R. dose in experiment 2, (for organic Se n =2, for inorganic Se n= 1). 44 in urine declined more slowly during the first 35 hrs (p<0.01). The difference between the final values due to selenium source was sig- nificant only in milk (p<0.05). Interaction be- tween the selenium source and time was sig- nificant both in milk, urine (p< 0.001), and faeces (p<0.05). Urine was the primary route for 75Se excre- tion after the I.V. dose in IO goats through- out the experiment and in OR goats later than 6 hrs after dosing. In OR goats the excretion of 75 Se in milk contributed 70 % of total ex- cretion during the first 6 hrs, but thereafter it was exceeded by excretion in urine. The proportion of 75Se excreted in milk tended to plateau at 6 % and 22 % (p<0.05), and in urine at 71 % and 55 % of total excretion in IO and OR goats, respectively. The propor- tion of 75 Se excreted in faeces increased rapidly and reached 30 % of total excretion at 33 hrs post dosing, thereafter declining slightly towards a plateau at 23 °/a of the total excretion (Figure 16). Differences between the selenium sources in milk and urine (p<0.05), and the effect of time on milk (p<0.05) and faeces (p< 0.001) as well as the interaction be- tween the selenium source and time in milk (p<0.01) were significant. 5.2.2.2. Rate constants Due to rapid excretion of 75Se in milk and urine all rate constants could only be deter- mined for faecal excretion. The goats voided urine infrequently and thus only two of rate constants were determined in urine (k 2 and k 4). In milk the highest activities were counted in the first samples and in spite of a one hour sampling interval, the fastest rate constant was incalculable. The turnover time for compo- nent k, was 4.7 hrs for IO goats and 2.4 hrs for OR goats (p>0.05). A total of four rate constants were determined for milk (Appen- dix 1). Selenium source had a significant (p<0.05) effect on k 2 and k 4. 5.2.2.3. 75 Se in plasma, erythrocytes and hair More than 80 % of the dose was recovered in plasma one minute post dosing. Thereafter Figure 14. "Se in plasma and erythrocytes after oral and I.R. dose in experiment 2, (for or- ganic Se n = 2, x±SE, for inorganic Se n= 1). 45 46 Figure 15. Excretion of 7!Se after I.V. dose a) in milk b) in urine c) in faeces in experiment 2, (n =2, x±SE). 75Se activity in plasma decreased rapidly in OR goats, but more slowly in IO goats (p< 0.01). At one hour post dosing the 75 Se ac- tivity began to rise again until 4 hrs. 75Se ac- tivity in plasma of IO goats was higher until 10 days whereupon it was exceeded by OR goats. The slope of the linear regression was steeper (p < 0.05) in IO goats than in OR goats Figure 16. Proportional excretion of 75 Se after I.V. dose in experiment 2, (n =2). Figure 17. 75 Se in plasma and erythrocytes after I.V. dose in experiment 2, (n =2, x±SE). 47 after 3 days post dosing. There was, however, no effect due to selenium source on final values at day 28 (Figure 17). At one minute post dosing 75Se activity in erythrocytes was in IO goats 5.7 % and in OR goats 3.7 % of the dose (p>0.05). Thereafter 75Se activity in erythrocytes decreased until one hour and then increased slightly and ex- ceeded 2 % of the dose in both groups at 28 days post dosing. More 75 Se was retained in erythrocytes in IO than in OR goats dur- ing the time interval from two minutes to 27 hrs (p<0.05). The proportion of 75Se in erythrocytes exceeded that of plasma on day 14. 75Se was retained more efficiently in hair when dosed as selenomethionine than as selenite. At 7 days post dosing the difference between the IO and OR goats was not great (0.4 % vs. 0.6 % of dose), but 28 days after dosing 0.6 ±0.03 and 2.0 + 0.14 % of dose (p<0.01) was calculated to be in hair in IO and OR goats, respectively. 5.3. Experiment 3 5.3.1. Blood parameters Average plasma parameters, blood hemo- globin and hematocrit values in each group during the seleniumsupplementation periods (periods II - V) are given in theTable 4. Most of the values are similar among groups, but plasma glucose and plasma Ca level were higher (p<0.01) in the groups given organic Se than in the groups given no Se supplemen- tation or supplemented with inorganic Se. Plasma ASAT and CK activities, which indi- cate cell damage in muscles and liver, were lower in the groups given organic Se (p< 0.01). Alpha-tocopherol was the only plasma tocopherol which was within the detection limit. Alpha-tocopherol content in the begin- ning of the experiment was 9.7, 9.8, 9.0 and 8.5 mg/1, and at day 259 was 4.3, 4.1, 5.1 and 5.4 mg/1 in the groups 1,2, 3 and 4, respec- tively. The difference in the a-tocopherol level between sampling times reflects seasonal differences, since the first sample was taken in Septemberand the latter one in May. Plas- ma a-tocopherol content tended to be posi- tively correlated to plasma and whole blood selenium content as well as to GSH-Px con- tent in plasma and whole blood. 5.3.2. Plasma selenium Plasma selenium concentration was signifi- Table 4. Plasma parameters, blood hemoglobin and hematocrit during periods lI—V in cows fed various Se diets (for group explanation see Table 1, p. 29). Group 12 3 4 Plasma Ca (mmol/1) 2.36“ 2.40“ b 2.50 b 2.45ab P (mmol/1) 1.621.69 1.641.63 ns. Mg (mmol/1) 0.940.98 0.970.93 ns. glucose (mmol/1) 3.53“ 3.52“ 3.86b 3.83b cholesterol (mmol/1) 5.215.55 5.415.33 ns. urea (mmol/1) 3.554.01 4.064.03 ns. total protein (g/1) 73.972.3 73.974.8 ns. albumin (g/1) 38.439.8 39.438.9 ns. ASAT (lU/1) 52.8“b 63.1» 49,0“ 48.6“ y-GT (lU/1) 19.020.6 20.318.5 ns. CK (lU/1) 79.0“ 113.l b 69.6“ 68.8“ Whole Blood hemoglobin (g/1) 121.6121.1 116.2119.6 ns. hematocrit (%) 36.135.8 34.635.4 ns. Means not sharing a common letter are significantly different ab (p<0.05), ns. (non-significant). 48 Table 5. Plasma selenium content (mg/1) in cows fed var- ious Se diets (for group explanation see Table 1, p. 29). Period Group 12 3 4 0.031 0.037 0.035 0.038 ns. 0.024» 0.052» 0.085' 0.091' 0.031» 0.067» 0.084' 0.086' 0.034» 0.084b o.loo= o.lll' 0.031» 0.073" 0.087»' 0.102' 0.033» 0.058» 0.082' 0.089' 0.018» 0.020» 0.027» 0.030' ! ll* ll** 111 IV V VI * after 28 days supplementation ** after 56 days supplementation For significances see Table 4, p. 48. 5.3.3. Erythrocyte selenium Selenium source did not affect erythrocyte selenium content, even though the organic Se source tended to increase erythrocyte Se con- tent more rapidly (Table 6). The interaction between group and period was significant both when increasing (p< 0.001) and decreasing (p<0.01) dietary Se level indicating more ef- ficient uptake and retention of Se in erythro- cytes when feed Se was provided to the animals. cantly affected by dietary treatment (p < 0.05) and by time (p< 0.001) (Table 5). The inter- action between group and period affected plasma Se content (p< 0.001) both when die- tary Se level was increased and when it was decreased. This indicates that feed Se is taken up more efficiently by plasma and when cows are depleted plasma Se also remains higher in animals fed feed Se than in those fed inorganic Se. The proportion (total plasma Se : daily Se intake) decreased with increasing dietary Se content, but there was no effect of dietary selenium source. During the selenium sup- plementation periods total plasma Se concen- tration was 1.43, 0.48, 0.54 and 0.38 times the daily Se intake in the groups 1,2, 3 and 4, respectively. Only group 1 differed from the others (p< 0.001). Age of cow, lactation stage or milk production had no effect on plasma Se content. Plasma albumin concentration (p< 0.001) but not plasma total protein con- tent affected plasma Se content. The following equation explained 68.1 % of the variation in plasma selenium content: Y= -0.040 + 0.1150 X, -0.1041 X,2 + 0.1886 X 2-0.1199 X2 2 +0.0019 X 3 where, Y = plasma Se (mg/I) X, = mineral Se intake (mg/kg DM) X 2 = feed Se intake (mg/kg DM) X 3 = plasma albumin (g/1). Table 6. Erythrocyte Se concentration (mg/1) in cows fed various Se diets (for group explanation see Table 1, p. 29). Period Group 12 3 4 I 0.247 0.259 0.288 0.273 ns. ll* 0.214 0.221 0.225 0.190 ns. ll** 0.173* 0.239» 0.327* 0.342* 111 0.155* 0.360» 0.416»* 0.443* IV 0.213* 0.537» 0.549» 0.630» V 0.209* 0.366» 0.441» 0.536* VI 0.096* 0.187» 0.221»* 0.237' • after 28 days supplementation ** after 56 days supplementation For significances see Table 4, p. 48. Both milk production and lactation stage were related (p< 0.01) to erythrocyte selenium content, but the effect seems to be due to lac- tation stage rather than actual production. Erythrocyte Se content was lowest two months post partum and reached maximum at seven months post calving whereupon it started to decrease. This indicates preferred use of Se for milk production and fetal growth. Neither age of the cow nor hematocrit affected erythro- cyte Se content (p > 0.05). Erythrocyte Se con- tent plateaued at a 0.63 mg/1 at dietary Se level of 1.82 mg/kg DM and there was no clear difference between the selenium supplemen- tation groups. The following equation ex- plained 62.6 % of the variation in erythrocyte content: 49 Y = 0.130 + Lj + 0.1408 X,+2.4075 X 2, where Y = erythrocyte Se (mg/1) X, =dietary Se intake (mg/kg DM) X2 = plasma Se content (mg/1) L, = lactation stage i: weeks I—l 3 = 0.289 weeks 14—30 = 0.350 weeks 31— =0.351 dry period =0.319 Lactation weeks I—l 31—13 differed from lacta- tion weeks 14—30 and 31- end (p < 0.05).The proportion of total daily Se intake (mg/d) which was retained in erythrocytes decreased with increasing Se intake (p< 0.001). During the selenium supplementation periods erythro- cytes retained an average of 5.32, 1.43, 1.86 and 1.36 the daily Se intake in the groups 1, 2,3, and 4, respectively. Only group 1 differed from the others (p< 0.001). Lactation stage affected the proportion of dietary Se retained in erythrocytes even more than it affected ac- tual erythrocyte Se concentration. It was cal- culated that after eliminating the effect of Se intake erythrocytes retained 1.59, 2.62, 4.68 and 3.57 times the daily Se intake during lac- tation weeks 1—l3, 14—30, 31 and dry period, respectively. Lactation weeks 1 —l3 differed from lactation weeks 31 end and the dry period, and lactation weeks 14—30 differed from lactation weeks 31 end (p < 0.05). 5.3.4. Erythrocyte and plasma GSH-Px activity Erythrocyte GSH-Px activity in group 1 differed from that in Se supplemented groups and also differed between groups 2 and 4 (p<0.05, Table 7). Selenium source had no significant effect. Se intake alone accounted for only 41.1 % of the variation in erythro- cyte GSH-Px activity even though both Se source and linear, quadratic and cubic com- ponents were included in the model. Erythro- cyte Se content explained 61.2 % of the vari- ation in GSH-Px activity. Interaction between group and period in erythrocyte GSH-Px- Table 7. Erythrocyte GSH-Px activity (ukat/1) in cows fed various Se diets (for group explanation see Table 1, p. 29) Period Group 12 3 4 845 815 897 903 ns. 785* 861 ab 1017ab 1056» 666» 922 b 1047b 11llb 501» 1244b 1125b 1253» 974» 2818 b 3005b 3175» 1345» 2189» 2584bc 2954' 931» 1352» 1684»' 1828' I ll* ll** in IV V VI * after 28 days supplementation ** after 56 days supplementation For significances see Table 4, p. 48. activity was significant (p< 0.001) both on in- creasing and decreasing dietary Se levels in- dicating better bioavailability and retention of feed Se as erythrocyte GSH-Px when com- pared to sodium selenite. Erythrocyte GSH- Px activity did not reach a plateau within the range of experimental dietary Se content. Age of the cow, lactation stage or milk pro- duction had no effect on erythrocyte GSH-Px activity, nor did any of the other determined minerals, trace elements or blood parameters. The activity of GSH-Px-enzyme (kat/mol GSH-Px) was calculated using a molecular weight of 88 000 U and four Se atoms per unit (Ladenstein 1979). Furthermore the assump- tion that 75 % of erythrocyte Se exists in GSH-Px-enzyme was made (Oh et al. 1974, Sunde et al. 1978). This activity was 2464, 2208, 2155 and 2101 kat/mol GSH-Px in the groups 1,2, 3 and 4, respectively. Only group 1 differed from the others (p<0.05). Plasma GSH-Px activity averaged 2.7, 3.2, 3.2 and 3.8 |ikat/l in the groups 1,2, 3, and 4, respectively (p > 0.05). Proportion of whole blood GSH-Px-activity associated with plas- ma was 0.89, 0.54, 0.46 and 0.55 % in groups 1,2, 3 and 4, respectively. Only group 1 differed from the others (p<0.01). Plasma GSH-Px activity did not correlate with plas- ma Se, erythrocyte Se or GSH-Px, or Se in- take, but it was negatively correlated with lac- tation stage (p<0.01). 50 5.3.5. Milk selenium Milk Se content differed (p< 0.001) be- tween groups, dietary organic Se being more effective (p < 0.001) in raising milk Se content than inorganic Se (Table 8). Calculated linear functions for the relationship between milk Se content (Y, mg/1) and dietary Se content (X, mg/kg DM) were for mineral Se and for feed Se Y = 0.007 +0.0160 X and Y =0.011 + 0.0256 X. The second degree polynomial func- tions were Y= 0.007 + 0.0240 X-0.000008 X 2 and Y =0.011+0.0441 X-0.000015 X 2, re- spectively. The difference in linear compo- nents due to Se source was highly significant (p<0.001). The following equation explained 91.4 % of the variation in milk Se content: Y =0.003 + P, + 0.0131 b, + 0.1227 b 2 -0.1549 b2 2 + 0.0654 b 2 3 where Y = milk Se content (mg/1) Pi = effect of experimental period (i = 1..6) b, = dietary mineral Se content (mg/kg DM) b 2 = dietary feed Se content (mg/kg DM) Dietary Zn content was negatively corre- lated to milk Se content in cows fed inorganic Table 8. Milk Se content (mg/1) in cows fed various Se diets (for group explanation see Table 1, p. 29). Period Group 12 3 4 I 0.006 0.008 0.007 0.007 ns. 0.006" 0.009» 0.017" 0.019" 0.008» 0.011» 0.020" 0.025" 0.008» 0.016" 0.029= 0.034" 0.008» 0.020" 0.040= 0.051 d 0.008» 0.011" 0.023= 0.027" 0.005 0.005 0.006 0.006 ll* I!** HI IV V VI * after 28 days supplementation ** after 56 days supplementation For significances see Table 4, p. 48. Table 9. Proportion of dietary Se in milk (%) in cows fed various Se diets (for group explanation see Table 1, P. 29). Period Group 12 3 4 4.43.3 4.04.3 ns. 11.6b 7.1» 9.6» 8.3» 19.7° 4.4» 9.4 b 5.7ab 18.3b 3.3» 4.1» 3.2» 35.0C 13.3» 28.7 bc 16.6»b 14.0» 17.8ab 17.5ab 20.2 b i n 111 IV V VI For significances see Table 4, p. 48. Se (p<0.01). Dietary S in cows fed inorganic Se (p<0.01), and dietary P both in cows fed inorganic (p<0.05) and organic Se (p<0.001) were positively related to milk Se. An average of 0.050, 0.047, 0.059 and 0.061 % of plasma Se circulating through the mammary gland was extracted and secreted into milk in the groups 1,2, 3 and 4, respec- tively. Only group 2 differed from groups 3 and 4 (p<0.05). Plasma Se (p<0.001), plas- ma Mg (p<0.01) and plasma cholesterol (p<0.05) were negatively, and milk Se (p< 0.001) and plasma P (p<0.05) positively related to the transfer of plasma Se into milk. Thus a lower percentage of plasma Se was transferred into milk with increasing plasma Se content. There was no difference between the Se sources. Neither lactation stage, milk production nor age of a cow affected the Se transfer from plasma into milk. The proportion of dietary Se transferred in- to milk was the greatest in group 1 (Table 9). The proportion of Se transferred into milk de- creased with increasing dietary Se content, and with inorganic Se this decline was greater than with organic Se source (p<0.01). Dietary minerals and trace elements, milk production or composition, lactation stage or age of a cow were not related to the efficiency of Se trans- fer from diet into milk. 51 6. DISCUSSION This investigation attempts to elucidate me- tabolism of Se originating from inorganic and organic sources. In the present discussion feed sprayed with selenite before cutting is con- sidered to be organic Se even though chemi- cal form of Se in feed was not identified. Results from other investigations have re- vealed, however, that selenite-Se is incorpo- rated into amino acids and further into plant proteins in less than 30 minutes (e.g. Peter- son and Butler 1962, Shrift 1969, Gissel- Nielsen 1976). Results from goats are generally regarded applicable to cows even though Debski et al. (1987) reported some discrepancies in sele- nium metabolism. However, goats were dosed with radioactive tracer in the present study, because the use of dairy cows in this part was beyond the facilities. Because the intraruminal dose of selenite in phase I of experiment 2 failed in one of the goats, only one observation for this treatment is available. These results should therefore be considered a demonstration of the principles prevailing in selenium metabolism, even though vigorous statistical evaluation is not possible. Both experiments involved principal- ly the same treatments and were well in line thus increasing reliability of the results. The length of the follow-up periods and sampling frequency were not identical in two goat ex- periments. Several tissue Se pools contribute to excretion of Se in milk, urine and faeces. Thus the length of follow-up period and sam- pling frequency significantly affect the rate constants obtained. Shorter sampling intervals towards the end of the follow-up period would have improved the accuracy of calculated rate constants, but probably would not have af- fected total excretion values, because most of 75 Se-label was excreted during the first days after dosing. Radioactive measurements from milk, urine and faeces were counted in triplicate, from plasma in duplicate and from erythrocytes in singlet. Erythrocyte 75Se values are thus the least reliable particularly the samples that had to be diluted to obtain equal geometry in counting. Also plasma contamination may have interfered with determined 75 Se activi- ties in erythrocytes during the first days after dosing. In experiment 2 attempt was made to avoid inaccuracy due to plasma contamination by measuring hematocrit in the erythrocyte suspension. Counting errors for 75Se activity ranged mainly between one and two percent and only in a very few cases exceeded five per- cent. Accurate figures were thus available for calculations. Roughage was fed to the cows as a group, which caused some inaccuracy in estimating dietary mineral and trace element intake. Therefor no significances concerning diets are given, although average values are suspected to represent true intake. The cows were sam- pled for blood parameters and milk Se con- tent only once during each experimental pe- riod except during the first two periods when they were sampled twice. Variation in samples taken during the period I were insignificant, but during the period II parameters describing Se status of the cows changed considerably be- tween the two samples in cows supplemented with Se indicating that the length of Se sup- plementation has to be considered carefully. Unequal length of supplementation periods 52 may thus have biased the calculated relation- ship between dietary Se content and the parameters describing Se status of the cows. More detailed investigations including the identification of chemical forms of seleno compounds in the course of metabolism should be conducted to confirm the conclu- sions made from the present data. 6.1. Absorption of Se The apparent absorption of Se in this study varied from 46.6 to 62.5 % for feed Se and 31.2 % to 61.5 % for selenite, in accordance with several other studies. Values of 46 % (Neathery et al. 1987) and 51 % (van Ryssen et al. 1987) have been found in calves fed low selenium diets, and values range from 36 % to 51 %in dairy cows fed supplemented ra- tions (Harrison and Conrad 1984a, 1984b). In sheep apparent absorption of Se has been reported to vary from 44 % to 51 % (Pope et al. 1979, Shariff and Khrisnamurti 1987, Zanetti et al. 1987). Results of apparent selenium absorption vary considerably be- tween studies and values as low as 11 % have been reported in dairy cows (Koenig et al. 1989) with 0.19 mg Se/kg DM intake. This low value was, however, obtained with a tech- nique which differs from those used most commonly. On the other hand as high as 97 % apparent absorption of Se in depleted sheep has been reported (Shariff and Khrisnamur- ti 1987). Though the available data on sele- nium absorption are relatively abundant, most of the results are not applicable to ruminants. Absorption values obtained for monogastric animals and humans mainly range between 90% and 100 % (e.g. Brown et al. 1972, Thomson et al. 1975, Rahim et al. 1986). However, lower values in long term experi- ments have been also reported (Robinson et al. 1978). True absorption of Se was similar from both inorganic and organic Se sources as well as from various dietary Se levels. True absorp- tion in experiment 1 was 64.5 %. In experi- ment 2 true absorption for feed Se was 63.3 %, but for inorganic Se was only 37.0 °/o. However, the value for inorganic Se in experi- ment 2 is based on data from only one goat and thus may not be reliable. This value was significantly lower (p<0.01) than the value obtained in experiment 1 for inorganic Se. Thus, if the result from experiment 2 for in- organic Se represents the real situation, the low absorption of inorganic Se could be due to a reduction of selenite to elemental Se or other insoluble forms of Se, such as metal selenides (Pope et al. 1979). The total amount of 75Se excreted during the follow-up period for this goat (92.1 °7o of the dose) was well above other types of dosing (range from 43.5 to 83.2 %) indicating that this might be the case. Langlands et ai. (1986) speculated based on their results in sheep that Se entering fae- ces had been reduced to unavailable forms, such as elemental Se during passage through the rumen. The quantity of Se reduced de- pends on the quantity of organic matter fer- mented in the rumen and is thus related to or- ganic matter intake. This is partly supported by the findings of Peter et al. (1985), but not by results from Harrison and Conrad (1984b). In the present study dry matter in- take was on the average 77 g/kg W075 in both experiments and thus cannot be the cause for the differences in absorption. An inactive carrier was used in experiment 2, but not in the experiment 1. Apart from using carrier, the procedures in the experiments were very similar. The total amount of intraruminally dosed Se was less than half of the total daily Se intake in experiment 2 and should thus not have disturbed absorption of selenite. It would have been expected to decrease reduction of labeled selenite to selenides in the rumen rath- er than the other way around. However, bene- fits and disadvantages of using carrier in this kind of study have to be carefully considered. Another possible reason, even though unlike- ly, for lowered Se absorption of 75Se-selenite with selenite feeding might be a saturation of the selenite absorption system. However, ab- sorption of selenite is primarily by passive 53 diffusion (Wolffram et al. 1985, 1986), and Lopez et al. (1968) did not find any decrease in Se absorption when feeding selenite diets containing Se up to 5 ppm to lambs. The goats in experiment 2 excreted almost three times as much of the intravenously dosed 75Se in faeces as the goats in experiment 1 (p< 0.001). However, there was no difference in faecal excretion due to the selenium source. In experiment 1 selenium deficient and sup- plemented goats excreted equal amounts of in- travenously dosed 75Se in faeces. The differ- ent length of the follow-up period does not explain the discrepancy between the experi- ments because about 95 % of excretion oc- curred during the first two weeks. Endogenous faecal excretion was similar to that obtained in most other studies with values of 11 %to 23 °7o in sheep (Langlands et al. 1986), 5 % in lambs (Lopez et al. 1968) and 8 to 17 % in cows (Symonds et al. 1981a). Earlier results from monogastric animals are around 10 % (e.g. Burk et al. 1972, Thomson and Stewart 1973, Hansen and Kristensen 1979, Kristen- sen and Hansen 1980) varying less than results from ruminants. In most studies dietary Se level has not af- fected proportion of endogenous Se excreted in faeces (Lopez et al. 1968, Burk et al. 1972), but Shariff and Khrisnamurti (1989) reported increased endogenous faecal excre- tion in Se supplemented sheep. The source of Se affected endogenous excretion of Se in dairy cows (Symonds et al. 1981a), but not in rats (Thomson and Stewart 1973). Arsenite has been reported to increase dramatically selenium excretion in bile in rats (4 to 40 °Io) (Levander and Baumann 1966), but not in sheep (Langlands et al. 1986). The main route for endogenous Se secretion in sheep is reported to be saliva (Dejneka et al. 1979) with other important routes being bile and digestive juices (Hansson and Blau 1963, Imbach and Sternberg 1967, Hidiroglou and Jenkins 1974, Symonds et al. 1981b). The an- terior part of the small intestine is reported in some studies to be the main site for Se secre- tion (Wright and Bell 1966, Hidiroglou and Jenkins 1974, Langlands et al. 1986). In the present study the transit time for 75 Se in faeces was shorter after an intravenous dose than after oral or intraruminal doses sup- porting the conclusion that secretion mainly occurs in anterior part of the small intestine. 6.2. Selenium in blood and hair In cows fed selenited feed a plateau in plas- ma Se content was calculated to occur at 0.113 mg Se/1, but in cows fed inorganic Se plasma plateaued at a slightly lower level. Unequal in- takes of inorganic and organic dietary Se caused some discrepancies in calculating the dietary level at which plasma Se plateaus, but after equalizing the range of dietary Se intake a plateau was calculated to be reached at ap- proximately 0.5 mg Se/kg DM. Previous re- search reports that a plateau occurs in dairy cows between 0.10 and 0.12 mg Se/1 plasma (Waite et al. 1975, Maus et al. 1980). Feed- ing cows Se up to 100 mg/d as selenite raised this level only to 0.14 mg/1 (Fisher et al. 1980). Stevens et al. (1985) reported an ex- tremely high value (0.78 mg/1) in grazing cattle. Values in that range, however, are not supported by any other investigations. In the present study goats fed Se at levels of 0.34 and 0.38 mg kg/DM had an average plasma Se content of 0.16 mg/1. The propor- tion of plasma Se excreted in milk and urine decreased rapidly during the first two days and slowly thereafter. The subsequent change to predominantly faecal excretion was not so clear due to long retention time in gut. How- ever, plasma Se removed to faeces was better related to endogenous excretion of Se than erythrocyte Se removed to faeces. In cows erythrocyte Se content decreased during the first weeks of the first Se sup- plementation period, which probably was due to the slight decrease in dietary Se content which was necessary to adhere to standard recommendations during the two months preceding the experiment. Because the turn- over in erythrocyte Se content is known to be slow (Whanger and Beilstein 1986a), the ef- 54 feet of the dietary changes appeared after a lag period. Depressed erythrocyte Se content towards the end of gestation observed in the present study is probably due to retention of Se in fetus, which is reported to sequester blood Se from the dam with higher priority than dam's own tissues (Koller et al. 1984). Even though differences in erythrocyte GSH-Px between the groups fed either inor- ganic or feed Se were not significant, interac- tion between the Se source and dietary Se level indicated that feed Se maintains erythrocyte GSH-Px activity better than inorganic Se when cows are depleted. The slight difference between the sources when increasing dietary Se levels were fed may be due to unequal Se intake in the groups. Pehrson et al. (1989) have demonstrated in heifers that organic Se increases erythrocyte GSH-Px activity more efficiently than inorganic Se. The opposite has been reported in pigs (Sankari 1985). During the periods V and VI when Se in- take was gradually reduced the activity of GSH-Px enzyme (activity kat/mol of GSH- Px) was markedly increased indicating either changes in the enzyme activity itself or pre- ferred retention of Se in the GSH-Px enzyme instead of other selenoproteins. The main rea- son for poor correlation of Se intake with erythrocyte GSH-Px activity was the time re- quired for depletion during the periods when decreasing dietary Se levels were fed, even though these periods lasted about four months. Butler et al. (1990) reported that in the rhesus monkey erythrocyte GSH-Px activi- ty was saturated in 4.5 months when animals were fed selenomethionine, but not within 8 months when selenite was the source of sele- nium. When rats were depleted, GSH-Px ac- tivity decreased more slowly in those animals which had been supplemented with seleno- methionine than in those supplemented with selenite (Beilstein and Whanger 1986a). A correlation between dietary Se and erythrocyte GSH-Px activity has been demonstrated in sheep (Oh et al. 1976, Whanger et al. 1977), in cattle (Thompson et al. 1976, Hoffman et al. 1978), in pigs (Chavez 1979) and in rats (Hafeman et al. 1979), but not in humans (Butler et al. 1982). Generally when dietary Se intake is low, the correlation between die- tary Se and erythrocyte GSH-Px is poor (Whanger et al. 1977). The proportion of total GSH-Px activity in blood associated with plasma, ranging in the present study between 0.5 % and 0.9 %, is well in line with earlier studies which report the proportion to be about one per cent (Carlström 1979). In experiment 1 only 75Se activity in hair was measured, but no attempt was made to estimate the growth of hair so it is not pos- sible to calculate deposition. There were no differences due to dietary Se level in hair 75Se activity and, because of the fact that growth of hair occurs within the skin, comparing of sources during this short-term experiment may not be valid. In experiment 2, 75Se retention in hair was similar whether goats were dosed orally selenited grass or I.R. selenite, but af- ter I.V. dose more of 75Se was retained in hair when dosed as selenomethionine than as sodium selenite. In rats selenomethionine was two to three times more efficient than sodium selenate in raising Se content in hair, but die- tary methionine content affected retention of Se in both sources (Salbe and Levander 1990). 6.3. Excretion of Se in urine 75Se excretion in urine was low in depleted goats (5.7 %of a dose and 11.4 %of excreted Se) which is in agreement with results from other studies (Hopkins et al 1966, Lopez et al 1968, Burk et al. 1972). A value as high as 50 % has been reported (Langlands et al 1986), but the high excretion value was ob- tained by using a different technique from the present one. Goats dosed with 75Se-selenite excreted about twice as much of the dose in urine as goats dosed with an organic source of 75Se, whether the calculation was based on actual excretion or on the excretion of truly absorbed 75Se. 55 56 Differences due to selenium source in uri- nary Se excretion have been evident both at low and high Se intakes (Thomson and Stewart 1973, Nahapatian et al. 1983, Fos- ter et al. 1986b). On a low Se intake (0.05 ppm) urinary excretion of Se in rats was about 4 % and 13 % of orally administered seleno- methionine and selenite, respectively. Excre- tion of intravenous doses was slightly higher (Thomson and Stewart 1973). With adequate Se intake (0.25 ppm) about 30 % of seleno- methionine, 40 % of selenocysteine and 45 % of selenite were excreted in urine in rats (Na- hapatian et al. 1983). In the present study, excretion of in- travenously dosed 75Se-selenomethionine in urine increased linearly with increasing dietary Se content (p<0.05). Excretion of feed 75Se did not increase above a dietary Se content of 0.22 mg/kg DM, whether the calculations were based on actual excretion or on excre- tion of truly absorbed 75Se. The excretion pattern for selenite was similar when the cal- culations were based on actual excretion, but not when they were based on the excretion of truly absorbed 75Se. Excretion of truely ab- sorbed selenite increased linearly with in- creasing dietary Se content. This is probably more accurate because the lowered absorption with the high selenite intake obtained in ex- periment 2 leads to underestimated actual ex- cretion values. Increased urinary excretion of 75Se with in- creased Se intake is in agreement with several other studies reporting that urinary Se excre- tion was 40 to 67 °?o of the dose at high Se intakes (e.g. Burk et al. 1972 and 1973, Lo- pez et al. 1968, Hansen and Kristensen 1979, Alfaro et al. 1987). Results in rats vary concerning the effect of the Se dose on urinary Se excretion. Some of the studies report no dose-dependency (e.g. Hopkins et al. 1966, Palmer et al. 1970, Kristensen and Hansen 1980), while others show dose-dependency at physiological levels but not at higher levels (Burk et al. 1972). Nahapatian et al. (1983) reported an absence of dose dependency for selenomethionineand selenocysteine, whereas selenite excretion in urine decreased with increasing Se dose. These discrepancies are most likely due to volatili- zation of Se at high doses. Thus a very high dose may result in a rather low percentage of injected Se being excreted in urine. Most of Se dose is exhaled as dimethylselenide, which is an intermediate product in the TMSe path- way. TMSe is the main urinary Se metabolite when Se is fed at high levels (Palmer et al. 1969). Based on regression analysis of data from dosing with organic sources of Se, excretion of intravenously-given Se in urine exceeded that of orally-given Se at a dietary Se level of 0.13 mg/kg DM. Plasma Se plateaus at about this dietary Se level and thus the ability of plasma to bind Se may be exceeded with the excess Se being filtered by the kidneys. How- ever, with increased dietary Se, a large propor- tion of the Se excreted in urine was excreted towards the end of follow-up period. Burk et al. (1973) studied the dietary thresholdfor uri- nary excretion and found it to be between 0.05 and 0.08 ppm above which the percentage of the dose excreted in urine increased up to at least a level of 1 ppm (Burk et al. 1972). They speculated that the increase in urinary excretion is probably related to metabolic pro- duction of substances such as TMSe or that therenal threshold for these metabolites may be altered. This is not supported by Nahapa- tian et al. (1983) using selenomethionine, selenocysteine and selenite. In the present study 75Se clearance from plasma to urine was dependant on a dietary Se level and time, but for the most part not on Se source. The most distinct discrepancy between the sources occurred after the in- travenous dose at a high dietary Se level with much higher clearance of selenite than seleno- methionine (550 vs. 150ml/h) during the first 8 hours post dosing. After 24 h the clearance for both sources was equal, about 30 ml/h. When dietary Se level was increased from 0.05 mg/kg DM to 0.22 mg/kg DM the rate of clearance increased 4 to 6 times, but there was no further increase above the Se level of 0.22 mg/kg DM. After an oral or intraruminal dose the plasma clearance in supplemented goats decreased from about 230 ml/h during the first hours to about 24 ml/h and in de- pleted goats from about 100 ml/h to 4 ml/h. In depleted goats after I.V. dose of seleno- methionine plasma clearance never exceeded 75 ml/h and in 8 hrs it fell below 5 ml/h reaching a level of about 2 ml/h few days later. Thus regardless of dosage route depleted goats cleared much less Se from plasma than supplemented goats. The dose of 75 Se contained less than 10 ng of Se which contributed roughly 10 % excess Se to plasma in depleted goats and even less in supplemented goats. Se which exceeds the binding capacity of plasma is rapidly taken up by tissues, most notably liver and kidney, and is processed to compounds which may be re- tained in the body and released later in the continuous turnover of body Se pool (Sy- monds et al. 1981b, Beilstein et al. 1984). Zeisel et al. (1987) speculated based on their results in the rat that production of TMSe is dose dependant, but excretion of a single dose was dependant on the previous Se intake. This is not supported by the findings of the pres- ent study, probably because Zeisel et al. (1987) were operating at relatively high Se in- takes, their lowest Se level being about at the same as the highest level in the present study. Above the level of 0.2 mg Se/kg DM there was a tendency for decreasing plasma clearance to urine after both oral and intraruminal doses, but not after intravenous doses. Thus the speculation of Zeisel et al. (1987) could be valid, but only when intake of Se is in a range which more closely approximates require- ments. 6.4. Excretion of selenium in milk In goats neitheractual excretion of feed in- corporated 75 Se in milk, varying from 6.7 % to 8.1 % of the dose, proportion of total 75 Se excretion excreted in milk, varying from 11.3 % to 13.2 %, nor excretion of truly ab- sorbed 75 Se, varying from 10.2 % to 12.7 %, were dependant on dietary Se level. The ac- tual excretion of I.V. dosed 75Se-selenome- thionine in milk also was not dependant on Se intake. However, when expressed as a proportion of total excretion, relatively less 75Se was excreted in milk with increasing dietary Se level due to higher urinary 75 Se losses (76.4 %, 52.8 % and 38.7 °/o at dietary Se levels 0.05, 0.22 and 0.34 mg Se/kg DM, respectively). Higher 75Se excretion in milk after an I.V. dose than after an oral dose was due primarily to excretion very shortly after the I.V. dose. With higher Se intake more of dosed Se is metabolized to compounds which are not excreted to milk, but are excreted in urine (Nahapetian et al. 1983). Maximum excretion in milk tended to oc- cur earlier with increasing dietary Se level both with feed-incorporated 75Se and 75Se-selenite. There was no difference in k, or k 2 due to dietary selenium levels, but the slowest com- ponent (k 3 or k 4) tended to be faster with in- creasing selenium intake. Length of sampling period and sampling interval play an impor- tant role in determining rate constants in this kind of study, where several pools are con- tributing to the sampled pool. Selenium con- tent in different tissues varies greatly and a given tissue may contribute to several pools (Burk et al. 1972, Behne and Höfer-Bosse 1984). A longer follow-up period would en- able determination of the slowest turnover rate, and more frequent sampling in the early phases would allow closer determination of k, and k 2. After an oral dose of 75Se-labeled grass in depleted goats, milk 75Se activity only slight- ly exceeded that of plasma for the first four hours; thereafter 75Se activity in milk was lower than in plasma. At dietary Se levels of 0.22 and 0.34 mg Se/kg DM, milk 75Se ac- tivity was above plasma 75Se activity until 24 and 30 hrs, respectively. This indicates that or- ganic selenium is at least partly transferred into milk by some active process thus con- firming results from Jenkins and Hidiroglou (1971). The ratio of 75 Se in milk to 75Se in plasma was dependant on dietary selenium 57 level. Generally changes in milk 75Se : plasma 75Se ratios during the first 12 hrs were not great. A plateau was reached at 6, 8 and 9 days post dosing at ratios 0.09, 0.10 and 0.15 for dietary Se levels of 0.05, 0.22 and 0.34 mg/Se kg DM, respectively, (p<0.01). Plasma 75Se was transferred into milk on the average 1.5 times more efficiently after an I.V. dose than after an oral dose of 75Se. At highest Se intake 75Se activity was 25 times higher in milk than in plasma one hour after dosing with I.V. 75Se-selenomethionine. In depleted goats the maximum concentration in milk was only four times higher than in plas- ma. Milk 75 Se : plasma 75 Se ratios, however, decreased rapidly after the I.V. dose and 20 hrs post dosing were less than one at each Se level. Plateaus in these ratios were reached in depleted goats 10 days post dosing, but in sup- plemented goats not until two weeks post dos- ing. The heights of these plateaus were about 1.5 times higher than after the oral dose. Enhanced efficiency of plasma Se transfer into milk with increasing dietary Se content might reflect saturation of plasma Se concen- tration which is reported to occur about at the level of 0.1 mg/1 (Waite et al. 1975, Maus et al. 1980). However, this saturation was not found in the present goat experiments; goats fed 0.34 mg organic Se/kg DM and 0.38 mg inorganic Se/kg DM had average plasma Se concentrations of 0.17 mg/1 and 0.16 mg/1, respectively Excretion of intraruminally dosed 75Se- selenite in milk also was similar (5.3 % to 6.6 %) at all Se intakes when calculated based on truly absorbed 75 Se. Maus et al. (1980) reported milk Se content to be linearly related to dietary Se intake up to the level of 0.3 mg/kg, but not above that. This was, how- ever, because of their use of an experimental ration which contained a high level of natu- ral feed Se and this diet was supplemented with sodium selenite. Excretion of 75 Se-selenite in milk after an intravenous dose was similar to that after an intraruminal dose. However, excretion of in- travenous 75Se-selenite in milk was only about 10 % that of selenomethionine. When inorganic selenium was administrat- ed intraruminally, the lowest milk 75Se : plas- ma 75 Se ratios were reached at 1, 4 and 6 days at dietary Se levels 0.05, 0.22 and 0.38 mg Se/kg DM, respectively. The ratios were lower for depleted goats than for supple- mented goats. After reaching minimums the ratios began to increase until 14 days, prob- ably due to the release of selenocompounds from some slow turnover pool and having greater km -value for excretion in milk. A milk-Se plateau was reached at 20 hrs af- ter the I.V. dose of 75Se-selenite. The increase in milk 75 Se: plasma 75 Se ratios found after the I.R. dose was not discernible after the I.V. dose of selenite. Enhanced transfer of plas- ma Se into milk with increased dietary Se level was greater when organic forms of Se were used. In cows the correlation between dietary Se content and milk Se content was high (p< 0.001) both with organic (r: 0.85) and inor- ganic Se (r: 0.79). This is in agreement with results from Perry et al. (1977) on selenite (r: 0.94) and Conrad and Moxon (1979) on selenite (r: 0.77) and on brewers grain (r: 0.89) (Conrad and Moxon 1979). Fisher et al. (1980), did not find a correlation between dietary Se content and milk Se content, but the 8 days supplementation period in that study was probably insufficient. Plateaus in milk Se content were reached at 0.025 and 0.043 mg/1 in cows fed mineral and feed selenium, respectively. It was anticipated that a plateau in milk Se would be reached at a dietary level of 1.5 mg Se/kg DM with both Se sources. However, in the group receiving both mineral and feed selenium supplemen- tation at high levels (1.8 mg/kg DM) milk Se content exceeded the expected plateau level. This suggests either some disturbance in Se secretion in milk as speculated by Fisher et al. (1980) or that secretion of inorganic and organic selenium into milk does not share the same route and thus saturation occurs at higher level. Derivation of third degree poly- nomial functions fitted on the data indicated 58 that some kind of anomaly occurs in the trans- fer of Se into milk, because derivatives nei- ther for organic nor for inorganic Se have any solution. A second degree polynomial func- tion fitted on the data, gives a plateau for both Se sources, but fails to explain the effect of using both forms of Se-supplementation. In cows fed organic selenium blood Se cir- culating through the mammary gland was re- moved about 1.5 times more efficiently than in cows receiving inorganic supplementation. The difference was thus more apparent in goats than in cows. This could be due to differences in the distribution of Se in milk of goats and cows (Debski et al. 1987). Maus et al. (1980) concluded that cows fed sufficient Se up to a level of 0.6 ppm excrete 15 % of dietary Se in milk, but above that level only 7—lo %. They were, however, sup- plementing a diet containing 0.33 mg Se/kg DM natural selenium, which thus resulted in relatively high concentrations in milk. If the basal diet had contained less natural feed Se, much less of the Se would have been trans- ferred to milk (e.g. at a level of 0.77 mg/Se kg DM about 1.5 % of added Se). Based on their results they concluded that milk Se con- tent plateaus at 0.06 ppm. Fisher et al. (1980) did not notice this plateau in their experiment in which they were operating at Se dietary levels up to 10 ppm. They concluded that above toxic Se levels secretory controls may be overridden and more Se may be transferred into milk. They regarded selenite as a safer supplementation form for dairy animals, be- cause milk Se content was less responsive to dietary selenite unless a toxic amount of selenite was used. Milk Se content was positively related to plasma (r: 0.78, p<0.001) and erythrocyte Se content (r: 0.83, p<0.001) and to milk pro- tein content (r: 0.23, p<0.01). Milk Se is bound almost entirely to proteins (Jenkins and Hidiroglou 1971, Khirwar and Arora 1977). All correlations were higher in organic Se than in inorganic Se groups. Calcium intake was positively correlated to milk Se content, but this is due at least partly to high Ca content in the inorganic Se sup- plement. However, this correlation was also found in animals not receiving the selenium preparation. Dietary Ca content may affect Se absorption (Harrison and Conrad 1984b). With organic selenium Zn intake was negative- ly (r: -0.37, p<0.01) correlated to milk Se content. Sword et al. (1984) did not, howev- er, find any effect of Ca and Zn on whole blood Se content or utilization of Se. 59 7. GENERAL CONCLUSIONS True absorption from inorganic and organic sources of selenium is similar and is not af- fected by dietary selenium level. Organic selenium is more efficient in in- creasing plasma selenium content and sele- nium-supplemented animals maintain plasma selenium levels longer when depleted. Inor- ganic and organic selenium sources are simi- lar in their ability to increase erythrocyte selenium content. The efficiency of inorganic and organic selenium in increasing erythrocyte GSH-Px- activity is similar, but previous organic sele- nium supplementation results in higher GSH- Px-activity whenanimals are depleted. Eryth- rocyte GSH-Px-activity did not reach a pla- teau with selenium supplementation which was below toxic levels. About twice as much selenium was elimi- nated in urine after dosing with inorganic selenium as after dosing with organic sele- nium. Organic selenium supplementation results in greater seleniumcontent in milk than inor- ganic selenium supplementation. When sele- nium-supplemented cows were depleted, ani- mals fed natural selenium containing feed maintained milk selenium better than animals fed inorganic selenium. In order to raise milk selenium content to the level of 0.020 mg Se/1, which was regarded by the group to be sufficient, animals must be fed either 0.7 mg/ kg DM inorganic sele- nium or 0.1 mg/kg DM organic selenium. Organic selenium supplementation is safe and more efficient than inorganic selenium and does not risk toxic selenium intake by con- sumers, because milk selenium content pla- teaus with high dietary levels. 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Nutr. 117:1609—1614. 68 SELOSTUS Seleeniaineenvaihdunta maitoa tuottavalla vuohella ja lehmällä Pentti Aspila Helsingin yliopisto kotieläintieteen laitos 00710 Helsinki Tutkimuksen tarkoituksena oli selvittää epäorgaanisen ja orgaanisen seleenin aineenvaihduntaa maitoa tuotta- villa vuohilla ja lypsylehmillä useilla seleenitasoilla. Tutkimus koostuu kahdesta vuohilla ja yhdestä lehmillä suoritetusta kokeesta. Vuohille annettiin kerta-annoksena joko viikoa ennen niittoa Na 27S Se03:llä ruiskutettua ruo- hoa, pötsinsisäisesti (1.R.) Na 2 75 5e0 3 :ä tai suonensisäises- ti (1.V.) Na2 75 5e0 3 tai 75 Se-selenometioniinia. Seuranta- jaksojen pituus oli 15—28 d. Käytetyt seleenitasot olivat 0.05, 0.22, ja 0.34 mg Se/kg ka. Tulokset esitetään 7! Se imeytymisestä, erittymisestä maidossa, virtsassa ja son- nassa sekä 75 Se-aktiivisuudesta plasmassa, erytrosyyteissä jakarvassa. Lehmillä suoritettu koe kesti 539 d ja siinä 48 lehmälle annettiin joko Na2 Se0 3 :ä tai säilörehua, jolle oli viikkoa ennen korjuuta ruiskutettu Na2Se0 3:ä. Diee- tin seleenipitoisuus vaihteli välillä 0.03 ja 1.8 mg Se/kg ka. Tulokset esitetään maidon, plasman ja erytrosyyttien seleenipitoisuudesta sekä plasman ja erytrosyyttien GSH- Px -aktiivisuudesta. 75 Se todellinen imeytyminen oli 63 %ja6s % sekä erittyminen maidossa 4 °7o ja 7 % vuohilla, joille annet- tiin Na2 75 5e03 I.R. tai 75Se:llä leimattua ruohoa. Dieetin seleenipitoisuudella ei ollut vaikutusta 7! Se:n imeytymi- seen tai erittymiseen maidossa. Kun 75Se annettiin LV., 3.6 % seleniitistä ja 33 % selenometioniinista erittyi mai- dossa. Na 2 75 5e0 3 erittyi pääasiassa virtsassa. Lehmillä, joilla Na2 Se0 3 oli seleenin lähteenä, maidon seleenipitoisuus oli 0.011, 0.011, 0.016 ja 0.020, kun die- tin seleenipitoisuus oli 0.11, 0.17, 0.42 ja 0.68 mg Se/kg ka. Selenoidun säilörehun ollessa seleenin lähteenä mai- don seleenipitoisuus oli 0.023, 0.020, 0.029 ja0.040 mg/l dieetin seleenipitoisuudenollessa 0.09, 0.20, 0.45 ja 1.20 mg/kg ka. Selenoitu säilörehu nosti maidon seleenipitoi- suutta voimakkaammin (p<0.001) kuin Na 2Se0 3 . Kun seleenitäydennyksen jälkeen eläinten seleenin saantia ra- joitettiin aleni maidon seleenipitoisuus nopeammin leh- millä, jotka olivat saaneet Na2 Se0 3:ä kuin lehmillä, jot- ka olivat saaneet selenoitua säilörehua. 69 Appendix 1. Rate constants, retention time and half-life in experiments 1 and 2. Experiment 1. Oral dose of 75 Se-labeled grass (Phase I). Milk Urine Faeces D 1 S 2 D S D S k, 1.623 1.919 N.D. N.D. 1.484 2.406 k 2 0.778 0.849 0.664 0.389 0.961 1.040 k 3 0.061 0.096 0.062 0.065 0.081 0.039 TMRT 18.7 12.6 18.3 22.5 52.2 28.1 T* 11.5 7.4 11.6 13.7 34.7 18.2 1 Goats fed diet containing 0.05 mg Se/kg DM (n =2) 2 Goats fed diet containing 0.22 mg Se/kg DM (n =2) N.D., not determined Intraruminal dose of 75Se-labeled sodium selenite (Phase II). Milk Urine Faeces D S D S D S k, N.D. 2.707 3.742 2.127 2.637 2.169 k 2 1.462 1.380 0.898 1.105 0.909 1.026 k 2 0.027 0.048 0.019 0.068 0.139 0.102 TMRT 38.4 23.7 53.0 17.5 9.9 12.0 T* 26.1 15.5 35.7 11.0 5.3 6.8 Intravenous dose of 75 Se-labeled selenomethionine (Phase III) Milk Urine Faeces I) s I) S I) S k, k; k 3 1.546 4.959 0.627 1.204 0.090 0.100 13.7 13.0 7.7 8.2 11.238 9.952 0.858 1.716 0.032 0.043 35.8 31.4 23.8 20.4 4.183 5.171 0.042 0.074 25.5 16.3 12.8 10.1 TMRT T 70 71 Appendix 1 (continued). Experiment 2. Oral dose of 75Se-labeled grass (OR) or intraruminal dose of 7! Se-labeled sodium selenite (IO) (Phase I). Milk Urine Faeces lO' OR2 I O OR IO OR k, 2.654 5.348 1.518 1.779 0.237 0.364 0.054 0.049 2.997 2.099 2.655 1.027 0.112 0.094 1.386 1.327 0.891 0.685 0.078 0.082 TMRT 38.4 23.7 26.1 15.5 53.0 17.5 35.7 11.0 9.9 12.0 T 5.3 6.8 1 Goats fed sodium selenite 0.38 mg Se/kg DM (n= 1) 2 Goats fed selenited barley 0.34 mg Se/kg DM (n =2) Intravenous dose of 75 Se-labeled selenomethionine (OR) or 75 Se-labeled sodium selenite (IO) (Phase II). Milk Urine Faeces lO' OR 2 IO OR lO OR k, k. 5.060 9.902 1.009 1.837 0.274 0.324 0.068 0.059 19.6 20.7 10.2 11.8 3.460 2.894 1.700 2.252 0.073 0.068 1.240 1.976 0.082 0.090 TMRT 13.7 15.2 9.7 10.4 13.5 12.3 T 8.4 7.8 1 Goats fed sodium selenite 0.38 mg Se/kg DM (n =2) 2 Goats fed selenited barley 0.34 mg Se/kg DM (n = 2) 72 Appendix 2. Mineral composition of feeds in experiment 3. Feed Control silage Selenited silage Grass Hay Phase II 111 IV VI II 111 IV V lI—III IV V—V Ca, g/kg DM 9.7 8.0 5.7 8.2 7.9 7.5 4.7 11.4 7.7 3.1 2.4 P, g/kg DM 2.5 3.1 3.0 4.0 2.9 3.1 3.5 4.2 3.1 2.3 2.5 Mg, g/kg DM 2.7 2.4 1.7 2.2 2.1 2.3 1.5 3.0 1.6 1.0 1.0 S, g/kg DM 1.9 1.9 1.5 2.2 2.3 2.1 2.3 2.6 1.5 1.1 1.1 Fe, mg/kg DM 518 1023 330 549 180 490 260 1865 218 98 124 Zn, mg/kg DM 30 32 30 57 29 25 38 68 27 21 26 Se, mg/kg DM 0.03 0.03 0.02 0.02 0.20 0.55 1.69 0.03 0.02 0.01 0.02 Mineral composition of feeds (continued) Feed Concentrate Protein Selenited Mineral mixture Selenite concentrate barley preparatio Phase 11—III IV V—VI 11—IV V—VI V lI—III IV V—VI lI—V Ca, g/kg DM 6.5 6.5 7.5 23.1 20.9 0.4 193 193 206 370 P, g/kg DM 5.8 5.7 6.8 9.8 10.5 4.6 64 64 82