Effects of barley-bound organic selenium compared with inorganic selenite on selenium concentration and structure of tissues in pig

. A study was made of the effects of barley feeds containing varying levels of natural grain selenium derived from fertilizer and of the effects of sodium selenite on selenium concentrations in the tissues of growing pigs and on the histological structure of myocardium and longissimus muscle. The results indicated that organic grain selenium affects the selenium levels of porcine tissues significantly more than sodium selenite supplementation of the same amount. The organic selenium concentrations of feed and organs were found to be linearly correlated. Spleen, lung and heart were the most reliable indicators of tissue selenium level in pigs. The selenium concentration ofmuscles of pigs fed on naturally seleniferous feed (270 gg Se/kg DM) was at the internationally level (500 gg Se/kg DM in meat), whereas the same amount of selenite in feed increased the selenium level of muscle only slightly (to 200 /ig Se/kg DM). The histological structures of heart and muscle of pigs fed on naturally seleniferous feed were normal, whereas in the selenite group there were signs of muscle dystrophy when the selenium level was below 200 gg Se/kg DM. In addition, the pigs given selenite were subse-quently placed in a lower, fat-containing carcass class. Feed with a natural organic selenium level of 150—200 gg Se/kg DM is preferred to selenite selenium supplement in pork produc- tion.


Introduction
The low selenium content in Finnish fodder plants (Oksanen andSandholm 1970, Kurkela 1982) makes it necessary to add selenium compounds to feeds during their production. Inorganic selenium supplements in feed have not always had the desired effect. Nutritional muscular dystrophy (NMD) has occurred even when the selenium content of the feed has been 0.1 mg Se/kg DM.
Selenium-containing fertilizers can also be used to elevate the selenium content of feed. The changes in the selenium contents of Index words: pig, sodium selenite, organic selenium JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND animal tissues resulting from the use of these fertilizers for growing feed grain should be investigated and the effects of elevated tissue selenium contents examined from the point of foodstuffs hygiene.
The object of the present study was to investigate the effects of feed with varying levels of organic and inorganic selenium on health organ selenium content macroscopic and histological structure of the muscles in the pig.

Animals
Twenty-five Yorkshire pigs with an initial weight of 25 ± 0.5 kg were used in this study. They were divided into four groups (6 + 6 + 7 + 6). Each group received the test feed shown in Table 1 over a period of 110 days. In addition, samples for comparison were taken from the slaughterhouse from healthy pigs and from pigs with NMD.

Feed and feeding
Kemira Oy had grown barley with different selenium levels by fertilizing it one month after sowing with 0 to 1.0 kg sodium selenite per hectare (Korkman 1980). The feeds were mixtured from these barleys and protein mineral concentrate containing 40 % raw protein and 18.5°7o minerals (Table 2) and from sodium selenite (Table 1). All the feeds used in the trial contained 30 HJ/kg of vitamin E. All animals were given the group 1 feed for the four weeks leading up to the trial. The pigs were kept in sties with concrete floors. They obtained test feed and water freely from automatic dispensers.

Follow-up
The animals were examined clinically once a week. They were slaughtered after 110 days. The carcasses were examined visually at the time the meat was inspected and classified in the slaughterhouse.

Samples
Blood and faeces samples were taken at the end of the trial. Samples of urine and bile were taken after slaughtering, together with tissue samples from the spleen, liver, muscle (dorsal longissimus muscle), lung, kidney, pancreas, brain, CNS, eye and coccyx. The tissue samples were taken by cutting a sagittal or otherwise representative sample from the organ or from one of its lobes. A sample of myocardium was taken from the papillary muscle and the left ventricle wall. Samples from equal-sized control pigs of the same strain from the same slaughterhouse used as random sample material were taken from the longissimus muscle and the myocardium both from clinically healthy animals and from those with nutritional muscular dystrophy (NMD).
The samples were frozen immediately and kept at -2O°C until analysed. Histological samples from the muscle, myocardium, liver and kidney were stored in 10 % formalin.

Chemical analysis and histological examination
The method employed in the selenium determination was Saari and Paaso's (1980) modification of the hydride method coupled with atomic absorption spectrophotometry. The lowest measurable selenium content by this method is 10 fig Se/kg.

Statistical analysis
The following methods were used in the statistical analysis of the results: The differences in tissue trace element contents between the feed groups were tested for homogeneity of variances by Bartlett's test, differences between means by one-way variance analysis and significantly different means were discriminated by Tukey's test. the dependence of tissue selenium level on feed selenium level was tested by linear regression analysis with feed selenium content as the systematic variable. Both the logarithmic function and the linear model were tested in the analysis.
The correlations between the Se-levels of different organs were calculated using a correlation coefficient.
Quality classification of the carcases and slaughtering weights were tested using Fischer's accurate four-field test, one-way variance analysis and Tukey's test.

Results
The test pigs were clinically healthy during the tests. The mean slaughtering weights in the different groups are shown in Figure 1. No statistical differences were found in these.
In the quality classification, five pigs out of six in Group II were found to be fatty and were placed in class I -. The rest of the pigs were classified as belonging to class I ( Figure 1).
The mean selenium contents of samples taken from test animals are shown in Table 3.
It was found that sodium selenite supplement in the basic feed (270 Se/kg DM) in Group II did not statistically significantly elevate the selenium content of the muscle and other organs, whereas a corresponding selenium supplement in organic form (Group III) resulted in a statistically significant increase. In the muscle this difference in the elevation of the selenium contents was about 250 %.
The change in the organ selenium contents in Groups I, II and IV was linear. The dependence of muscle selenium content on the level In the test groups, the selenium content of blood correlated best with the selenium con-tents of the myocardium, liver, CNS and bile (0.998 < r < 1). Blood selenium content was only indicative of the selenium content of muscle. The most reliable indicator organs of the body selenium content were found to be  the spleen and the lung, followed by the heart and the brain (  nutritional muscular dystrophy 641 ± 159/tg Se/kg DM, i.e. no statistically significant differences were found. The mean selenium content of the meat of pigs with clinical muscular dystrophy was 378 n g Se/kg DM, which deviated greatly from the corresponding means in Groups I, II and IV, while the mean selenium content of the myocardium, 641 /ig Se/kg DM, differed significantly from the mean selenium contents of Groups 111 and IV.
High biliary selenium contents were found in Groups 111 and IV, whereas in Group II it was the selenium content of the urine and faeces that was high. The biliary selenium contents were in agreement with the blood selenium contents and correlated significantly with the selenium contents of the myocardium and other internal organs.
Macroscopically, no changes were observed in the meat or organs of the test animals. Histologically in Groups I and II incipient degradation of the the myofibres was often seen in the longissimus muscle and the myocardium, as characterized by swelling of the myofibrils, absence of the sarcoplasm and myolysis. The myocardium showed loss of cross-striation and segmentation of myofibres (Figures 4,5, 6). These degenerative muscular and myocardial changes were manifested progressively with increasing severity when the muscular selenium content fell to 200-150 /xg Se/kg DM or below. In pigs with clinical nutritional muscular dystrophy, these histological changes were found in all samples of the longissimus muscle in which the selenium content was 200-450 /xg Se/kg DM.
Progressively increasing degradation changes were observed in the myocardium of the pigs in Groups I and II and of the clinically healthy control animals when the myocardium selenium content fell to 600-700 /xg Se/kg DM or below. The selenium content of the myocardium of pigs with clinical nutritional muscular dystrophy was 600-850 /xg Se/kg DM.

Discussion
The clinical condition of the test animals was good during the trial. The selenium contents used did not adversely affect the health of the pigs. This was as expected, because the selenium contents of the test feeds were clearly between the selenium contents causing deficiency states (50 /xg Se/kg DM) reported by Latshaw et al. (1977), and the content found to be toxic in pigs (5000) /xg Se/kg DM). The selenium content of the feed given to Groups II and 111, 270 /xg Se/kg DM, was of the same order as the mean natural selenium content of feed grain grown in the selenium-rich area of the USA (Kubota et al. 1967).
The different feeds used resulted in different selenium contents in the pig tissues. In Group I and in the healthy control group the selenium contents were 133 ± 23 /xg Se/kg DM and 150 /xg Se/kg DM, corresponding to the selenium content of Finnish pork reported by Nuurtamo et al. (1980), which in turn is lower than the selenium contents of foreign  (Lindberg 1968, Morris and Levander 1970, Ku et al. 1972, Norrman 1977. In Group II (feed containing 270 fig Se/kg DM as sodium selenite) the selenium content of the pork was 200 ng Se/kg DM. The elevation is small although the selenium content of the feed was three times greater than in Group I. In Group 111, in which the feed contained the same amount of organic selenium of cereal origin, the selenium content of the muscle rose to 500 fig Se/kg DM and in Group IV to 1000 fig Se/kg DM. The selenium level in the muscles was closely related to the organic dietary Se levels the pig received (p < 0.001).
The differences could also be seen in tissue selenium determinations in different organs: the difference between the biological effect of sodium selenite and organic selenium of cereal origin is shown in the selenium contents of Groups II and 111. The different chemical compounds of selenium, organic and inorganic, have different metabolic pathways in the organism (Cary et al. 1973). Selenomethionine and selenocysteine are biologically very active in pig (Ku et al. 1972).
In plants such as wheat and barley, most of the selenium is incorporated into protein as selenomethionine (Olson et al. 1970). In fish meal, selenium is known to be present as selenocysteine, the bioavailability of which has been found to be lower than that of selenomethionine.
Monogastric mammals cannot incorporate selenite selenium directly into cysteine or methionine, although the bacteria of intestinal tract, e.g. E. coli, can synthesize organoselenium compounds from selenite, which may explain the conversion of Se to nonlabile reduced forms in vivo (Allaway 1973).
The selenium bound in wheat or barley in the form of selenomethionine has a better bioavailability than the same quantity of selenium bound in meat or fish meal as selenocysteine (Ku et al. 1972). This is probably one reason for the low selenium content of Finnish pork, since the selenium content of Finnish feed grain is low, 5-20 fig Se/kg DM, and the pig feed mixtures used contain meat and fish meal as the source of protein.
In the USA, soya is usually used instead of these (Ku et al. 1972). It seemed that selenite selenium, selenomethionine and seleniferous plants (non accumulator species) may follow different metabolic pathways leading to differences in tissue concentrations of selenium, even though all these forms of Se are about equally effective in preventing selenium response diseases (Cary et al. 1973, Jaakkola et al. 1982. Variance analysis showed the selenium levels in the blood samples to be highly significantly dependent (p 8 0.001) on the feed selenium content, but in the regression analysis the dependence of blood selenium level on feed selenium content was only suggestive (90 %, regression coefficient 1.4). Contrary to the results obtained with chickens (Kääntee and Kurkela 1980), blood was not among the most reliable tissue indicators of body selenium content in the pig. The spleen, lungs, heart and brain were more reliable. The selenium contents of all these tissues rose quite high due to organic selenium in the feed.
In all test groups, the mean selenium contents of the organs were the highest in the kidneys, varying between 5200 and 8600 bone. The greatest differences in the selenium contents between different test groups were found in the heart, liver, brain, spinal cord and bone.
The low selenium content of Finnish feeds is compensated during manufacture by the addition of sodium selenite, which has a low biological availability. This is shown by the incidence of histologically verified nutritional muscular dystrophy in Groups 1 and 11, but also in healthy and unhealthy animals from the slaughterhouse. High selenium contents were found in both the muscle and myocardium of these unhealthy animals. This may be due either to the selenium administered to the animals in the form of medication or to mobilization of selenium from the body to the diseased areas (Toikka 1978). Since neither the international selenium content of pork nor even the selenium level required for production of histopathologically healthy meat is obtained by the addition of sodium selenite, even up to 270 fig Se/kg DM, the study supports the idea that the only way to elevate the selenium content of Finnish pork to the international standard is to raise the organic selenium content of pork feed to at least 150-200 fig Se/kg DM (i.e. 0.15-0.2 ppm). This can be done safely by fertilizing the feed grain by selenium-containing fertilizers or by foliar spraying with solutions containing selenium (Korkman 1980). Table 2, and the average pork consumption of about 71 grammes per person per day in Finland (Nuurtamo et al. 1980) the range selenium per pig diet studied in the present investigation accounts for a difference of not more  (NBH Circular No. 1762/1981. The toxic level could be exceeded with a daily consumption of more than about 1.1 kg pig kidney or more than about 2.1 to 2.6 kg liver, from pigs whose diet contains between 100 and 700 fig Se/kg DM (0.1-0.7 ppm).

Summary
An investigation was made into the effects of barley feeds with varying levels of natural organic selenium introduced by means of selenium-containing fertilizer and of inorganic sodium selenite on the concentrations of selenium in the tissues of growing pigs (blood, spleen, bone, muscle, heart, liver, lung, kidney, pancreas, brain, spinal cord and eye), and on the histological structure of myocardium and longissimus muscle.
The results indicated that natural organic grain selenium affects the selenium levels of tissues in pig significantly more (muscle 250 *7o) than sodium selenite supplementation of the same amount. The natural selenium concentrations of feed and organs were linearly correlated. Spleen, lung and heart were the most reliable indicators of tissue selenium levels in pigs.
When the feed selenium level was supple- In the quality classification of the carcasses, 5/6 pigs in the group receiving sodium selenite were placed in Class I-because of their high fat content.
The study supports the idea that natural organic selenium of grain origin of 150 -200 /xg Se/kg DM (0.15 -02 ppm) is the most both as regards the health of the animals and suitable for pork production and more effec-the elevation of the selenium content to the live than selenite-induced selenium content normal international level.