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Comparative metabolic and immune responses of chickens fed diets containing inorganic selenium and Sel-PlexTM organic selenium

Published: January 1, 2002
By: L. Leng1, R. Bobcek2, S. Kuricová3, K. Boldizárová1, L. Grešáková1, Z. Ševcíková3, V. Révajová3, M. Levkutová3, M. Levkut3
INTRODUCTION Selenium is well known to be an essential trace element with many vital functions in humans and animals. In nature, it can be found in the elemental form as well as incorporated into both inorganic and organic compounds. Apart from supplementation, farm animals receive selenium mainly in the form of selenoamino acids (selenomethionine) in vegetable feed ingredients (Schrauzer, 2000a). Selenium in the form of another amino acid, selenocysteine, is the central structural component of specific selenoenzymes including glutathione peroxidases, iodothyronine deiodinases, thioredoxin reductases, selenophosphate synthetase and many others. To date, about 30 selenoproteins have been identified, but the precise function of most of them is not yet known. The best understood selenoenzyme is cytosolic glutathione peroxidase (cGSH-Px, EC 1.11.1.9), which works as the antioxidant by removing reactive oxygen species (Behne and Kyriakopoulos, 2001). Adequate intake of selenium is needed for immunocompetence. It is well known that deficiency results in the insufficient cell and humoral immune response of humans and animals (Rayman, 2000). On the other hand, supranutritional doses of selenium have been found to have anticarcinogenic effects in humans (Schrauzer, 2000b). Consequences of insufficient selenium intake in farm animals including nutritional myodystrophy (white muscle disease), exudative diathesis, disorders of liver and pancreatic functions and many other syndromes have been well described. Associated production problems are poor animal performance, reproductive disorders and considerable economic loss due to morbidity and mortality. Another situation might be induced by marginal selenium deficiency and/or by the use of solely inorganic selenium sources to supplement the feedstuff. In such cases delayed immune system development of young chicks, poor feathering with associated energy losses, increased mortality and culling, reduced egg production and increased embryonic mortality are noted. In most countries the natural selenium content of grain and forages, which consists mainly of the selenoamino acids selenomethionine and selenocysteine in plant proteins, is only 0.03–0.12 mg/kg with values more commonly at the lower end of this range. Intake of such feeds can result in serious selenium deficiency and health problems, especially in highly productive animals. For this reason, feedstuffs are routinely supplemented with various selenium sources at 0.2-0.3 mg Se/kg of dry matter. Until recently, inorganic forms of selenium have been used almost exclusively in animal feeds. Apart from the known antioxidant role of selenium in physiology, basic research has clearly shown that selenite per se has undesired pro-oxidative features (Spallholz, 1997). This finding is especially alarming in the light of recent results of Kobayashi et al. (2001). These researchers showed that selenium bound in the form of selenide (H2Se) to albumin is susceptible to oxidation, yielding selenite (SeO3 -2) in vitro. They suggested that this process might also occur in vivo. It should be understood that selenide is a key product of sodium selenite metabolism before selenium can be incorporated into the specific selenoproteins. Newly-formed selenide continually produces reactive oxygen species in the body during very rapid diffusive recycling between plasma and red blood cells. This could be one reason why occasional overdosing with organic forms of selenium does not result in the acute toxicity seen when inorganic forms are overdosed. Dietary selenoamino acids, though also converted to selenide before eventual incorporation into selenoproteins, can alternatively be taken up and used in synthesis of other proteins. As a result, the harmful pro-oxidative effects of selenide-selenite recycling are almost eliminated when organic selenium forms are in the diet. Objectives of this work were to compare the effects of supplementing feed with sodium selenite and organic selenium in the form of selenium from yeast (Sel-PlexTM, Alltech Inc.) on metabolism of selenium, its incorporation into tissues and on the activities of the selenoenzyme glutathione peroxidase (GSH-Px) in blood of chickens from hatch to seven weeks of age. Selenium balance and retention were measured when birds were in the fifth week of age. The immunohistochemical and flow cytometry analysis of cellular and humoral responses to organic selenium were done on samples of duodenum, caecal tonsils, bursa of Fabricius and spleen tissue. MATERIAL AND METHODS ANIMALS, DIETS AND TREATMENTS Two hundred and twenty female chickens of the laying strain Isa Brown were obtained on the day of hatching from LP–Párovské Háje a.s., (Slovak Republic). They were divided into four groups of 55 birds and kept in large pens with wood shavings. From the first day of life till the age of seven weeks the chickens were fed treatment diets, which differed only in the total content and/or form of selenium. The first group was given the basal diet (Table 1) containing 0.12 mg Se/kg of dry matter (DM) arising only from the natural occurrence of selenium in the dietary components. The second received the basal diet to which sodium selenite was added to supply 0.2 mg/kg Se resulting in a final selenium level of 0.32 mg/kg. The third group received the basal diet supplemented with 0.2 mg/ kg Se in the form of selenium yeast (Sel-PlexTM) yielding the same final selenium level (0.32 mg/kg) as in the diet fed the second group of birds. The fourth group received the basal diet containing an added 0.7 mg/kg Se from selenium yeast for a total dietary concentration of 0.82 mg/kg Se. Diets fed groups 1-3 were supplemented with non- Se yeast extract (NuPro™, Alltech Inc.) to supply amounts of ‘yeast’ equal to that added to the fourth diet (63.7 g in 100 kg of feed). Mineral and vitamin premixes were supplied by Biotika a.s. (Slovak Republic). Chickens were reared starting with a lighting regimen of 23L:1D, which was adjusted to 15L:9D after three weeks of age. The initial room temperature (32–33°C) was reduced every week by 3°C to a final temperature of 23°C. All animals had free access to water and feed. SAMPLE COLLECTION AND ANALYSIS Each week six chickens from each treatment group were sacrificed for collection of blood and tissue samples. Following IP injection of pentobarbital (Spofa, Czech Republic, 60 mg/kg body weight), blood was collected by intracardial puncture and placed in heparinised tubes or in tubes containing EDTA solution. Following euthanasia, samples of liver, heart, breast muscle, spleen, lungs, gizzard and kidney tissue were collected and stored at –24°C. The selenium balance and retention measurements were obtained using the bag technique for quantitative collection of excrement (Grešáková et al., 2003). Briefly, the feathers surrounding the cloaca were clipped and a polyethylene bag (microten PE-HD 02) was sealed firmly around the cloaca using double-sided adhesive tape. The chickens were housed individually in plastic boxes (length 60 cm, width 40 cm, height 35 cm) for 10-12 days with water and feed freely available. Each box was furnished with one vessel containing tap water and another containing precisely preweighed feed. The vessels were placed firmly in the opposite corners of boxes to prevent mutual contamination. A period of at least three days was allowed for adaptation to boxes and bags. Excrement was collected for analysis daily for six days. Each bird was weighed daily after removal of the bag for sample collection. Spilled feed was carefully collected and weighed along with any remaining feed. Excrement samples were dried at 55°C to a constant weight for 48 hrs and ground in preparation for selenium analysis. For the precise balance measurement, selenium was analysed in feed samples for each bird daily in order to obtain the true values of selenium intake. SAMPLE ANALYSIS Blood GSH-Px activity was determined as in Paglia and Valentine (1967) using the Ransel kit (Randox, UK). Haemoglobin was analysed using a kit from the same manufacturer. The levels of selenium in feed, excrement, blood and tissues were analysed in triplicate by the fluorimetric method of Rodriguez et al. (1994). Selenium concentration in blood was measured at 3, 4, 5, 6 and 7 weeks of age. Dry matter content of tissue, feed and excrement was determined by drying samples at 105°C. For immunohistochemical analysis of lymphocyte differentiation the monoclonal antibodies (Moab, Scandic, Czech republic) against chicken CD (cluster of differentiation) 3 T-cells were used in duodenum and caecal tonsil, CD4 T-cell subpopulation in the bursa of Fabricius and CD8 T lymphocytes in the duodenum (Table 2). The indicated organs were collected from 1, 4 and 7 week-old chickens. Frozen sections from the caecal tonsils, duodenum and bursa of Fabricius (7 µm thick) were immersed in cold acetone, rinsed with phosphate buffered saline (PBS) and incubated with 1% skim milk for 30 min to reduce non-specific binding. Sections were then incubated for 1 hr with primary monoclonal antibodies. Biotin-Streptavidin amplified (B-SA) peroxidase detection was used to detect the lymphocytes (Biogenex, USA). Instead of primary antibody, the PBS was used for a negative control. The specific colour reaction was induced by 3,3 diaminobenzidine in Tris-buffered hydrochloric acid. Lymphocytes in the duodenum and caeca were counted at the base of the villi as well as in the medullar part of bursa of Fabricius follicles. In the duodenum lymphocytes were counted in the areas of the villus epithelium and lamina propria from the lamina muscularis mucosa toward the point of the villi. Six appropriate areas were chosen at random from each of these sites in the gut and 100 areas in the bursa of Fabricius. Measurements were taken by light microscope at 1000x magnification in the duodenum and caecal tonsils and at 400x in the bursa of Fabricius. The positive lymphocyte-stained cells within each randomly selected area were counted using a calibrated ocular graticule LTD 0.25 mm IdxGrd (Electronmicroscopy, UK). The appearance of positive lymphocytes is expressed in numbers per square millimetre. For flow cytometry, the spleen was obtained from 1 and 4 week-old birds. Spleen lymphocytes were removed by teasing through a 70 µm mesh screen (Heller and Schat, 1987) and isolated on a density gradient – Telebrix (Sevac, Czech Republic). After separation, the lymphocytes were washed twice with PBS. Fifty µl of cellular suspension (1 x 106 lymphocytes in PBS) and 50 µl of specific MoAb (Table 2) were mixed and incubated at 4°C for 30 min. After incubation the cells were washed twice in PBS and pellets were mixed and incubated with 25 µl of secondary antibody (FITC-conjugated goat antimouse immunoglobulin, Dakopatts, Germany) in a 1:50 dilution and incubated in the dark as described above. The cells were washed twice in PBS and resuspended in 0.5 ml of 1% paraformaldehyde in PBS. Samples were analysed by FACScan flow cytometer (Becton Dickinson, Germany). Data on 1 x 104 viable cells were collected using the Cell Quest programme of Becton Dickinson. STATISTICAL PROCEDURE Statistical analysis was conducted using one-way analysis of variance with the post hoc Tukey and Dunett tests used to separate means where appropriate. RESULTS With the exception of week 4, blood selenium concentrations were significantly higher in birds given organic selenium than in birds given selenite (Figure 1). Blood GSH-Px was highest in birds given the highest amount of selenium. This finding confirms the linear correlation between blood selenium content and activity of this selenoenzyme in blood. No differences in blood GSH-Px were noted between sources of selenium administered at 0.2 mg/kg, however there was a trend for higher values in birds given organic selenium after six weeks. A sharp decline in blood GSH-Px activity was noted during the first two weeks in birds given the unsupplemented basal diet. It is during this period that young birds are most sensitive to stress factors. Remarkable also was the finding that blood GSHPx in this group did not recover even to levels measured at hatch in the unsupplemented group. This observation clearly demonstrated that feedstuffs mixed from ingredients grown in Central Europe do not provide sufficient selenium for adequate GSH-Px activity of chickens. Liver selenium content followed a pattern similar to selenium content of blood with highest selenium levels reached in animals given the largest supply (Figure 2a). Significant differences between liver selenium content of birds given inorganic vs organic selenium appeared after six weeks. The most striking response to selenium form was noted in breast muscle (Figure 2b). Selenium content of this tissue was significantly higher during the entire experiment in birds given 0.2 mg/kg Se from the organic source compared to birds given the same amount from sodium selenite. The highest muscle selenium content was recorded in birds given an added 0.7 mg/kg Se from Sel-Plex. The selenium contents of other tissues at the end of experiment (7 weeks) are presented in Table 3. The lowest values were recorded in birds maintained on the unsupplemented basal diet. Birds given Sel-Plex™-supplemented diets had significantly higher selenium levels in heart, lung and gizzard tissue compared to those given the same amount of inorganic selenium. Again, the highest values were determined in birds supplied with the largest amount of organic selenium. Selenium balance data indicated positive balance in all groups with quantities dependent on the selenium content of feed (Figure 3). Though the difference between the groups given inorganic or organic selenium at 0.2 mg/kg Se fell just short of statistical significance, the trend was toward larger values in birds given the Sel-Plex™, which supports higher bioavailability of organic selenium from Sel-Plex™. This trend is also supported by lower retention of inorganic selenium. Birds given 0.2 mg/ kg Se from selenite retained even less diet selenium than birds given the unsupplemented basal diet. Feed intake and body weight of four-week old Isa Brown female chickens during the balance experiment are summarised in Table 4. The immunohistochemical observations clearly demonstrated that the expression of avian T lymphocytes was correlated with the total intake of selenium (Figure 4a). Numbers of CD3 cells in the duodenum tended to increase with age. Values in seven-week-old chickens were significantly higher than in the birds at one week of age. Though differences among treatments were not significant, there was a trend for increased expression of CD3+ cells with higher selenium intake, which can be seen in birds at seven weeks of age. This pattern was also observed for duodenal CD8+ cell expression (Figure 4b). Expression of CD3+ cells in caecal tonsil also showed a dose-dependent pattern after seven weeks (Figure 5a). On the other hand, this relationship has already been found in CD4+ cell numbers in the bursa of chickens at four weeks of age. Flow cytometry analyses of CD3+ cells in the spleen revealed no differences in expression in chickens at 1 and 4 weeks. On the other hand, data in birds at four weeks clearly showed a selenium dependent increase in numbers of CD8+ cells (Figures 6a and b). DISCUSSION This experiment showed that blood GSH-Px in young chickens is dependent on amount, but not form, of selenium supplied. The explanation is based on the fact that all dietary selenium compounds must be converted to H2Se before de novo synthesis of selenocysteine for incorporation into the active centre of the selenoenzyme (Schrauzer 2000a). Interestingly, even the highest amount of selenium supplied (0.82 mg/kg) did not appear to fully saturate the activity of blood GSH-Px. In contrast, we have recently found that increasing selenium intake beyond that present in an unsupplemented diet did not affect cGSH-Px activity in chicken liver (Holovská et al., 2002). This finding suggests that synthesis of different types of GSH-Px in various tissues might be saturated at diverse levels of selenium entry. To date there are five types of GSHPx described: cytosolic or classical cGSH-Px, gastrointestinal GI-GSH-Px, plasma pGSH-Px, phospholipid peroxide PH-GSH-Px and sperm nuclei snGSH-Px (Behne and Kyriakopoulos, 2001). The main function of these peroxidases is the removal and/or detoxification of hydrogen peroxides and lipid hydroperoxides to less harmful alcohols (Ursiny et al., 1997; Mates and Sanchez-Jimenez, 1999). The direct effect of dietary selenium form on blood GSH-Px activity has been determined in other animal species, but it seemed to be just a consequence of apparent selenium deficiency (Yoshida et al., 1999). Our results unequivocally confirmed the more efficient utilization of the organic form of selenium in chickens as reflected by its higher levels in tissues, particularly in muscle. The most striking finding in birds given 0.2 mg/kg Se organic selenium was the fact that muscle selenium over the entire experiment was twice that of birds given 0.2 mg/kg Se from selenite. Another noteworthy result was that muscle selenium of those given 0.2 mg/kg Se from selenite did not differ from that of chickens given the unsupplemented basal diet. Moreover, muscle selenium level in 7 week-old birds given inorganic selenium fell to half that at hatch (from 11.8±1.3 to 5.5±0.2 µmol/kg, P<0.001). This means that selenite is unable to supply selenium in a form that allows tissue deposition. When selenium is supplied in organic form, muscle tissue becomes the most significant storage deposit of selenium in a form of selenomethionine. The reason is that muscle mass represents about 52–56% of body weight. The uptake of selenomethionine by muscle proteins is very important from the point of view of increased transfer of selenium from the dam to eggs and embryos; which is an important determinant of subsequent immunocompetence and health of young birds. The practice of supplementing organic selenium to poultry diets benefits human consumers. The human population in many countries, especially the young and middle aged, face marginal to medium severe selenium deficiencies with consequent health disorders such as decreased nonspecific immunity, disorders of thyroid metabolism, heart and vascular diseases and cancer (Kvicala, 1996; Rayman, 2000). Higher utilisation of organic selenium compared to the inorganic form is also reflected in the retention data from balance measurements (Figure 3). Even the low selenium intake from the basal diet (0.12 mg/kg Se, solely in the natural organic form) resulted in higher selenium retention than when 0.2 mg/kg Se was added to the same diet in the form of sodium selenite. These differences failed to reach statistical significance owing to low numbers of experimental animals in these two groups (5 vs 7). Nevertheless, the data demonstrate the trend favouring higher retention of organic selenium. It is well known that selenium is necessary for optimum performance of the immune system. To our knowledge, the immunohistochemical approach has not been used to characterize selenium effects on development of the T lymphocyte subpopulation in young growing chickens. The effects of selenium deficiency on chicken splenocyte proliferation and cell surface marker expression were well defined using flow cytometry by Chang et al. (1994). In general, the correlation between selenium dose and T-cell expression has been determined. This phenomenon appeared predominantly in the second half of the experiment during the fourth and seventh weeks. A trend toward increased expression of CD8+ cells in the duodenum, CD4+ in the bursa of Fabricius and CD3+ cells in the spleen after organic selenium addition to the diet could be seen in the same experimental period. While the selenium effects on duodenal and spleen lymphocyte numbers appeared after the seventh week, only the changes in bursal T-cells have previously been observed in four week-old birds. This is obvious because the development of the bursa of Fabricius is accomplished during weeks 4-12 of life (Glick, 1991). CD3 presents a complex of proteins acting as signal transducers and it is found on all T-cells. That is why these cells have been used for detection of all T lymphocyte expression (Tizzard, 2000). The function of CD4+ helper cells as coordinators of immune function includes responsibility for generation of antibody responses by providing assistance to B-cells. They can also influence cytotoxic responses. Thus CD4+ cells have a central role in the avian immune system and their activation is a prerequisite for responses by other types of cells. CD8+ cells are known to be effector cells in cytotoxic response, by killing infected target cells (Artsila et al., 1994). Selenium has been shown to stimulate the transformation of T lymphocytes into cytotoxic cells (Kiremidjian-Schumacher et al., 1994). This experiment was carried out without the presence of infection. The data showing an increase in T-cell expression with selenium supplementation offers new knowledge about the density and functionality of cells responsible for cellular immunity. Our results demonstrated that selenium improves chicken immune status by increasing ability of immunocompetent cells to respond to antigen stimuli. Finally, supplementation of a low selenium basal diet with Sel-Plex™ selenium yeast revealed a trend toward improved protection against pathogens. The basic difference between utilisation of inorganic and organic selenium after absorption from the digestive tract is the substantially higher bioavailability of this essential microelement from the organic sources. This is derived from two principal features of the selenoamino acid selenomethionine, which is the main Se-containing compound in Sel-Plex™ selenium yeast. The first specific feature of selenomethionine when absorbed from the digestive tract is that the fraction not immediately used for synthesis of specialised selenoproteins is incorporated non-specifically into the structural proteins of muscle, gizzard, heart and other organs. Selenomethionine substitutes for the amino acid methionine, which is structurally similar except for the presence of sulphur instead of selenium. This is possible because tRNAMet is not able to differentiate between these two amino acids. This serves to create selenium reserves against future stress, improve resistance to disease and moreover for the transfer of selenium from the dam for reproductive processes (Edens, 2001; 2002; Paton et al., 2002). The larger selenomethionine content of embryonic tissues together with selenium supplied by milk or from the yolk sac provides an easily metabolisable source of selenium for neonates during the most critical period of early life (Surai, 2000). The better utilisation of organic selenium is documented also by the balance experiment, which showed higher total body selenium retention from the organic than the inorganic source. Another great advantage of selenomethionine incorporation into muscle proteins is that selenium in this form is not involved in selenide-selenite recycling in blood with its consequences of reactive oxygen metabolism formation (Kobayashi et al., 2001). Our recent work demonstrated that 4-week old chickens did not respond to feed with the very high supply of organic selenium (+0.7 mg/kg Se) by the higher production of thiobarbituric acid reactive substances (TBARS) in liver than the birds on the basic diet with 0.12 mg/kg of selenium (Holovská et al., 2002). The measurement of TBARS levels in tissues is a widely accepted tool for the assessment of reactive oxygen species production (Gutteridge, 1984). The second specific feature of selenomethionine involves its renal handling. It is well known that contrary to inorganic selenium compounds, all amino acids including selenomethionine are almost completely reabsorbed from kidney glomerular filtrate in the proximal tubule. Selenomethionine shares the active transport mechanism with sulphur-containing methionine in this nephron segment. Reabsorbed selenomethionine is captured in kidney capillaries and re-enters whole body metabolism via the blood stream. In other words, there are practically no urinary losses of selenium in the form of selenomethionine. On the other hand, selenite selenium not engaged in synthesis of specialised selenoproteins does not have metabolic route for incorporation into tissue proteins. Within a few minutes, selenite absorbed from the gut is metabolised into selenide (H2Se), which forms nonspecific bonds with plasma albumin (Suzuki et al., 1997). After multiple recycling of selenium via the selenide-to-selenite transformation pathway and its methylation, surplus inorganic selenium is rapidly excreted via urine. Though the glomerular filtration of H2Se seems to be limited due to its albumin bond, the rapid urinary elimination of selenium of inorganic origin is another significant disadvantage in comparison to selenoamino acids (Boldizárová et al., 2001). CONCLUSION This study clearly demonstrated the benefits of supplementing poultry diets with organic selenium in the form of yeast (Sel-Plex™) due to its ability to support formation of tissue selenium stores. Integration of organic selenium into the food chain provides substantially greater transfer of selenium in highly utilizable form for human consumers. In addition to positive effects on production parameters and immunocompetence, selenomethionine from yeast added to animal feed could aid in improving the health of the human population, which is subject to widespread selenium deficiency in Central Europe as well as many other parts of the world. REFERENCES Artsila, T.P., O. Vainio and O. Lassila. 1994. 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