Long-Term Supplementation of Laying Hen Diets with Various Selenium Sources as a Method for the Fortification of Eggs with Selenium

-e following study focuses on a comparison of the effectiveness of egg content enrichment with selenium (Se) via application of sodium selenite (Na-selenite), selenium-enriched yeast (Se-Yeast), or selenomethionine (Se-Met) in laying hen diets. Two hundred sixteen laying hens were divided into four treatments, each comprising eighteen replications, and each with three hens per cage. Animals were fed a basal diet without Se supplementation (control: selenium content 0.058mg/kg), with the addition of Na-selenite, Se-Yeast, or Se-Met in amounts equivalent to 0.3mg/kg of added selenium. -e egg quality, the selenium content in eggs after the third and the fifth months of using Se supplementation, and the selenium level in the liver were determined. Enrichment of egg content with selenium was the most effective (382 μg/kg) via application of dietary Se-Met. Application of Na-selenite and Se-Yeast led to a similar effect on Se-accretion in egg content (255.9 and 258.9 μg/kg, respectively). Additionally, the calculated average Se concentration in one fresh egg was also higher in eggs from hens that received selenium additives in their diet and was far higher, almost three times higher for Se-Met addition, than the concentrations in controls. Se-accretion in the liver wet tissue was greater following application of Se-Yeast in the diet than following other treatments. -ese results indicate that the use of selenomethionine in the laying hen diet is the best method of enriching eggs with this micronutrient. In turn, the eggs obtained in this way can be an excellent source of highly bioavailable selenium in the human diet.


Introduction
e vital role of selenium (Se) originates from the fact that it is incorporated in several key enzymes and selenoproteins that are crucial for numerous biological activities, i.e., antioxidation, anti-inflammation, thyroids, fertility, DNA synthesis, and reproduction [1][2][3][4].e best known selenium-dependent enzymes are glutathione peroxidases (GSH-Px); however, there is also a known seleniumindependent glutathione peroxidase [5].Some forms of gastrointestinal GSH-Px, plasma GSH-Px kinase, and iodothyronine deiodinases are synthesized and activated when the Se level in a diet is optimal [2,3,[6][7][8].Selenium protects the liver against necrosis and prevents muscular degeneration, neurodegenerative disorders, pancreatic fibrosis, microcytic anemia, degeneration of the testis, and, in birds, against the poor hatchability of eggs [9].e best known disorder in livestock associated with Se deficiency is white muscle disease, with clinical signs that include lesions in skeletal and/or heart muscles.A low intake of Se in the human diet may lead to a number of diseases such as severe heart diseases, hypothyroidism, reduced male fertility, a weakened immune system, and enhanced susceptibility to infections, and it may even lead to cancer [10][11][12][13].Dietary, daily reference values for selenium in human nutrition found in available reports vary greatly.
e proposed adequate intake of selenium for children ranges from 15 to 70 μg/day, for adults from 60 to 70 μg/day, and for pregnant and lactating women, that value ranges from 70 to 85 μg/day.e tolerable upper intake level for adults, including pregnant and lactating women, ranges from 200 to 500 μg/day [14].Selenium is also known to be chronically toxic, and selenosis has been reported both in humans and in animals.Human chronic selenosis may occur when dietary selenium intake is above 1,000 μg/day.However, toxic levels of selenium are difficult to establish since selenium toxicity is affected by the kind of selenium compounds present in the food as well as mutual interactions with other components of the diet [14][15][16].Unfortunately, as has been stated in many countries across the world, human food ingredients can contain low levels of selenium and selenium deficiency in human nutrition has become a global phenomenon [17][18][19].
ere are several potential options to improve human selenium intake, including dietary modification and diversification or direct Se supplementation.In the last few years, a dynamic increase has occurred in terms of the attention paid to the creation of functional or designer foods containing more biologically and physiologically active components than standard food.
ese products have prosalubrious properties and prevent diseases connected with nutritional factors [2,20].erefore, the production of selenium-enriched (biofortificated) animal-origin products such as eggs, meat, or milk, where selenium is present in higher amounts than in standard products, as well as Se occurrence in varied available forms, is the best way to increase both daily selenium uptake and the prevention of disorders related to its deficiency in the diet.e enrichment of products of animal origin with selenium may take place by increasing its concentration in the diet of animals (not exceeding the maximum levels permitted in their feed) or by selenium forms in animal nutrition which are characterized by high bioavailability.Selenium-enriched eggs are perfect candidates for the category of functional foods because eggs are affordable and are considered a traditional form of food in most countries, as well as being consumed by people of all ages more or less regularly [19,21].Selenium-enriched eggs contain superstandard levels of Se in the form of the essential amino acid, selenomethionine (Se-Met).e level of Se in a standard egg is about 11 μg, while the selenium-enriched egg contains around 32.6 μg of Se; therefore, consumption of two selenium-enriched eggs a day may satisfy over 70% of the Se daily dosage recommended for humans [22].
An inorganic selenium compound-sodium selenite (Na-selenite) or selenate, the major commercial Se supplements-has been used in animal feed as a source of selenium for the last few decades [3,23].Currently, selenium may be added to feed mixtures or to premixes both in an inorganic form and in organic forms: selenium-enriched yeasts (Se-Yeast), selenocysteine (Se-Cys), selenomethionine (Se-Met), or selenocystine [6,7,24].Selenium in seleniumenriched yeast is typically in the form of the selenoamino acids: Se-Met (approx.60-85% of total Se species) or Se-Cys (approx.to 2-4% of total Se) and as an inorganic selenium (IV) ion (less than 1% of total Se) [16].Selenoamino acids that are used as selenium additives are the natural organic bonds present in animal feedstuffs [1,3,25,26].Major dietary ingredients contain selenium exclusively in organic forms and Se-Met that may comprise even more than 50% of total selenium, as well as smaller amounts of Se-Cys and selenate [27].e bioavailability of Se from various forms applied in poultry feed is relatively high and mainly depends on the absorption pathways.Selenium from Na-selenite is passively absorbed by diffusion from intestinal content [28] and that from Se-Met is actively absorbed via methionine transport mechanisms.Noy et al. [29] evaluated the selenium absorption coefficient from poultry diets at a level from 60 to 86%.However, the effectiveness of Se-Yeast as a source of selenium closely depends on the kind of yeast [30], mainly Saccharomyces cerevisiae strain, that is used in the production of selenium-enriched yeast.
e level of selenium requirement recommended for laying hens varies and ranges from 0.05 to 0.08 mg/kg according to NRC standards [31], and the maximum allowable level given by the Association of American Feed Control Officials is 0.3 mg of Se/kg of complete feed mixture [32].Usually, to meet Se requirements in poultry, supplemental Se in commercial feed is delivered with premixes at a level of 0.2-0.3mg/kg complete feed, while the Se amount in the feed ingredients is not considered [23,33].However, complex poultry fodder mixes are recommended to include an Se supplement of 0.5 mg/kg of the fodder mix, which is in accordance with the maximum tolerated dietary concentrations of selenium given by EFSA [22,34].
Given the above information about issues concerning the production of functional foods enriched in selenium, especially eggs, the objective of this study was to investigate the level of selenium transfer into the egg content of hens fed with diets supplemented with Se at the recommended level of 0.3 mg/kg.e sources of selenium in the diets prepared for experimental purposes were sodium selenite, seleniumenriched yeast, or selenomethionine.Considering the age of hens and the period of Se-inclusion into the diet may impact the transfer of selenium into eggs, a second experimental factor in the present studies was the length of the period selenium application.Additionally, some characteristic egg parameters and selenium liver concentrations were determined, as these parameters are also known to be modified by selenium supplementation.

Animals and Management.
Two hundred sixteen Lohmann Brown pullets at the age of eighteen weeks were housed in battery cages and, before laying, were fed with the standard mixture for that stage of maturity that contains about 145 g/kg of crude protein and 11.5 MJ/kg of metabolizable energy.Directly after the laying of first eggs at the age of twenty weeks, hens with an average 1.27 kg body weight were allocated to cages according to body weight and laying at the start time.Afterwards, the cages were randomly divided into four treatments, each comprising eighteen replications and each with three hens per cage (fifty-four hens per treatment).Free access to water (nipples) was ensured.In that period, hens were fed with complete mixture containing about 180.3 g/kg of crude protein and 11.6 MJ ME/kg (according to Lohmann Brown recommendations).According to experimental assumptions, the amounts of selenium added to the feed mixture with premixes were the same (0.3 mg/kg) and the only factor that differentiated particular feeding groups was the selenium source.Selenium was added in the form of sodium selenite, selenium-enriched yeast, or selenomethionine.e environmental conditions in the autumn-winter period were controlled.
e room temperature varied between 16 and 18 °C, and the lighting program was set up for fourteen hours of light daily.e management was in accordance with the European Union guidelines, and the local Ethics Commission for Experiments with Animals accepted all experimental procedures.

Diets and Feeding Program.
e dietary composition (Table 1) was based on our own chemical analyses of feed components and was calculated using linear optimization.
e mixture of feed components prepared according to our recipe was determined for selenium content-this amounted to 0.058 mg/kg.e basal diet was divided into four parts, three of which were enriched with selenium using either Na-selenite, Se-Yeast, or Se-Met in amounts appropriate to the anticipated selenium supplement at the level of 0.3 mg of Se/kg of full mixture (Table 2).All the feed mixtures were prepared prior to the experiment.A homogenous mixture of selenium within the feed was obtained by preparation of premixtures with a selenium-free mineral-vitamin premix that was made for the experiment.According to the Lohmann Brown recommendations, the feed full mixture diet contained 180.3 g/kg of crude protein and 11.6 MJ ME/kg and was given at the dose of 110 g per hen per day.

Egg Characteristics: Data Collection.
e number of eggs was registered per cage daily, and all eggs from each cage were weighed.After the third and the fifth months of feeding with experimental diets, thirty eggs from each treatment were randomly sampled for determination of egg quality characteristics using a PM 600 RX processor apparatus (Technical Service and Supplies Ltd., York, England).Each measurement was repeated twice.Egg yolk color was determined using the La Roche scale (max.15 points).For determination of selenium content, thirty-six eggs from each treatment were randomly collected (two eggs from replicate/cage), and the selenium content was determined in eighteen pooled samples for each treatment (two eggs per pooled sample).At the end of the experiment, after twelve hours starvation from each treatment, eight hens were randomly selected, weighed, and killed by cervical dislocation.In the sampled livers, selenium content was determined.

Analytical Methods.
e chemical compositions of the feed compounds and complete mixtures were determined according to standard methods [35].Nitrogen content was assayed using a Kjeltec 2300 Foss Tecator apparatus (Häganäs, Sweden, AOAC, 976.06); crude protein, by multiplying the nitrogen content by 6.25 (AOAC, 2003.06);crude fiber, by the Henneberg-Stohmann method using a Fibertec Tecator apparatus (Häganäs, Sweden, PN-EN ISO 6865).For mineral determination, feed samples and experimental mixtures were mineralized with nitric acid (HNO 3 ) using a MarsX apparatus (CEM Corporation Matthews, USA).Phosphorus was analyzed after prior wet mineralization with nitric acid (HNO 3 ) and perchloric acid (HClO 4 ) according to the ammonium vanadomolybdate method using a Specol 11 spectrophotometer (Carl Zeiss, Jena) at a wave length of 470 nm.Calcium and sodium were determined by atomic absorption spectrophotometry using an AA 240 FS apparatus with SIPS 20 (Varian, Mulgrave, Australia).For the determination of sulfur-containing  amino acids, the feed samples were oxidized (0 °C, 16 h) with formic acid and hydrogen peroxide (H 2 O 2 ) (9 : 1 (v : v)) prior to HCl hydrolysis.After oxidation, samples were hydrolyzed with 6 M hydrochloric acid (HCl) for 24 h at 110 °C, and then, sulfuric amino acids were separated according to the Moore and Stein method [36] using an AAA 400 Ingos analyzer (Prague, Czech Republic).Selenium in feed components, complete mixtures, eggs, and samples of liver was determined by hydride-generation atomic absorption spectrometry (HG-AAS).e egg's whole content and liver samples from experimental animals were freeze-dried and homogenized.Se concentrations in eggs and livers were determined after prior mineralization with HNO 3 .Afterwards, the solvent was evaporated and the residue dissolved with 25% H 2 SO 4 .Selenium content was assessed using a Vapor Generation Accessory 77 (Agilent Technologies, Inc., USA) for atomic absorption.A selenium hollow cathode lamp was used as a source of linear radiation (NARVA, Berlin, Germany).Data obtained for the freezedried material were converted, and selenium content was expressed on a fresh-weight basis.

Statistical Analysis.
All collected data were evaluated using one-or two-factorial ANOVA within StatSoft Statistica ® computer software [37].e differences between parameters were tested according to the following statistical model: y ij � µ + α i + e ij (for differences between treatments) or y ijk � µ + β j + (αβ) ij + e ijk (for Se supplement and period of analyzed parameters in eggs), where y ij or y ijk is the variance associated with parameter α; µ is the overall mean; α i is treatment effect; β j is the time of analyzed parameters; (αβ) ij is the interaction effect; and e ij or e ijk is an error term.

Results and Discussion
A significant (P < 0.01) effect was found for all selenium additives used in the experiment on the concentration of this element in laying hen eggs (Table 3).
In comparison with eggs received from hens from the control group (156.2 μg•Se/kg) and in eggs from hens fed a diet containing Na-selenite, the selenium concentration increased by 64% (255.9 μg•Se/kg), for the Se-Yeast-treated group by 65% (258.9 μg•Se/kg) and for Se-Met by about 144% (382.2 μg•Se/kg).e length of the period for which hens were fed supplemented diets had no significant influence on the accretion of selenium in the eggs.Additionally, the calculated average Se concentration in one fresh egg was also higher in eggs from hens that received selenium additives in their diets, and this was the highest-almost three times higher in the case of Se-Met addition-when compared with the control.e data are in agreement with results previously published by Delezie et al. [38], who also stated that dietary Se-Met is more effectively transferred to the egg than Se-Yeast or Na-selenite.Our results also agree with those obtained by Gajčević et al. [39].e distinctive influence on Se transfer to egg embryo was obtained by Cantor et al. [40].Payne et al. [41] and Reis et al. [42] suggested that Se-Yeast inclusion into hen diets is a useful strategy to improve the nutritional status of egg and embryo.As reported by Invernizzi et al. [43], when selenium was added as Se-Yeast or in an inorganic form, the Se egg content either doubled or increased by 74%, respectively.Se-Yeast increased Se egg content by 47% more than the increase observed in Na-selenite.e great variety of data characterizing Se concentrations in egg, egg white, or yolk content presented in the literature may have resulted from the various kinds of Se supplements used, the levels of added selenium, the genotypes of laying hens, the laying phases, the periods of Se addition, the kind of diets, and many other factors.According to Pappas et al. [26], the maintenance systems modulating the selenium content in eggs also have a meaningful input.A similar effect to that stated in our own experiments was confirmed by Zia et al. [44] who reported that egg selenium concentration was improved in the eggs of the animals receiving both the organic (Se-Yeast) and the inorganic (Na-selenite) selenium sources, whereas the effect of Se-Yeast treatment was superior to that of Na-selenite.Bargellini et al. [20] found the Se content of egg yolk increased by 39-61% when using 0.1 mg/kg of Na-selenite or Se-iodine, with the suggestion that this addition may enhance the Se absorption.According to literature data, it may be stated that the main reason for the increased Se deposition in eggs via Se-Yeast addition is that the majority of Se in Se-Yeast is Se-Met, a Se analog of methionine [45].Se-Met is deposited in the egg to a greater extent than selenium from Na-selenite and is actively absorbed and incorporated into eggs as effectively as methionine [46].Given this, it is clear that the highest level of selenium is to be found in the egg content of hens fed with diets enriched in Se-Met.
e estimated parameters of egg characteristics (Table 4) revealed that use of selenium feed additives does not cause significant changes, except for yolk color which was clearly higher according to the La Roche scale (P < 0.01) in eggs derived from hens fed with diets supplemented with selenium.
In the presented experimental data, selenium source did not influence egg characteristics.e data are comparable with the results obtained by Invernizzi et al. [43]-these authors stated that addition of selenium into hen diets had no effect on shell thickness, which ranged from 0.359 mm for eggs received from hens from the control group (without selenium addition) to 0.369 mm for eggs received from hens fed with diets with Se-Yeast.
ese authors additionally stated that the kind of selenium additive applied significantly affected shell strength-this parameter, in their experiment, was the highest for egg shells in the group that received an addition of Se-Yeast, and this amounted to 39.03 N. In the presented data (Table 4), shell strength was also the highest in eggs from animals that received Se-Yeast addition, but these differences were not significant statistically.Heinz and Hoffman [47] did not find any significant differences in the mass or thickness of the eggshell in eggs from Mallard ducks, as a result of the kind and level of selenium.Only one parameter, the yolk color index, was influenced by Se addition.A similar effect of selenium addition was reported by Mohiti-Asli et al. [48].e effect of selenium on yolk color may be explained by its antioxidative properties.e color of egg yolk is produced by xanthophyll pigments (oxycarotenoids) derived from the diet.Oxycarotenoids, when oxidized, lose their pigmenting power; therefore, it may be assumed that selenium, as well as vitamin E, has a beneficial effect on egg color stabilization when added to laying hen diets [48].e length of the period of supplementation of Se influenced changes in the egg characteristics and caused an increase in the share of egg yolk and a decrease in the share of egg white.No available data were found on the effect of selenium on yolk and white share in eggs.Our result may be an effect of the laying period (phase) and physiological changes in egg composition including white and yolk share.
e accumulation of selenium in the liver varied between treatments from 313.5 to 462.6 μg•Se/kg (Table 5).
e highest Se concentration in hen livers was found in birds fed with diets containing Se-Yeast (462.2 μg/kg), but the lowest were in treatments in which hens were fed with Se-Met diets (P ≤ 0.01).Selenium content in wet liver tissue, as presented in numerous publications, changed significantly; however, in general, the presented data from these experiments indicated that application of organic forms of selenium significantly increased selenium concentrations in the liver.Pan et al. [49] found that, when using Na-selenite, the Se level in terms of the wet weight of the liver amounted to 645 μg/kg and for Se-Yeast, the level was 669 μg/kg (P < 0.05) in comparison to the control (582 μg/kg).In fact, our data are in agreement with these findings concerning selenium-enriched yeast application because the content of selenium in the liver of birds receiving diets with Se-Yeast was significantly higher (P < 0.05) when compared to the control and Na-selenite groups of animals; however, unexpectedly, no statistical differences were stated between the control and Na-selenite groups.In our data, the content of selenium in the liver of hens fed with diets enriched with selenium in the form of Se-Met was significantly (P < 0.01) lower than in the remaining groups.Our data do not agree with those contained in most publications on this topic.A significant effect on liver selenium concentration of Se-Met addition into hen diets was reported, among others, by Wang et al. [50], who found Se concentrations in the liver of 315 μg/kg when Na-selenite was added and 596 μg/kg when Se-Met was included in the diet.Also, Windisch et al. [51] stated that, by using Se-Met, the cumulation of this trace element was significantly (P < 0.05) greater than that by the use of Na-selenite or Se-Cys. is obtained result, according to the authors, may have various causes depending on the selenium form and on the selenium metabolism pathways used.According to Suchý et al., Se-Met, in comparison to the inorganic form, is absorbed more quickly in the small intestine and is resorbed independent of its levels in the organism and, additionally, this substance is not stored in the liver and is extensively used and reused [22].Selenomethionine is nonspecifically incorporated into proteins (Se-Met in place of methionine), particularly in organs with high rates of protein synthesis [52].Given this fact, in laying hens this form of selenium is incorporated to a greater extent into egg proteins.is was confirmed in the presented experiment by the higher content of selenium in eggs from Se-Met supplemented hen diets.4).

Conclusions
In conclusion, the best method for the enrichment of egg content with selenium is the application of dietary Se-Met as the source of selenium in laying hen diets.Selenium content in eggs from this group of animals was higher than that in a standard egg (11 μg): 17.20 μg/egg.Consumption of such selenium-enriched eggs may increase daily selenium intake and may be considered to be safe.Consumption of two selenium-enriched eggs a day satisfies about 60% of the daily Se dose recommended for humans.

Table 1 :
Ingredients and nutrient levels of the basal/experimental diets.

Table 2 :
Scheme of selenium supplementation.

Table 4 :
Effects of dietary selenium supplements on egg characteristics.
1 La Roche scale: max.15 points.Means within a column marked with different superscripts differ significantly with A and B by P < 0.01.

Table 3 :
Se content in fresh egg content (μg/kg).P values: 0.348 for time of supplementation; 0.059 for interaction treatment × time.Means within a row marked with different superscripts differ significantly with A and B by P < 0.01.* Calculated on the basis of egg characteristics (Table