Effects of Dietary Selenium Source and Storage on Internal Quality of Eggs

A 4-week experiment was carried out on 360 laying hens of the Hy Line Brown hybrid. Laying hens were divided into three groups (C, E1 and E2) with 120 hens in each group and kept in 24 cages. Hens were fed layer diets containing 18% of crude protein and 11.60 MJ ME. Hens in the control group C were fed diets that contained 0.2 mg/kg of inorganic selenium (sodium selenite). Experimental groups E1 and E2 were given diets with increased concentrations of selenium as follows: E1 = 0.4 mg/kg of selenium (sodium selenite), E2 = 0.4 mg/kg of organic selenium (SelPlex). Selenium concentration in diets affected significantly the content of selenium in albumen (P < 0.001) and yolk (P < 0.05). The highest concentration of selenium was determined in albumen and yolk of eggs produced in group E2 (345 ng/g and 783 ng/g, respectively), then in eggs of group E1 (230 ng/g and 757 ng/g, respectively), and group C had the lowest concentration of selenium in albumen and yolk (181 ng/g and 573 ng/g, respectively). After 28 days of storage at 4 °C, the eggs containing organic selenium had more freshness (VN: C = 32.9, E1 = 2.60, E2 = 2.11). It was concluded that higher concentration of organic selenium in eggs was a limiting factor in metabolic processes, which positively affected the indicators of egg freshness. Organic selenium, inorganic selenium, albumen, yolk, egg freshness Selenium is found in two forms in nature: inorganic and organic. Inorganic selenium refers to different minerals such as selenite, selenate and selenide, and organic selenium is related to amino acids, methionine and cysteine. Outdoor living animals that eat plants take selenium in the form of selenomethionine (SeMet) in concentrations that depend on selenium concentration in soil, which can vary considerably according to area (Reilly 1996). Klapec et al. (2004) reported low concentrations of selenium in soil on the territory of Croatia because of which the selenium content in plant and animal foodstuffs was relatively low (egg = 52.5 ng/g; chicken meat = 115.3 ng/g; onion = 12.4 ng/g; potato = 7.2 ng/g). As a microelement, selenium has manifold importance in animal feed. Its antioxidant properties help protect animals from free radicals caused by oxygen metabolism. Moreover, selenium strengthens the immune system and boosts growth and feathering. As a dietary supplement, selenium reduces negative effects of stress in chickens kept in intensive production conditions, reduces mortality rate of one-day chickens and enhances quality of poultry products. One of the possibilities of enriching feed with selenium is to supplement it with plants fertilized with selenium (MacLeod et al. 1998). Another possibility is to supplement selenium preparations to commercial mixture fed to animals in order to design animal products that differ in their nutritive values from other conventional products available on the market. The aim of our study was to determine the effects of supplementation of organic and inorganic selenium in increased concentrations to the hens’ diet and to assess deposition of selenium in egg yolk and albumen as well as the effects of selenium on preservation of egg freshness. Materials and Methods The experiment was carried out on 360 Hy Line Brown laying hens of the age of 26-30 weeks. Hens were divided into three experimental groups; each group consisted of 24 repetitions. Hens were kept in cages by groups of five and fed commercial mixture containing 18% of crude protein and 11.60 MJ ME. The experiment was set up in three different feeding treatments. The control group had diets supplemented with 0.2 mg/kg of selenium ACTA VET. BRNO 2009, 78: 219–222; doi:10.2754/avb200978020219 Address for correspondence: Prof.Dr.Sc.Dr.h.c. Gordana Kralik Faculty of Agriculture Josip Juraj Strossmayer University of Osijek Trg svetog Trojstva 3, 31000 Osijek, Croatia Phone. +385 31 224 241 E-mail: gkralik@pfos.hr http://www.vfu.cz/acta-vet/actavet.htm from an inorganic source (sodium selenite). Groups E1 and E2 were given 0.4 mg/kg of selenium (E1: inorganic source of selenium; E2: organic source of selenium, selenomethionine Sel-Plex ®, Alltech, Inc.). On the 30th day of experiment five eggs were selected randomly from each group. The eggs were broken, and yolks and albumens were prepared for selenium analysis. The analysis was carried out as follows: The egg was broken and albumen and yolk were separated carefully from each other. Separated egg parts were homogenized. Around 0.5 g of homogenized sample was put in a Teflon flask for destruction. The sample was mixed with 5 ml of concentrated HNO3 and 1 ml of H2O2. After 15 min, the tubes were closed and heated in a microwave oven (CEM, Mars 5 model). After digestion, the tube content was transferred to a 25-ml flask and filled up with distilled water up to the line. Prior to sample preparation, all laboratory utensils were kept in a 10%-solution of HNO3 for 24 h. Selenium content in egg albumen and yolk was determined by the method of electrothermic atomic absorption spectrometry (Perkin Elmer, AAnalyst 600). Egg freshness was assessed over three different time periods (fresh eggs, eggs stored at 4 °C for 14 and 28 days) by determining values of the ageing rate (AR) and value number (VN) using the method of Kralik et al. (1990). The AR was obtained by the following mathematical expression: AR = ( 1.4184 – y) · 1000; where: y = refractive index of examined yolk, 1.4184 = refractive index of standard yolk obtained from a sample of 20 eggs at a temperature of 25 °C. The VN was calculated by the following expression: VN = 1000 · (y – a); where: y = refractive index of examined yolk, a = refractive index of examined albumen. Yolk and albumen refraction was determined using Refracto 30PX device. In order to calculate AR and VN, 75 eggs from each group were analyzed over three time periods. The influence of the main factors (concentration and source of selenium) on the assessed traits was determined using MANOVA (Main effects ANOVA). When the selenium source or concentration had a significant (P < 0.05) effect on the assessed traits, differences between the groups were tested by the Fisher’s LSD-test. Analyses were performed using Statistica v.7.1 software (StatSoft, Inc., 2005). Results and Discussion Data presented in Table 1 show that the source and concentration of selenium supplemented to the hens’ diet affected selenium deposition in albumen (P < 0.001). The content of selenium in albumen of eggs of group E2 was higher than in groups E1 and C (345 ng of selenium/g: 230 ng of selenium/g: 181 ng of selenium/g, respectively). The concentration of selenium in the hens’ diet has greater influence (P < 0.001) on the selenium content in egg yolk. The source of selenium had slightly less but still significant influence (P < 0.05) on the selenium content in egg yolk. Sample analysis showed that the eggs of the control group contained less selenium in yolks than groups E1 and E2 (573 ng of selenium/g : 757 ng of selenium/g : 783 ng of selenium/g, respectively). Based on the results stated above it was concluded that plants should be enriched with selenium in order to provide animal diets with higher concentrations of organic selenium as it is absorbed through membranes of the intestinal tract and actively accumulated in the organism cells. Humans and animals cannot synthesize selenomethionine in their organisms, so it is necessary to ensure selenium intake through diets (Schrauzer 2000). Producers usually supplement sodium-selenite (inorganic source of selenium) to animal feed; however, inorganic selenium cannot be fully used by an organism and is mostly excreted. Therefore, it is important to emphasize advantages of organic selenium in comparison to inorganic selenium. 220 Table 1. Content of selenium in egg albumen and yolk (n = 5) a, b P < 0.05; A, B, C P < 0.001 Values within the row differ significantly C = 0.2 mg/kg of inorganic selenium, E1 = 0.4 mg/kg of inorganic selenium, E2 = 0.4 mg/kg of organic selenium Content of Groups ( x ± SD) P value selenium C E1 E2 Source Concentration

Selenium is found in two forms in nature: inorganic and organic. Inorganic selenium refers to different minerals such as selenite, selenate and selenide, and organic selenium is related to amino acids, methionine and cysteine. Outdoor living animals that eat plants take selenium in the form of selenomethionine (SeMet) in concentrations that depend on selenium concentration in soil, which can vary considerably according to area (Reilly 1996). Klapec et al. (2004) reported low concentrations of selenium in soil on the territory of Croatia because of which the selenium content in plant and animal foodstuffs was relatively low (egg = 52.5 ng/g; chicken meat = 115.3 ng/g; onion = 12.4 ng/g; potato = 7.2 ng/g). As a microelement, selenium has manifold importance in animal feed. Its antioxidant properties help protect animals from free radicals caused by oxygen metabolism. Moreover, selenium strengthens the immune system and boosts growth and feathering. As a dietary supplement, selenium reduces negative effects of stress in chickens kept in intensive production conditions, reduces mortality rate of one-day chickens and enhances quality of poultry products. One of the possibilities of enriching feed with selenium is to supplement it with plants fertilized with selenium (MacLeod et al. 1998). Another possibility is to supplement selenium preparations to commercial mixture fed to animals in order to design animal products that differ in their nutritive values from other conventional products available on the market.
The aim of our study was to determine the effects of supplementation of organic and inorganic selenium in increased concentrations to the hens' diet and to assess deposition of selenium in egg yolk and albumen as well as the effects of selenium on preservation of egg freshness.

Materials and Methods
The experiment was carried out on 360 Hy Line Brown laying hens of the age of 26-30 weeks. Hens were divided into three experimental groups; each group consisted of 24 repetitions. Hens were kept in cages by groups of five and fed commercial mixture containing 18% of crude protein and 11.60 MJ ME. The experiment was set up in three different feeding treatments. The control group had diets supplemented with 0.2 mg/kg of selenium from an inorganic source (sodium selenite). Groups E 1 and E 2 were given 0.4 mg/kg of selenium (E 1 : inorganic source of selenium; E 2 : organic source of selenium, selenomethionine -Sel-Plex ® , Alltech, Inc.). On the 30 th day of experiment five eggs were selected randomly from each group. The eggs were broken, and yolks and albumens were prepared for selenium analysis. The analysis was carried out as follows: The egg was broken and albumen and yolk were separated carefully from each other. Separated egg parts were homogenized. Around 0.5 g of homogenized sample was put in a Teflon flask for destruction. The sample was mixed with 5 ml of concentrated HNO 3 and 1 ml of H 2 O 2 . After 15 min, the tubes were closed and heated in a microwave oven (CEM, Mars 5 model). After digestion, the tube content was transferred to a 25-ml flask and filled up with distilled water up to the line. Prior to sample preparation, all laboratory utensils were kept in a 10%-solution of HNO 3 for 24 h. Selenium content in egg albumen and yolk was determined by the method of electrothermic atomic absorption spectrometry (Perkin Elmer, AAnalyst 600). Egg freshness was assessed over three different time periods (fresh eggs, eggs stored at 4 °C for 14 and 28 days) by determining values of the ageing rate (AR) and value number (VN) using the method of Kralik et al. (1990).
The AR was obtained by the following mathematical expression: AR = ( 1.4184 - y ) · 1000; where:  y = refractive index of examined yolk, 1.4184 = refractive index of standard yolk obtained from a sample of 20 eggs at a temperature of 25 °C.
The VN was calculated by the following expression: VN = 1000 · ( y - a ); where:  y = refractive index of examined yolk,  a = refractive index of examined albumen. Yolk and albumen refraction was determined using Refracto 30PX device. In order to calculate AR and VN, 75 eggs from each group were analyzed over three time periods. The influence of the main factors (concentration and source of selenium) on the assessed traits was determined using MANOVA (Main effects ANOVA). When the selenium source or concentration had a significant (P < 0.05) effect on the assessed traits, differences between the groups were tested by the Fisher's LSD-test. Analyses were performed using Statistica v.7.1 software (StatSoft, Inc., 2005).

Results and Discussion
Data presented in Table 1 show that the source and concentration of selenium supplemented to the hens' diet affected selenium deposition in albumen (P < 0.001). The content of selenium in albumen of eggs of group E 2 was higher than in groups E 1 and C (345 ng of selenium/g: 230 ng of selenium/g: 181 ng of selenium/g, respectively). The concentration of selenium in the hens' diet has greater influence (P < 0.001) on the selenium content in egg yolk. The source of selenium had slightly less but still significant influence (P < 0.05) on the selenium content in egg yolk. Sample analysis showed that the eggs of the control group contained less selenium in yolks than groups E 1 and E 2 (573 ng of selenium/g : 757 ng of selenium/g : 783 ng of selenium/g, respectively). Based on the results stated above it was concluded that plants should be enriched with selenium in order to provide animal diets with higher concentrations of organic selenium as it is absorbed through membranes of the intestinal tract and actively accumulated in the organism cells. Humans and animals cannot synthesize selenomethionine in their organisms, so it is necessary to ensure selenium intake through diets (Schrauzer 2000). Producers usually supplement sodium-selenite (inorganic source of selenium) to animal feed; however, inorganic selenium cannot be fully used by an organism and is mostly excreted. Therefore, it is important to emphasize advantages of organic selenium in comparison to inorganic selenium.  Cantor et al. (1996) studied the effects of selenium (inorganic and organic; 0.3 mg/kg) supplemented to the hens' diet on the selenium content in egg and reported a significant effect of selenium concentration in diet on its content in egg, which corresponds with our results. Moreover, Payne et al. (2005) pointed out that different sources of selenium (inorganic or organic) and concentrations of selenium in the feed (0; 0.15; 0.3; 0.6 and 3 mg/kg) significantly affected (P < 0.01) content of selenium in egg. The authors stated that the concentration of selenium in eggs was increased in proportion to the concentration of selenium in the diet (P < 0.01). Their results are also in accordance with ours. Surai (2000) stated that supplementation of organic selenium to the hens' diet could significantly increase the selenium content in egg yolk and albumen (P < 0.01). He also reported a high correlation coefficient between selenium concentrations in the hens' diet and in eggs. Cantor (1997) and Paton et al. (2000) also confirmed relations between source and concentration of selenium in diets and selenium concentration in eggs.
Egg freshness is related to its quality. It depends on the egg storage period (counted in days) and on conditions under which the eggs are stored (temperature and relative air humidity). There are many indicators that show changes occurring as a consequence of egg storage. Longer period of storage increases AR and decreases VN. Table 2 shows changes in AR and VN of eggs of all three groups over the period of 28 days. In our experiment, the AR values were increased over the 28-day period; however, the differences between groups in were not significant (P > 0.05) for the first two periods (1 st and 14 th day). After storing eggs at 4 °C for 28 days, the highest AR was determined for group C (3.29), followed by group E 1 (2.60), and the least AR was exhibited by group E 2 (2.11). Determined differences regarding AR of investigated eggs after 28-day storage were significant. The source and concentration of selenium in hens' diets influenced the determined differences in AR (P < 0.05 and P < 0.01, respectively). In the course of egg ageing, the concentration of egg mass is reduced. Water contained in albumen permeates the yolk, and some nutrients contained in yolk can permeate albumen. These osmotic tracks and changes in albumen and yolk concentrations can be measured by the refractometric method. Metabolic processes in eggs of group E 2 were less intensive, so AR in that group was the lowest over the whole storage period, and the VN was the highest. Kralik et al. (1990) measured the mean of AR of fresh eggs 0.92, which corresponds to AR of group E 2 . Rotruck et al. (1973) proved selenium to be a part of glutathione peroxidase enzymes (GSH-Px), which confirmed its role in cellular antioxidative metabolism. Wakebe (1999) emphasized positive effects of dietary supplementation with organic selenium at the concentration of 0.3 mg/kg of feed on the activity of GSH-Px enzymes. Its presence reduces occurrence of free radicals that intensify metabolic processes in cells (oxidation of lipids and proteins) and reduce activity of enzymes of polymerase, nuclease, ligase, etc. Changes that occur through intensified metabolic processes in the egg have negative effects on preservation of egg freshness. In our study, the highest VN was measured in fresh eggs of group E 2 , then of group E 1 , and C group had the least VN (60.60 : 60.50 : 60.20, respectively), which is in accordance with the results of Kralik et al. (1990). On the 14 th and 28 th day of egg storage, VN was still the highest in group E 2 . The influence of source and concentrations of selenium were not significant (P > 0.05) regarding VN of the examined groups. Sources and different concentrations of selenium in hens' diets resulted in the increase of the selenium content of egg albumen (P < 0.001) and yolk (P < 0.05 and P < 0.001, respectively). Analysis of AR and VN of eggs exhibited more favourable values for eggs produced by hens that were fed diets containing organic selenium compared to eggs from hens fed diets with inorganic selenium. The source and concentration of selenium in hens' diets did not influence the differences in VN between groups, however, both factors had a significant effect on AR of eggs after they were stored for 28 days at 4 °C (P < 0.05 and P < 0.01, respectively).