Consumers' preference for egg selection has now been shifted from yolk cholesterol content or fatty acid profile to its colour [29]. Dietary supplementation of carotene plays a vital role in egg yolk colour intensification [30], pigment (carotene) synthesis in hen eggs is feasible with its supplementation via diet’s ingredients [31]. Microorganisms like algae, fungi some bacteria, and plants were reported to synthesized carotene pigments [32]. In the present study, egg yolk colour assessment was estimated by RYCF and CIELAB photo-calorimetric determination system of L* (lightness), a* (red), b* (yellow), chroma (saturation or colour intensity), and hue (colour tone). The results revealed dietary supplementation of organic selenium increased (p < 0.05) egg yolk yellowness and reduced yolk brightness, with no significant (p > 0.05) effect on egg yolk redness among all the treatment groups and for both the fresh and post stored eggs, respectively. Similarly, hens received organic Se supplementation showed increased values of yolk colour (fresh and stored) observed by Yolk Colour Fan® (Roche) scale, though; only determine the sequence number of a stripe range. Research on the effects of Se on egg yolk colour traits measured by a Chroma Meter is wanting.
Some literature findings contained antioxidant are used to support or otherwise of these findings. An investigation by Gouveia et al. [33] confirms absorption of xanthophylls via the intestinal tract by chickens, assimilated into triglyceride-rich lipoproteins (chylomicrons) released into the blood (circulatory system), and transported to the yolk [34]. Since, in the current study, hens were offered the same basal diet except for Se supplementation (inorganic or organic), the enhanced yolk colour observed in the treated group might be induced because of xanthophylls being accumulated in the yolk.
Table 4: Total antioxidant capacity of pre- and post-stored egg yolk and breast tissue for 14 days at 4 ± 2 0C
Assay | Parameters | Days | Treatments |
| | | Con | Na2SeO3 | Se-Yeast | ADS18 | P-value |
Phosphomolybdenum Assay (Antioxidant activity, mg AAE/g) * | Egg yolk | Initial | 0.82 ± 0.07 | 0.87 ± 0.04 | 0.81 ± 0.04 | 0.86± 0.06 | 0.8073 |
Fresh | 0.83 ± 0.05d | 1.12 ± 0.02c | 1.40 ± 0.04b | 1.81 ± 0.04a | < .0001 |
Stored | 1.50 ± 0.15c | 1.55 ± 0.11bc | 1.93 ± 0.16ab | 2.11 ± 0.06a | 0.0127 |
Breast meat | NA | 0.77 ± 0.05 | 0.74 ± 0.08 | 0.77 ± 0.06 | 0.73 ± 0.05 | 0.957 |
Ferric Reducing Antioxidant Power (FRAP) assay (Antioxidant activity, mg GAE/g) ** | Egg yolk | Initial | 0.84 ± 0.02 | 0.88 ± 0.01 | 0.85 ± 0.01 | 0.84 ± 0.01 | 0.2238 |
Fresh | 1.73 ± 0.07b | 1.78 ± 0.06b | 1.90 ± 0.08b | 2.23 ± 0.04a | 0.0005 |
Stored | 3.16 ± 0.09 | 3.01 ± 0.07 | 3.08 ± 0.05 | 3.12 ± 0.06 | 0.4896 |
Breast meat | NA | 1.90 ± 0.10 | 2.06 ± 0.13 | 2.27 ± 0.18 | 2.13± 0.11 | 0.294 |
1Con = control, Na2SeO3 = sodium selenite; Se-yeast = Selenium yeast; ADS18 = Stentrophomonas maltophilia. Initial = day 3, fresh = day 95 and stored = day 109 for 2 weeks. *: Antioxidant activity evaluated as phosphomolybdenum reducing power and express in ascorbic acid equivalent (AAE). **: Antioxidant activity evaluated as ferric reducing power and expressed gallic acid equivalent (GAE); a − c Mean in the same row with different superscripts are significantly different (p < 0.05). NA = not applicable |
Table 5: Effects of different Se sources on oxidative stability of pre- and post-stored egg yolk, breast and thigh muscle
Parameters | Days | Treatments1 | P-value |
| Con | Na2SeO3 | Se-Yeast | ADS18 |
Egg yolks, µg MDA/Kg | D 3 | 0.093 ± 0.03 | 0.092 ± 0.032 | 0.084 ± 0.006 | 0.088 ± 0.004 | 0.4105 |
D 46 | 0.129 ± 0.004a | 0.117 ± 0.002a | 0.102 ± 0.004b | 0.082 ± 0.006c | < .0001 |
D 60 | 0.133 ± 0.007a | 0.111 ± 0.007b | 0.092 ± 0.004c | 0.084 ± 0.004c | < .0001 |
D 74 | 0.118 ± 0.004a | 0.109 ± 0.002b | 0.102 ± 0.001c | 0.096 ± 0.002c | < .0001 |
D 95 | 0.114 ± 0.004a | 0.104 ± 0.003a | 0.086 ± 0.003b | 0.077 ± 0.003b | < .0001 |
D 109 * | 0.148 ± 0.010a | 0.127 ± 0.005b | 0.105 ± 0.004c | 0.084 ± 0.002d | < .0001 |
Breast meat, µg MDA/g | D 0 | 11.46 ± 0.51a | 10.05 ± 0.40b | 9.85 ± 0.09b | 8.72 ± 0.10c | 0.0001 |
D 1 | 10.69 ± 1.40ab | 12.15 ± 1.17a | 8.23 ± 0.92b | 7.59 ± 0.55b | 0.0211 |
D 5 | 17.79 ± 1.60a | 16.31 ± 1.17ab | 15.64 ± 0.74ab | 13.62 ± 0.53b | 0.0854 |
Thigh, µg MDA/g | NA | 27.00 ± 0.89a | 26.39 ± 1.38a | 22.62 ± 1.21b | 18.95 ± 0.84c | 0.0001 |
Serum, nmol MDA/ml | NA | 0.184 ± 0.006a | 0.179 ± 0.006ab | 0.169 ± 0.002bc | 0.159 ± 0.003c | 0.0046 |
1Con = control, Na2SeO3 = sodium selenite; Se-yeast = Selenium yeast; ADS18 = Stentrophomonas maltophilia. Initial = day 3, fresh = day 95 and stored = day 109 for 2 weeks. *: D109; Eggs were stored for 14 days at 4 ± 2 0C prior to analysis a − c Mean in the same row with different superscripts are significantly different (p < 0.05). NA = not applicable |
The present results are consistent with previous results of [35], who reported a linear increase in egg yolk colour score (RYCF) with dietary incorporation of marine algae (Spirulina platensis) at 0.1 to 0.2% (6.3–7.6) and 1.5 to 2.5% (10.55–11.66) compared to the negative control, respectively. Similarly, [36] follow the same suits with [30] feeding 4.5% linseeds. Studies on the Se supplementation (inorganic or organic) on the egg yolk colour are lacking. However, regarding the calorimetric determination, a report by Omri et al. [30] revealed a decreased (p < 0.05) in the lightness of yolk colour with 2% tomato and red pepper mixture supplementation. Dietary addition of 130 g of dried tomato peel per kg was reported to enhance the egg yolk colour index from 8.5 to 14.6 [37]. Arpasova et al. [38] reported lighter in egg yolks colour from hens fed-lower Se than those fed higher. This resulted in a deeper egg yolk colour in the organic (ADS18 or Se-Yeast) treated group. Contrary, Omri et al. [32] reported an increase in egg yolk redness and decreased yellowness with colorimetric determination when evaluating the effects of Arthrospira platensis (spirulina) supplementation on laying hens. However, the latter author [39] reported an increase of yellowness and decreased redness of eggs stored at 4 0C for 30 days corresponding to linseeds-fed hens. Nonetheless, studies on the effect of storage on egg yolk colour are lacking. A trial on yolk pigmentation stability in omega 3 (ω-3) enriched eggs stored at room temperature (26.5 0C) and refrigeration (7.9 0C) for 35 d showed decreased yolk colour [40]. A similar pattern of significant (p < 0.05) differences was showed for fresh and stored egg yolks saturation (C*), however, no dietary influence on hue angle values to either of the treatment groups. The use of Se supplementation (organic) as antioxidants can be a solution substantially to minimize the use of synthetic pigments as feed additives in laying hens’ diets. Therefore, its stability over a defined period of storage needs to be of primary concern to researchers. It is important to fix undesirable changes (be it chemical, enzymatic, or physical) in appearance, colour as well as the quality of the nutrients contained in layers diet, as some can damage and lead to pigment losses during storage. Contrast comparison showed distinct differences (p < 0.05) between basal diet, Na2SeO3 and organic (ADS18 or Se-Yeast) in respect to brightness and yellowness of the egg yolk (fresh or stored), in which hens received supplemented Se source had higher values of their parameters. There were significant (p < 0.05) differences between the treatments over the trial period for Chroma (C*) with no difference (p > 0.05) observed for the Hue (H*) index.
Carotenoids are lipid-soluble compounds for color pigmentation of orange-yellow or sometimes red in plants, insects, birds, or aquatic animals that have resulted from a pigment known as carotene or carotenoids [41]. Egg yolk carotenoids solely depend on nutrients available in the feed, thus, varies with egg types [42]. Selenium as an antioxidant may play a role in improving egg yolk color. For instance, dietary supplementation of organic selenium or vitamin E enhanced egg yolk concentration [43]. Egg yolk color is influenced by oxycarotenoids (xanthophyll pigments) resulting from hen’s diet, as well as lost when oxidized [44]. They are connected to lipoproteins, and transported to egg yolk [45]. The stability of lipid-soluble carotenoids available in hen’s diet or body is influenced by yolk color response to antioxidants [46]. Furthermore, carotenoid is an antioxidant that acts as feather dye, vitamin A precursor, and other related endocrine and immune activity in poultry [47]. Even though this study measured total carotenoids. Therefore in addition to age-related macular degeneration, the two major egg carotenoids (lutein and zeaxanthin) play an important role as their mechanism of action by protecting light-induced oxidative damage in eye macular disease [48]. They also found their ability to attract blue light until its effects on photoreceptor cells to have passive antioxidant action [48]. Owing to their possession of double bonds, they have ability to produce a highly resonance-stabilized C-centered radical that help to scavenge hydroxyl and superoxide radicals [49]. Many of these effects are related to its function as biological antioxidant [50]. The organic bacterial protein (ADS18) posed superior total carotenoids than inorganic and basal diet groups in the present research. This is in line with previous findings by Karadas et al. [51] who documented a 22-fold increase in carotenoids in hens supplemented with carotenoids in eggs than control during pre- and post-hatch studies. In in vivo studies with dried tomato peel showed a 2.7-fold (p < 0.05) increase in β-carotene versus 1.7 µg/gDM in comparison [52]. There is a lack of data on the effect of storage of eggs in hens supplemented with similar treatment to ours. Similar to our findings, decreased total carotenoids egg yolk concentrations (28.55 vs 22.09 µg/g) and (28.55 vs 23.57 µg/g) were shown for eggs stored at room temperature (26.5 0C) or under refrigeration (7.9 0C) for 35 d [40]. Contrary to this, no decrease was observed in the total carotenoid concentration of eggs yolk for eggs stored at 2 0C for 56 d [53] and 4 0C for 28 d [30].
Total egg yolk and breast meat cholesterol was significantly decreased (p < 0.05) by supplementation with selenium. Selenium supplementation was reported by Poirier et al. [54] to reduce plasma lipids concentrations of total cholesterol, LDL-cholesterol, and VLDL-cholesterol in male Syrian hamsters. Selenium plays a vital role in hormonal (thyroid) balance of fat metabolism, as established earlier [55]. Selenium deficiency may be associated with increased 3-hydroxy 3-methylgluatryl CoA (HMG-CoA) reductase activity in liver microsomes [56]. Selenium could play a role in reducing cholesterol as an antioxidant form of the active center of GSH-Px [57]. In their review, Brown and Jessup [58] observed that, as antioxidant level increases in the diet, the cholesterol concentration decreases, and vice versa. Among the experimental treatments in the present research, organic (ADS18 > Se-Yeast) Se supplementation significantly (p < 0.05) reduced total egg yolk and breast meat cholesterol compared to inorganic (Na2SeO3) and basal diet fed hens. A linear reduction in egg yolk and serum cholesterol levels was recorded with an increase of 0, 5, 10, and 15 g MPM/Kg in Moringa oleifera pod (Lam.) meals, corresponding to 219.07, 216.88, 212.49, and 201.87 mg/100 g respectively [59]. Organic selenium and vitamin E supplementation has been shown to decreased serum and egg yolk cholesterol content (p < 0.01) [25]. Nano-selenium supplementation reported by Radwan et al. [57] lowers the total plasma and yolk cholesterol (153 mg/dl and 14.0 mg/g) at 0.25 ppm, respectively. Similarly, Attia et al. [60] and Łukaszewicz et al. [61] reported a significant reduction in plasma cholesterol at 0.25 ppm and 0.3 mg/kg, of dual-purpose breeding hens of Gimmizah and Japanese quails yolk fed-organic selenium. The lower cholesterol observed in fresh, stored egg yolk and refrigerated breast meat may be attributable to differences in cholesterol synthesis control enzymes in chickens [62]. The reason for the process of cholesterol decrease may be due to the inhibition of sterol biosynthesis by oxysterols. Selenium supplementation has been shown to increase 15d-PGJ2 (15-deoxy-Δ-12, 14 prostaglandin J2) production in response to oxidative stress-induced cells protection [63], a known peroxisome proliferator-activated receptor-γ ligand (PPAR γ) [64]. Activation of latter by troglitazone, regulate the concentration of sterol regulatory element-binding protein (SREBP)-2, resulting in a decreased cholesterol synthesis [65].
Abundant compounds show antioxidant properties in both egg white and yolk [10]. Egg proteins (ovalbumin, ovotransferrin, phosvitin), egg lipids (phospholipids), and micronutrients (vitamin E and A, selenium and carotenoids) have been classified as being high in antioxidants [10]. The type of flavonoids and phenolic acid that play a role in good antioxidant activity were the bioactive compounds in the egg, particularly the albumen [66]. The presence and activity of phenolic acids and flavonoids in the system may counteract the activity of free radicals effectively [66]. In the present study, dietary supplementation with Se increased the bioactive (phenolic) content of fresh, stored egg yolk and breast antioxidant. Phenolics are a major phytochemical class, which contains chemical compounds of one or more phenolic groups [67]. The resulting concentration of antioxidants depends on the phenol group and the double bond, i.e. the lower the concentration of antioxidants, the higher its activity [68]. Unlike the report from Siger et al. [69], that the binding ability to scavenge peroxyl radicals was unconnected with the flavonoid concentration because of the chances of the formation of the phenoxyl radicals. Simple phenolic acids are not easily deposited into chicken egg yolk due to their hydrophilic nature under natural conditions [70]. It is difficult to compare our findings with other literature studies because work is scarce in this regard. Untea et al. [71] have recently reported a significant increase in the total polyphenol content of egg yolk with dietary inclusion of bilberry and walnut leaves at 0.5% and 1.0%, respectively. From hens fed grounded mixtures of 4.5 and 2% of linseeds and fenugreek seeds, a significant increase in total phenol concentration in yolk was observed compared to 4.5% ground linseeds and 4.5% ground linseeds and 1% each of dried tomato and sweet pepper powder, respectively [37]. A phyto-additives (dried tomato peel) trial on laying hens’ yolk carotenoids and phenols showed an increase in total phenol content and correlated with cholesterol reduction results [72]. The inclusion of varying levels of dietary fennel seed did not, however have significant effect on the total phenol content of egg yolk from Cotunix cotunix japonica [73]. To the best of our knowledge, total phenol content in response to selenium supplementation is absent from data or literature on egg yolk and breast meat.
Flavonoids are forms of antioxidants that are water-soluble and have glucose groups in the side chain [74]. Subsequent concentration of antioxidants depends on the phenol group and the double bond presence, i.e. the lower the concentration of antioxidants, the higher its activity [68]. However, Siger et al. [69] reported that the binding ability to scavenge peroxyl radicals was independent of the flavonoid concentration due to the chances of the phenoxyl radicals formation. Our results from fresh and stored egg yolk and the flavonoid content of breast meat demonstrated that selenium supplementation did not affect this parameter. In this respect, literature is very scarce, making it difficult to compare our results. A study by Omri et al. [30], found no changes in flavonoids content of hens’ egg yolk supplemented with linseed alone or combined with dried tomato-red pepper mixture before and after storage, is close to our findings. Omri and Abdouli [37], however, reported an increase in the flavonoid concentration of egg yolk (1.53 to 2.96 mg CAE/g) and (1.53 to 3.02 mg CAE/g) with hens supplemented with sweet pepper and dried tomato and fenugreek seeds. Reports on the effect of selenium supplementation on the total flavonoid content of egg yolk (fresh and stored) and breast meat are scarce.
Eggs are considered an excellent source of dietary antioxidants [75]. The phosphomolybdenum method and Ferric reducing antioxidant power (FRAP) assay are the main methods applied to evaluate antioxidant effects [30]. Fresh and stored egg antioxidant activity measured by the reduction of MO6 + to MO5 + was affected by (p < 0.05) by selenium supplementation in this study. Whereas only stored eggs were affected by dietary treatments for ferric reducing power activity. These findings indicate that selenium supplementation has enhanced the antioxidant capacity of eggs. In laying hens, research in the selenium supplementation literature on egg antioxidant activity expressed as AAE or GAE per g are scarce. In agreement with our observations, Wang et al. [76] reported higher total antioxidant capacity in eggs from epigallocatechin-3-gallate (EGCG)-fed layers. Furthermore, Omri, et al. [30] noted an increase in antioxidant activity measured by phosphomolybdenum reduction of a mixture of a diet supplemented with ground linseed (4.5%), dried tomato paste (1%), and sweet pepper powder (1%), stored and slightly in the fresh egg for hens-fed (1%). No significant (p > 0.05) antioxidant activity of egg yolk or meat was found when measured in the above study with a ferric reduction antioxidant power assay. Similarly, in golden pheasants (Chrysolophus pictus) fed diets containing different levels of green vegetables, egg yolk total antioxidant activity was positively affected [77]. In the current study, organic Se was found to increase the carotenoid content and decreased cholesterol content of egg yolk, which may have led to the higher egg antioxidant capacity. It is therefore logical to assume that organic supplementation (ADS18 or Se-Yeast) will enhance the antioxidant capacity of egg yolk, probably because organic Se prevents oxidation of the carotene, maybe increasing its deposition. McGraw et al. [78] stated an increase in egg antioxidant status during hatching and fleeing in goldfinches may be beneficial. However, additional studies are necessary to explain the underlying mechanisms behind this response.
The freshness of eggs is one of the consistency parameters affected by storage time, temperature, and relative humidity [57]. In cells, free radicals can produce reactive substances, which in turn damages cells and tissues. Antioxidants may prevent this damage caused by oxidation. Oxidation intensity of lipids is one of the parameters used as an indicator to assessed the freshness of poultry products. MDA is one of the lipid peroxides metabolic products and negatively correlates with the activity of GSH-Px [79]. The degree of peroxidation of fatty acids (animal products) can be monitored by malondialdehyde (MDA) concentrations, i.e., the higher the MDA concentration, so also the degree of lipid peroxidation. A decrease of the content of MDA observed in egg yolk, breast muscle, thigh, and serum may be due to the increase in the activity of GSH-Px resulting from supplemental dietary form (organic vs inorganic). The advantageous effects of organic Se in layers are connected to its efficacy of being transferred to the egg [80]. Organic Se was found to improve the oxidative stability of eggs [25] by reducing the eggshell or fluid’s cellular damage. Generally, due to its antioxidant properties, Se provides fat and protein oxidation stability of in the eggs of laying hens fed a dietary Se diet [23]. A study to investigate the interaction between different Se sources and trace elements in relation to the antioxidant system of laying hens is consistent with our results [81]. Egg-laying hens receiving selenomethionine in stored eggs showed decreased lipid peroxidation, probably increasing the shell life of the eggs [82]. Selenium supplementation at 0.25 ppm showed significant decrease in MDA content in fresh and stored egg yolk compared with 0.10 ppm supplemented egg yolk [57]. Wang et al. [83] reported a significant increase in GSH-Px activity and decrease yolk MDA content when Se to Langshan layer hens were supplemented with 0.3 mg/kg. More egg freshness was observed by the latter author and Gajčević et al. [84] after a month of storage at 4 0C with 0.4 mg/kg of organic Se supplementation.
The noted increase in MDA content in stored eggs could be attributed to the storage temperature (4 ± 2 0C). However, organic Se-supplemented eggs had lower TBA values than inorganic Se and unsupplemented egg groups. Cimrin et al. [14] recently recorded lower yolk TBARS values in eggs of vitamin E-fed hens at room temperature, although refrigerated eggs did not notice any dietary impact. Susceptibility to lipid peroxidation and egg yolk decreased with a combination of increased Se and vitamin E concentrations in Hy-Line W-36 hens trials [20]. As a result, authors stated that with advanced storage, MDA would increase [85]. However, with egg storage at 20 0C and refrigerated for 7–14 days, [17, 25] reported significantly increased yolk lipid peroxidation and MDA content. The dietary inclusion of linseed mixture, dried tomato paste, and sweet red pepper on fresh egg yolk lipid oxidation stability has not improved [30]. Nimalaratne et al. [75] reported no changes to the malondialdehyde content of refrigerated egg yolk within six weeks of storage. Nadia et al. [86] observed a significant difference between the treatments with dietary natural antioxidants but not with storage time.
While hens (layers) are intended for the production eggs, their quality of meat is important to ensure oxidative stability after supplementing their diet with antioxidants. Therefore, it may be beneficial to measure lipid peroxidation by breast and thigh muscle MDA content. Poultry meat, due to its high polyunsaturated fatty acid, is typically susceptible to rapid deterioration. Our findings are consistent with the results reported by Ahmad et al. [79] of significantly decreased lipid peroxidation in fed-selenium yeast chicken breast meat. A significant decrease in malondialdehyde (MDA) concentration of 0.15 mg organic Se/kg each in broiler muscle (L-Se-Met or D-Se-Met) compared to inorganic sodium selenite group [87]. In comparison, dietary supplementation with Se did not affect the concentration of MDA (expressed as TBARS) in the lamb muscle over 9 days of storage [88]. A Significant (p < 0.05) effect was observed in the activity of GSH-Px in serum and liver as well as free radical inhibition which in turn reduced the content of MDA in broiler blood fed 0.30 mg/kg of nano-Se [89]. However, there was no significant impact of dietary supplementation with Se on serum concentration of MDA [87].
Lipid peroxidation is a complex pathway in which fatty acyl hydroperoxides form as a free radical chain process reaction between unsaturated fatty acids and reactive oxygen species [79]. Lipid degradation and oxidative rancidity have occurred due to the sequence of secondary reactions resulting from primary autoxidation that cause changes in flavour, loss of nutrition quality environmental pollution among others [90]. Over the trial period, organic selenium (ADS18 > Se-Yeast) supplementation decreased the MDA content in egg yolk, breast, and thigh muscle, and blood. The difference in the responses of different sources of Se may be due to their difference in metabolic pathway (inorganic or organic), as organic sources significantly preserved the integrity of muscle cells linked to lipid oxidation and oxidative stability [91].