3.1. Egg Physical Characteristics
Egg physical characteristics before and after storage at 4 °C for 30 days are reported in
Table 2.
As reported in our previous research study [
72], dietary supplementation of 4.5% of linseeds increased (
p > 0.05) fresh egg weight, and compared to the linseeds (L), tomato-sweet pepper mixture was without effect (
p > 0.05). However, stored egg weight was influenced (
p < 0.05) with dietary treatment and, for each of the four treatments, there was a slight loss of egg weight after storage at 4 °C. Fresh and stored egg yolk weights were not influenced (
p > 0.05) by dietary treatment. Dietary treatment did not affect (
p > 0.05) the fresh eggs’ albumen weight. However, LTP increased (
p < 0.05) the stored eggs’ albumen weight. Storage decreased (
p < 0.05) albumen weight of hens fed with the control diet. The shell weight of fresh eggs was not affected (
p > 0.05) by dietary treatment, but increased (
p < 0.05) by L and LTP addition for stored eggs. Storage decreased (
p < 0.05) egg shell weight of hens fed on the LTP diet. Shell thickness was affected (
p > 0.05) neither by dietary treatment nor by storage. In this regard, Yassein et al. [
83] showed that dietary addition of 5% of linseeds did not affect egg weight and albumen, yolk and shell percentages. Ahmad et al. [
84] found that feeding hens with 5% of linseed did not affect egg weight (59.93 g vs. 60.47 g), yolk weight (15.15 g vs. 14.81 g), albumen weight (9.05 g vs. 10.51 g), shell weight (8.13 g vs. 8.48 g) and shell thickness (0.39 g vs. 0.38 mm).
Concerning tomato, Akdemir et al. [
43] reported that the dietary supplementation of 0.5% and 1% of tomato powder did not influence yolk weight (16.59 g vs. 17.11 g), shell weight (6.79 g vs. 6.97 g) and shell thickness (0.399 mm vs. 0.393 mm).
Studies on the effect of egg storage on its physical characteristics are lacking. However, Niemiec et al. [
85] reported that the dietary addition of primrose, linseeds and rapeseeds at, respectively, 2.88%, 3.66% and 5%, with or without supplementation of 200 mg vitamin E/kg, did not affect egg weight after 20 days of storage at 12 °C. A significant reduction in the egg yolk weight after storage was found, from 24.23% (control group) to 23.35% (primrose, linseeds and rapeseeds) and 23.18% (primrose, linseeds, rapeseeds and vitamin E).
3.2. Egg Yolk Antioxidant Profile
Egg yolk antioxidants [
86] (α-carotene, β-carotene, β-cryptoxanthine, lycopene, zeaxanthine, total phenols and flavonoids) concentrations before and after storage at 4 °C for 30 days are reported in
Table 3.
Fresh eggs concentrations of all antioxidants were affected (
p < 0.05) by dietary treatment. Carotenoid concentrations of fresh eggs of hens fed with control diet (C) varied from 7.7 to 12.4 μg/g of yolk, respectively, for lycopene and zeaxanthine [
72]. These levels were in agreement with those (12.8 and 9.2 μg/g DM, respectively, for luteine and zeaxanthine) reported by Englmaierovà et al. [
49] of eggs from hens fed with corn, wheat, soybean meal and alfalfa meal. Our results, were higher than those reported by Hammershøj et al. [
46] who found that eggs of hens fed on a standard organic food (wheat, oats, peas, sunflower meal, fish meal) contained: 7.46 μg/g of lutein, 2.6 μg/g of zeaxanthine, 0.01 μg/g of α-carotene/g and 0.03 μg/g of β-carotene. Our results were also higher than those (7.09 μg/g of luteine, 0.85 μg/g of cis-luteine, 7.09 μg/g of zeaxanthine, 0.69 μg/g of cis-zeaxanthine and 1.07 μg/g of β-carotene) reported by Kotrbáček et al. [
87] for a diet containing 33% of wheat, 30% of corn and 24% of soybean meal. Studies in the literature with regard to this aspect are lacking. However, a total phenol content equal to 0.54 mg GAE/g DM was reported by Amar et al. [
88] and equal to 0.72 mg GAE/g (diet based on yellow corn) and 0.66 mg GAE/g DM (diet based on wheat) were found by Nimalaratne et al. [
14]. Dietary incorporation of linseeds increased (
p < 0.05) fresh egg yolk concentrations of carotenoid and total phenols and did not affect (
p > 0.05) egg yolk concentration of flavonoids.
Diet with dried tomato (1%) and sweet red pepper (1%) mix contained four times more carotenoid than diet with linseeds (L). However, fresh egg yolk carotenoid concentrations of hens fed with L and LTP were not different (
p > 0.05). Studies on the effects of linseeds on the egg yolk antioxidants profile are lacking. Concerning tomato, Habanabashaka et al. [
89] reported that dietary incorporation of 0, 3, 6 or 9% of tomato by-products increased egg yolk concentration of lycopene from 0.01 to 0.95 µg/g, lutein from 10.1 to 13.9 µg/g and of zeaxanthine from 9.4 to 12.9µg/g. However, Amar et al. [
88] reported that dietary incorporation of 0, 4, 7, 10 and 13% of tomato peel increased egg yolk concentration of lycopene from 26.5 µg/g DM (4%) to 42.8 µg/g DM (7%); 37.6 µg/g DM (10%) and 41.8 µg/g DM (13%) and of β-carotene from 6.5 µg/g DM (0%); 11.3 (4%) µg/g DM; 17.6 (7%) µg/g DM; 12.3 (10%) µg/g DM to 16.7 µg/g DM (13%). Akdemir et al. [
43] evaluated the effect of the dietary addition of 0.5%, 1% of tomato powder reported that an increase in egg yolk concentration of lycopene from 6.53 (0.5%) to 8.05 µg/g (1%), of β- carotene form 172 µg/g (0%) to 331 µg/g (0.5%) and 551 µg/g (1%) and of lutein from 6.85 µg/g (0%) to 7.23 µg/g (0.5%) and 9.03 µg/g (1%). Dietary incorporation of 0.1%, 0.2%, 0.4%, 0.8% of paprika extract increased egg yolk concentration of total carotenoid from 3.43 μg/g (0%) to 7.7 μg/g (0.1%), 10.86 μg/g (0.2%), 14.60 μg/g (0.4%) and 16.83 μg/g (0.8%) [
90]. Egg storage for one month did not affect (
p > 0.05) the egg yolk concentrations of all carotenoids (α-carotene, β-carotene, cryptoxanthin, lycopene and zeaxanthin) and decreased (
p < 0.05) the total phenol concentrations of each treatment.
Reports on the effect of storage of eggs of hens fed with similar treatments to ours diets are lacking. However, Barbarosa et al. [
91] reported that egg storage for 35 days at room temperature (26.5 °C) reduced egg yolk total carotenoid concentration from 28.55 to 22.09 μg/g. By contrast, egg storage for 35 days at 7.9 °C reduced total carotenoid egg yolk concentration from 28.55 to 23.57 μg/g. Gawecki et al. [
92] also reported that egg storage for 8 weeks at 2 °C did not reduce egg yolk total carotenoid concentration. However, after 15 weeks of storage, the total carotenoid concentration in the egg yolk decreased from 28.55 µg/g to 27.03 μg/g.
In the present study, egg yolk antioxidant capacity was evaluated by determining egg yolk capacity to reduce MO6+ to MO5+ and Fe3+ to Fe2+. Lipid oxidative stability determined by thiobarbituric acid reactive substances (TBARS) was even higher than the concentration of MDA, which was low.
Only fresh egg antioxidant activity determined by the reduction of MO
6+ to MO
5+ was influenced (
p < 0.05) by dietary treatment. Fresh egg antioxidant activity of the control group was equal to 4.48 mg AAE/g. Reports in the literature concerning egg yolk antioxidant activity expressed as AAE and as GAE are lacking. Fresh egg yolk concentration of MDA was not affected (
p > 0.05) by dietary treatment. Fresh egg yolk concentration of MDA of the control treatment was equal to 0.11 μg/g [
72]. This value was lower than the 1.17 μg/g reported by Englmaierovà et al. [
49] and the 0.7 μg/g reported by Venglovská et al. [
93] for fresh eggs of hens fed on corn, wheat and soybean meal. Our results were similar to those reported by Hayat et al. [
94] with a mean value of 0.1 μg/g for fresh eggs of hens fed on corn meal and soybean meal.
The dietary addition of 4.5% of linseeds increased (
p < 0.05) the egg yolk antioxidant capacity, but not its lipids oxidative stability (
p > 0.05). Thus, egg enrichment with fatty acids (polyunsaturated and polyunsaturated ω-3) through the dietary supplementation of linseeds did not reduce egg yolk lipid oxidative stability. This lipid protection against oxidation may be attributed to the increase of fresh egg yolk pigments and total phenol concentrations (
Table 3). In this regard, Hayat et al. [
94] reported that egg yolk MDA concentration increased from 0.1 to 0.23 μg/g (10% of linseeds flaxseed), 0.32 μg/g (10% of linseeds plus 50 IU of α-tocopherol) and 0.28 μg/g (10% of linseeds plus 150 mg of BHT). However, Boruta and Niemiec [
95] reported that the dietary addition of 3% of linseeds with or without supplementation of 200 mg/kg of vitamin E did not affect fresh egg yolk concentration of thiobarbituric acid-reactive substances (TBARS). The dietary inclusion of 1% of dried tomato and 1% of sweet red pepper in addition to the linseeds did not improve fresh egg yolk antioxidant activity and its lipid oxidative stability (
p > 0.05). This inefficiency of dried tomato and sweet red pepper mixture could be due to its low level of incorporation (2%) that did not affect egg yolk concentrations of pigments, total phenols and flavonoids when compared to diet with linseeds (
Table 4).
Akdemir et al. [
43] showed that the dietary addition of 0.5% and 1% tomato powder reduced egg yolk concentration of MDA from 0.33 µg/g (control) to 0.25 µg/g (0.5%) and 0.21 µg/g (1%). Sahin et al. [
96] also reported the dietary addition of 100 and 200 mg of lycopene/kg in Japanese quail diet reduced the egg yolk concentration of MDA from 0.86 (control) to 0.79 µg/g (100 mg of lycopene/kg) and 0.74 μg/g (200 mg of lycopene/kg). Dietary addition of carophyll, lutein or algae (chlorella) improved egg yolk the oxidative stability from 1.17 μg/g (control) to 1 μg/g (carophyll), 0.87 μg/g (lutein) and 0.90 mg/kg (algae) [
49].
Concerning stored eggs, only antioxidant activity measured by phosphomolybdenum reduction and lipid oxidative stability were influenced (
p < 0.05) by dietary treatment. Diet with linseeds (4.5%) plus sweet red pepper and tomato mix (2%) was associated with higher (
p < 0.05) lipid oxidative stability than diet with linseeds. However, egg storage decreased (
p < 0.05) yolk antioxidant activity but not lipid stability to the oxidation of the four treatments. In this regard, Pereira [
97] reported that MDA egg yolk concentration increased from 0.52 to 0.71 and 0.90 μg/g after storage at 4 °C for, respectively, 60 and 90 j. By contrast, Hayat et al. [
94] reported that egg storage at 4 °C for 20, 40 and 60 days did not affect MDA concentration. Shahryar et al. [
70] reported that ω-3 and ω-6 enriched eggs stored at 4 °C for 30 and 60 days increased MDA concentrations. Boruta and Niemiec [
95] evaluated the effect of dietary addition of 4% rapeseed, 3% linseeds and 2% primrose, with or without supplementation of 200 mg/kg of vitamin E, on egg antioxidant status and reported that egg storage for 3 and 6 months increased yolk MDA concentration. However, vitamin E supplementation decreased egg yolk MDA concentration after 6 months of storage.
3.3. Egg Yolk Coloration
Egg yolk coloration before and after storage at 4 °C for one month are represented in
Table 5.
Color scores determined by the Yolk Color Fan
® scale (RYCF) were affected by dietary treatment (
p < 0.0001). Fresh egg yolk score of the control group was the lowest with a mean value of 4.67 [
72]. Our values were not in agreement with 6.65 reported by Abdouli et al. [
98] and 8.64 found by Lokaewmanee et al. [
99] for eggs of hens fed with a similar diet to our control treatment. b* mean value showed that egg yolk yellow color was sufficiently intense. Our results were lower than 32.5 and 48.2 reported, respectively by, Abdouli et al. [
98] and Dvorak et al. [
100]. Negative mean value of a* indicated an absence of the red hue. Higher mean values ranging from 0.05 to 13.5 were reported by Dvorak et al. [
100]. L* and C* mean values showed that the egg yolks of hens fed with control treatment were characterized by an intense, light yellow color. Dietary incorporation of 4.5% of linseeds increased (
p < 0.05) the egg yolk color score (RYCF). The fresh egg yolk of hens fed with linseeds tended to have a slightly more yellow, redder and less light color than the eggs of the control group. Correlations between the yolk color scores (RYCF), parameters determined by Chroma Meter and pigments concentrations (
Table 6), showed that the yolk color scores (RYCF) and redness (a *) were positively correlated with all the determined pigments.
However, lightness (L*) and yellowness (b*) were negatively correlated with all the determined pigments. Dietary supplementation of the tomato and the red pepper mixture (2%) increased (
p < 0.05) the RYCF score and the red hue (a*), and decreased (
p < 0.05) the lightness (L*) without affecting (
p > 0.05) the saturation (C*). These results are in agreement with those reported by Akdemir et al. [
43] who found that dietary supplementation of 0.5% or 1% of dried tomato increased egg yolk color from 11.25 (control) to 13.08 (0.5%) and 13.58 (1%). The dietary addition of 130 g of dried tomato peel/kg DM increased the egg yolk color index from 8.5 to 14.6 [
88]. Habanabashaka et al. [
89] reported that the supplementation of 6% of tomato waste meal increased egg yolk scores from 4.66 (control) to 9.15 (6%). Salajedheh et al. [
101] found that dietary incorporation of 19% of dried tomato pomace had a significant effect on egg yolk color scores which increased from 7.25 to 9.38. Dietary addition of 50 and 100 g/kg of dried tomato powder increased egg yolk color from 6.7 (control) to 9.7 and 10.3 for respectively, 50 and 100 g/kg of dried tomato powder [
102]. Salajedheh et al. [
101], also reported that the dietary supplementation of 150 and 190 g/kg of dried tomato powder increased egg yolk color from 7.25 (control) to, respectively, 8.50 (150 g/kg) and 9.83 (190 g/kg). Studies on the effect of red pepper powder on the egg yolk color are lacking. However, Niu et al. [
90] showed that the dietary addition of 0.8% of paprika increased egg yolk color from 1.7 to 9.9. Li et al. [
42] also reported that dietary supplementation of 0.3 to 4.8 or 9.6 ppm and 0.8% of red pepper powder increased egg yolk color from 7.7 to 12.7.
Studies on the effect of linseeds on egg yolk color are lacking. However, Yassein et al. [
83] showed a decrease in yolk color related to linseed supplementation at a level of 5%. Reports on L*a*b* color space in response to dietary supplementation of carotenoid are also lacking.
Concerning stored eggs, with the exception of saturation (C*), all other parameters were influenced (
p < 0.05) by dietary treatment. Storage of eggs of hens fed with the control diet (C) did not affect (
p > 0.05) the yolk score and color parameters determined by colorimetry. By contrast, the storage increased (
p < 0.05) yolk lightness (L*) and decreased redness (a*) of eggs corresponding to linseeds treatment (L). However, stored eggs corresponding to the LTP- treatment were found to be less colorful (lower RYCF score) and lighter (higher L*) than fresh eggs. Studies on the effect of the storage on egg yolk color are lacking. However, Barbarosa et al. [
91] reported that storage of ω-3 enriched eggs for 35 days with or without refrigeration decreased the yolk color. This decrease became significant from the 28th day of storage for eggs stored at room temperature (26.5°). For eggs stored at 7.9 °C, egg yolk color reduction was not significant during the 35 days.
The use of dried tomato and red pepper as natural or biological antioxidants has been a new solution to reduce the supplementation of synthetic pigments as feed additives in laying hens’ diets. However, the stability of these antioxidants may be affected over a long period of storage. In fact, degradation reactions (chemical, enzymatic and physical) can cause undesirable changes in the appearance, color and texture as well as in the nutrient content of the laying hens’ diets. These reactions can be responsible for pigment losses during storage. These losses must be considered when formulating feed in order to avoid complaints. Also, the quality of these supplements may be affected by several factors such as tomato and pepper variety, soil, cultural practices and climate.