1. Introduction
The application of dietary plant extracts to poultry feeds is gaining recent interest, particularly for their use as an alternative to antimicrobial growth promotors [
1,
2,
3]. Plant extracts, also known as phytogenics, phytobiotics or botanicals, are products derived from plant origin and refer to a variety of ingredients including herbs, spices, essential oils, and oleoresins [
4]. Dietary phytogenics have been found to influence nutrient availability, growth performance, feed intake, endogenous secretions, and immune response [
1,
5]. They have also been found to possess antioxidative properties [
5,
6,
7,
8].
Stevia (
Stevia rebaudiana, Bertoni; STE) is a small, leafy, perennial shrub belonging to the
Asteraceae family. It is native to parts of South America, where, historically, it has been used medicinally by indigenous peoples. [
9]. Stevia is also well known for its use as a natural sweetener for human consumption. Steviol glycosides, such as stevioside and rebaudioside, extracted from the leaves of stevia, have been found to be approximately 300 times sweeter than sucrose [
10,
11]. In vitro work showed that stevioside, carotenoids, phenolic compounds and flavonoids in STE might also be involved in antioxidant defense mechanisms to help relieve stress [
12,
13]. Research by Pirgozliev et al. [
14,
15] showed that the lutein and zeaxanthin from STE can be accumulated in the liver of chickens, thus improving the antioxidative status of birds through enhancing dietary antioxidant availability and potentially increasing resistance to diseases. Lutein and zeaxanthin belong to the class of oxygen-containing carotenoids known as xanthophylls and can be found abundantly in green leafy vegetables, colorful fruit, maize, pepper, various animal tissues and egg yolk [
16]. An increase in the dietary intake of lutein and zeaxanthin in humans is associated with improvements in visual function [
17]. A daily intake of 5–10 mg/day of lutein/zeaxanthin can reduce the risk of age-related macular degeneration (AMD) [
17]. The daily consumption of those xanthophylls in developed countries, such as the USA, is relatively low, e.g., 1–2 mg/day, thus increasing the risk of reduced human macular pigment optical density [
17,
18]. In the EU alone, the annual costs associated with managing the effects of severe AMD is approximately EUR 89.46 billion [
19].
Eggs are an essential part of the human diet. Globally, the average consumption of eggs each year per capita ranges from about 62 in India to in excess of 358 in Mexico, as the average consumption is about 161 eggs per person per year [
20]. Ortiz et al. [
21] reported that the total xanthophyll content in the egg yolk of hens fed yellow or orange maize based diets, or free range hens may exceed 20 µg/g egg yolk. Comparable with other carotenoids, the bioavailability of lutein can be enhanced when consumed along with lipids [
22]. Bioavailability can also be influenced depending on the food source and matrix. For example, lutein derived from lutein-enriched eggs provides more bioavailable lutein than supplements or spinach [
23].
Stevia exhibits natural variation in carotenoids that are associated with visual function and overall human health benefits [
17]. These include lutein, zeaxanthin, β-cryptoxanthin, and β-carotene, as well as vitamin E and coenzyme Q10 [
14,
15]. The idea of the biofortification of hen’s eggs has been well applied [
21,
24,
25,
26], although information on the impact of feeding STE to laying hens on egg carotenoids is lacking.
Information on potential changes to the physical characteristics of stored eggs when feeding hens diets containing STE is also very limited [
27]. Niemiec et al. [
28] reported that the dietary addition of antioxidants did not affect egg weight after 20 days of storage at 12 °C, although Omri et al. [
27] found that feeding antioxidants reduced the losses of egg, albumen and the shell of eggs stored at 4 °C for 30 days. Whiting et al. [
29] also found no effect of dietary antioxidants on stored egg quality variables. In general, further information is required to determine the effect of dietary STE on the quality variables, chemical composition and antioxidant content of both fresh and stored hen eggs.
The aims of this study were to (1) establish whether increasing dietary levels of STE in hen feeds can increase the concentration of lutein, zeaxanthin and total carotenoids in hen eggs, (2) to examine the effects on egg and eggshell quality variables, and (3) to investigate the impact of dietary STE on the quality variables of stored eggs. It has been hypothesized that feeding STE to laying hens will increase carotenoid content in eggs without having an impact on egg production variables.
4. Discussion
This study aimed to investigate the impact of dietary stevia on yolk pigmentation, and carotenoid concentration when fed to Hy Line Brown laying hens at an early stage of production. Feed intake, egg chemical composition and egg production were also measured. The observed small differences between dietary calculated and determined composition is most likely due to the differences between the composition of the ingredients that were used in the present study and the values in the software used for dietary formulation for the same ingredients. Given the length of the study and the number of replications, it is not expected that this may have an impact on the study outcome. The production and egg quality results were within the expected range for eggs from Hy Line Brown laying hens at this stage of production. The lack of differences between the control and 2% STE regarding production variables suggest that STE can be incorporated in laying hen diets at 2%. However, the determination of an optimum inclusion level for both, antioxidant content in eggs and production variables requires more research.
The study compared the yolk pigmentation and carotenoid deposition of eggs produced from diets with the same proximate composition but with different dry stevia leaf inclusion. Yolk color has been previously found to reflect the content of carotenoids in hen feed, while yolk color has an aesthetic effect on consumer preference [
21,
41,
42]. The diets containing stevia resulted in egg yolks with a higher YF color and b* index values (indicating yellowness) compared with the control diet. Moreno et al. [
42] and Ortiz et al. [
21] obtained higher results in YF yolk color when feeding yellow or orange maize compared to our results. This may be explained by the high inclusion rate of maize in the diets (over 500 g/kg) compared to stevia (20 g/kg), although the stevia contained 55.8 µg/g carotenoids compared to 5.7 µg/g and 24.9 µg/g for yellow and orange maize, respectively [
21].
Although β-carotene and β-cryptoxanthin were present in the stevia leaves, they were not detected or found in negligible amounts in the eggs. When carotenoid compounds such as β-carotene and β-cryptoxanthin are converted into vitamin A, they lose their pigmenting properties which may have influenced this fraction in the yolk [
39,
43]. In the present study, the obtained range of total carotene fraction in the yolk is comparable to values reported in previous studies, irrespective of the dietary content or source [
21,
25,
39]. Kljak et al. [
40] also reported an initial increase in yolk carotenoids in a study, followed by a dip and further increase. Weekly fluctuations occurred among individual carotenoids, markedly so β-cryptoxanthin and β-cryptoxanthin compared with lutein and zeaxanthin. Despite the deposition of xanthophylls into egg yolk being a relatively quick process, it could take at least attain a stable response to dietary pigments [
40,
44,
45], thus supporting the results obtained in our study.
Lutein, zeaxanthin, and meso-zeaxanthin are the macular pigment carotenoids [
16]. The yellow color of the central retina (
Macula lutea) in human eye is due to the presence of the carotenoid pigments lutein and zeaxanthin [
18]. The high serum level of macular carotenoids is suggested to play a role in the protection of the retina against light-induced damage [
46]. However, the macular carotenoids cannot be synthesized
de novo and must be obtained through diet [
16]. The macular carotenoid concentration in yolks produced with the C + 2% STE diet was higher than in yolks produced with the C + 1% STE diets and the control in the reported study. It must be noted, however, that in the reported study, carotenoids were determined in the mix of yolk and albumen together, despite carotenoids being found in the yolk only. Since the yolk is about one-third of the egg without shell [
47] the actual carotenoid content in yolk is over three times higher than reported in the whole egg in this study. This means that the overall carotenoid content in the yolk at the end of the study for hens fed the control, C + 1% STE and C + 2% STE diets are about 9 µg/g, 14 µg/g and 17 µg/g, respectively. Thus, in the birds fed stevia, the carotenoid level was higher than the levels in eggs produced by hens fed white maize, but comparable to the eggs of free range, herb fed, colored maize or commercially available marigold fed birds [
21,
25,
39]. This also suggests that planting stevia shrubs in the areas of free-range reared hens may increase the carotenoid content in eggs. Thus, suggesting that further research on the impact of higher dietary stevia inclusion on egg carotenoid content is warranted.
Dietary STE had about 1.1 µg/kg or 6.2% less vitamin E compared to the control diet, thus suggesting an explanation for the higher vitamin E concentration in eggs laid at the beginning of the study, compared with the following time periods. Although numerically, only birds offered STE consumed less feed, which may be connected to inconsistent egg vitamin E concentration. Although diet is more influential than heredity in its effect on variability of egg composition, the content of protein and fat is directly influenced by birds’ genetics [
47], thus supporting the observed lack of differences in chemical composition of eggs in this study.
As expected, albumen height decreased with the length of storage while albumen pH increased [
48]. The reduced egg weight at the end of the four-week storage period results from water exchange between yolk and the egg white and from water and carbon dioxide loss through the eggshell pores [
20]. Egg yolks are known for their high fat content and are therefore susceptible to lipid oxidation. In this study, hens were able to deposit antioxidants in the form of carotenoids from diets into their egg yolks that could protect the lipids during egg processing. However, stevia addition in laying hen diets does not affect the impact of storage on egg weight or the internal quality of eggs produced. Usually, eggs are stored at room temperature and are considered as “fresh” up to 28 days after laying [
20]. In addition, research by Nimalaratne et al. [
49] has demonstrated that the antioxidant activity of egg yolk was globally unchanged during six weeks of retail storage. Thus, suggesting that the relatively short storage period and the controlled environment, i.e., 15 °C, may have contributed to the lack of interaction between dietary stevia and length of storage on the studied egg quality variables in this study. In addition, diets were supplemented with synthetic vitamin E (slightly exceeding NRC, 1994 recommendation of 12 IU/kg), thus providing enough for hens to deposit antioxidants from diets into their egg yolks that could protect the lipids during egg storage. This may further explain why STE addition in the laying hen diets did not affect the quality of stored eggs.