1. Introduction
Eggs enriched with one or more functional ingredients, namely, n-3 polyunsaturated fatty acid (PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), selenium, and lutein, have enhanced nutritional value within the context of human health [
1]. In recent years, functional nutrition of eggs has garnered the attention of many researchers [
1,
2,
3,
4,
5,
6,
7,
8,
9]. n-3 PUFA reduces the levels of plasma lipids and improves cardiovascular health, thereby reducing disease severity [
10] and serum concentrations of alanine aminotransferase and improving liver function in patients with non-alcoholic fatty liver disease [
11].
The consumption of eggs rich in n-3 PUFA is an effective way to acquire this functional ingredient [
12]. The most widely used method to produce eggs enriched with n-3 PUFA is to include sources of n-3 PUFA in the diet of hens [
13]. Among the different dietary sources of n-3 PUFA and α-linolenic acid (ALA) are present in plant-based oilseeds, whereas EPA and DHA are found primarily in marine oils and algae. In addition, EPA and DHA are also found in plant oils derived from certain genetically modified organisms, such as canola [
14] and arabidopsis seeds [
15]. However, the utilization of such oils containing EPA and DHA has not yet been commercialized. In one study, when the same amount of extra n-3 PUFA (120 mg per 100 g feed) was added to the diet, the deposition of n-3 PUFA was the highest in fish oil, followed by microalgae, and lowest in flaxseed [
16]. By adding the same amount of fish oil or flaxseed oil (FSO) (3%), more n-3 PUFA can be deposited in the egg yolk with FSO (7.60%) than with fish oil (4.17%), and the eggs from hens given feed with 3% fish oil were unacceptable to sensory panelists [
17]. Flaxseed is one of the most important oilseed crops, a rich source of ALA and is emerging as an important functional food ingredient [
18]. However, flaxseed contains linatine (a vita-min B6 antagonist) as well as anti-nutritional factors, including insoluble and soluble non-starch polysaccharides [
19]. Non-starch polysaccharides in flaxseeds decrease nutrient digestibility and can negatively impact egg n-3 PUFA incorporation [
20]. However, it was reported in some studies that the results obtained were differentiated due to some unknown reasons yet. A recent study reported that high content of flaxseed combined with another antioxidant source (sea buckthorn or grapeseed) had positive effects on production performances, fatty acids deposition, and health indexes [
21]. Research has shown that dietary FSO can be a viable option for enriching eggs with ALA. This is because FSO contains a higher amount of ALA than milled flaxseed, resulting in increased ALA deposition in yolks [
3,
18]. Furthermore, incorporating dietary FSO in the feed of laying hens can be an effective means of promoting the enrichment of n-3 PUFA in egg yolks [
22].
Selenium is known for its antioxidative properties, as it protects organisms from the harmful effects of free radicals and carcinogens [
23]. To improve human selenium status, foods such as meat, milk, and eggs that have been enriched with selenium can be consumed [
23]. In the poultry industry, it is common to supplement the diets of laying hens with selenium to enhance the selenium concentration in eggs and meat. Traditionally, sodium selenite has been the go-to source of selenium for animal feeds. However, in recent times, organic sources of selenium, such as selenium-enriched yeast (SEY), have gained traction as an approved means of increasing the selenium concentration in eggs and carcass meat [
6,
7]. As such, SEY supplementation can be implemented as part of selenium-enriched egg production [
7,
8].
Lutein has been shown to possess anti-inflammatory properties, making it useful in the treatment of a variety of inflammatory disorders, such as diabetes retinopathy, eye diseases, liver injury, and obesity [
24,
25]. Given that the synthesis of lutein within the human body is not possible, the only means of acquiring it is through the consumption of dark green leafy vegetables and egg yolks [
26,
27,
28]. Marigold flowers are an excellent natural source of lutein, and they are often utilized to enhance the color of the yolk and carotenoid content of eggs [
4,
29].
According to observations, it has been found that only a few studies have compared these egg-related indices across different breeds (i.e., within the context of dietary supplementation). It is important to compare the differences in the content of functional nutrients in enriched eggs from different breeds to produce eggs with enhanced nutritional profiles. Dwarf Layers are known for their good egg-laying performance, and their eggs have a pink shell color, which is preferred by Chinese consumers [
30]. Silky fowl, Beijing-you chicken and Shouguang chicken are local chicken breeds in China. Although they may not have high egg-laying performance, their eggs are priced higher. Considering the market potential for functional nutrition eggs, these breeds can be considered candidate chicken breeds. White Leghorn is a commonly used breed for functional egg research [
31,
32,
33]. Therefore, we have selected the above five breeds to explore the differences in the ability of different breeds to deposit functional nutrients. The aim is to provide some insights and references for the production of functional nutrition eggs.
2. Materials and Methods
2.1. Animal Ethics
The animal experiments in this study adhered to the Guidelines for Experimental Animals provided by the Animal Care and Use Committee of China Agricultural University, with permit number AW10803202-1-2. The experiments also complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
2.2. Experimental Materials and Feeding Management
The animal experiment took place at the Experimental Unit for Poultry Genetic Resource and Breeding, a facility belonging to China Agricultural University. The population used in the experiment was produced via pedigree mating, with each generation being the result of a random breeding process. The Dwarf Layer was developed by the Poultry Genetic Resources and Breeding Experimental Unit of China Agricultural University through 4 repeated backcrosses of the meat type dwarf ISA-Vedette to the female CAU brown egg layer, mainly used for egg production [
34,
35]. Silky fowl, Beijing-you chicken, and Shouguang chicken are all local chicken breeds in China. White Leghorn is a traditional and commonly used white shell layer. All chicken breeds used in the experiment are pure lines, among which only Dwarf Layers have dwarf genes.
In Trial 1, 150 laying hens (41 weeks old) of each breed were assigned at random to the FSO group, which was fed the basic diet mixed with 2.5% flaxseed oil and 0.016% vitamin E, and the CON group, which was fed the basic diet (n = 75 in each group). The breeds used were Dwarf Layer, White Leghorn, Silky Fowl, Beijing-you chicken, and Shouguang chicken. Flaxseed oil was obtained from Ningxia Qianhoufu Trading Co., Ltd. (Yinchuan, Ningxia, China). To avoid a negative change in appearance, a 2.5% inclusion level was implemented in this study based on findings from a prior publication, where 3% flaxseed oil inclusion resulted in an adverse effect on appearance [
17]. The addition of vitamin E is a traditional way to relieve PUFA oxidation and improve egg storage quality and oxidation stability [
36]. After 28 days of feeding, 30 eggs per breed and group were collected at random to confirm n-3 PUFA enrichment and egg equality. Weekly evaluations were carried out to measure their feed consumption, egg laying rate and egg weight for each group (n = 75 in each group). These measurements were recorded throughout the entire 28-day experimental period.
In Trial 2, 300 White Leghorn (24-week-old) and 300 Dwarf Layer (24-week-old) chickens were distributed within each breed into four groups: (1) CON; (2) FSO; (3) SEY, fed basic diet mixed with 0.02% selenium-enriched yeast (2000 mg/kg); 4) MFE, fed basic diet mixed with 0.2% marigold flower extract (1.5%). Flaxseed oil was obtained from Ningxia Qianhoufu Trading Co., Ltd. (Yinchuan, Ningxia, China). SEY was obtained from Yunnan Dongcheng Lvkang Biotechnology Co., Ltd. (Zhaotong, Yunnan, China). MFE was obtained from Hebei Meineier Biotechnology Co., Ltd. (Shijiazhuang, Hebei, China). After 28 days of feeding, 30 eggs per breed and group were collected at random to confirm n-3 PUFA enrichment and egg equality. Weekly evaluations were carried out to measure their feed consumption, egg laying rate and egg weight for each group (n = 75 in each group). These measurements were recorded throughout the entire 28-day experimental period.
The basic diet used in this study was formulated to meet the nutritional standards recommended by the National Research Council (1994) and the feeding standards for chickens (NY/T 33-2004). Please refer to
Table 1 for details on the composition and nutritional content of the basic diets. Metabolizable energy is calculated based on the Chinese feed composition and nutritional value table. The CON and FSO groups are illustrated in
Table 2, which displays the percentage composition of fatty acids. Throughout the 28-day study period, the chickens were kept individually in cages, with one chicken per cage. The recommended feeding schedule is twice a day (08:30, 14:30), and the eggs should be collected once a day (16:30). A nipple-type water dispenser is used to provide free drinking water for the hens. The light system consists of a morning and evening light supplement system, with a light duration of 16 h per day and a light intensity of 10 Lx. It is important to check the hens daily to ensure they have sufficient feed and water supply, as well as to monitor their health status.
2.3. Sample Collection
A plastic hand-held egg separator (Wuxi, Jiangsu, China) was applied to separate various egg components, such as yolk and albumen. In Trial 1, after the albumen of all groups was eliminated, the yolks were weighed and stored at −20 °C until subsequent analyses were performed. The albumen that had adhered to the eggshell was wiped with a paper towel, and then the eggshell was weighed after 12 h. For Trial 2, the albumen from the FSO, MFE, and a portion of the CON groups were removed, while the yolks were weighed and stored at −20 °C until further analysis. The egg yolks and albumen from another part of the CON group were mixed thoroughly and then stored at −20 °C for subsequent analysis. The egg yolks and albumen from the SEY group were mixed thoroughly and then stored at −20 °C for subsequent analysis.
2.4. Egg Quality Determination
The evaluation of egg quality involved collecting 30 eggs from each dietary group, which were then analyzed for various quality parameters such as eggshell thickness (EST), eggshell strength (ESS), yolk color (YC), egg weight (EW), egg yolk weight (EYW), albumen weight (AW), albumen height (AH), and Haugh units (HU). The EW, AH, HU, and YC measurements were obtained using a multifunctional egg tester (EMT-5200; Tokyo, Japan), while ESS was determined using a Model-II eggshell strength tester (Robotmation, Tokyo, Japan). EST was measured at three different positions on the egg (Blunt end, equatorial, sharp end) using a micrometer (Robotmation, Kyoto, Japan), and the average value was calculated for further analysis.
2.5. Nutrient Content Determination
Ten eggs from each group were used to determine the n-3 PUFA, lutein, and selenium concentrations in the egg. The separated egg yolks from CON and FSO groups were freeze-dried at −80 °C for 72 h with a vacuum freeze-dryer and weighed. Then, the lyophilized egg yolks were crushed into powder, and fatty acids were determined through gas chromatography (Agilent 6890, Agilent Technologies Inc., Santa Clara, CA, USA), following the national standard GB-5009.168-2016. In brief, 0.5 g of the sample was accurately weighed into a screw-top glass tube, and then toluene and acetyl chloride methanol solution (10%) were added. After mixing, the sample was allowed to stand in an 80 °C water bath for 2 h. Next, the reaction solution was transferred to a centrifuge tube, and the glass tube was washed with sodium carbonate solution. Finally, 100 μL of the upper clear liquid was taken, filtered through a membrane, and analyzed using a gas chromatograph to determine the content of fatty acids.
The samples from the CON and SEY groups were removed, and the selenium content was determined using a fluorescence spectrophotometer (AF 7500, Beijing Titan Instruments Co., Ltd., Beijing, China), following the national standard GB-5009.93-2017. The experiment involved digesting 1 g of the sample with a mixture of nitric and perchloric acids (v/v = 9:1), followed by adding hydrochloric acid and ethylene diamine tetraacetic acid solution, and then adding 2,3-diaminonaphthalene reagent. Following purification, the sample was subjected to measurement of fluorescence intensity, with an excitation wavelength of 376 nm and an emission wavelength of 520 nm.
The separated egg yolk from the CON and MFE groups was removed, and lutein content was determined through high-performance liquid chromatography (HPLC 1260, Agilent Technologies, Inc., Santa Clara, CA, USA), following the national standard GB-5009.248-2016. In summary, homogenized egg samples weighing 2 g were mixed with 0.2 g of butylated hydroxytoluene and 10 mL of anhydrous ethanol in a 50 mL polypropylene centrifuge tube. Then, 10 mL of 10% potassium hydroxide solution was added for the saponification reaction. After the reaction, the extract was obtained using a solvent extraction method. Then, after washing and concentration treatment, the extract was used for liquid chromatography analysis with 0.1% butylated hydroxytoluene ethanol solution as the base solution. For separation, a column (4.6 × 250 mm, 5 μm) was utilized with methanol/water and methyl tert-butyl ether mixture as the mobile phase, and the detection wavelength was set at 445 nm.
The contents of three functional nutrition ingredients in the egg were calculated as follows:
Fatty acid content (%) = [fatty acid content in the sample (mg/g)]/[total fatty acid content in the sample (mg/g)].
Selenium content (μg/egg) = the selenium content in the sample (μg/g) × egg weight (g).
Lutein content (μg/egg) = the lutein content in the sample (μg/egg) × egg yolk weight (g).
2.6. Statistical Analyses
All test data were all analyzed using the univariant program of the general linear model process of SPSS17.0, with two-way ANOVA for diets and chicken breeds. The Duncan multiplex test was performed to identify indicators with significant main effects. A predetermined level of statistical significance was set at p < 0.05. GraphPad Prism 8 was used as the analytical tool for data plot analysis.
4. Discussion
Research has indicated that incorporating n-3 PUFA sources into the diet of laying hens does not have a notable impact on either egg production or egg quality [
13,
20,
37,
38]. However, it has been observed that the inclusion of n-3 PUFA sources decreases EST and YC and increases AH and egg production [
39]. In Trial 1 of the present study, the effects of dietary FSO on egg production and quality parameters varied depending on the breed of chicken. There were no substantial effects of a diet containing FSO on some measures, such as ESS, YC, and AH. However, there were significant effects on other parameters, including the average daily feed intake of Dwarf Layer and White Leghorn, feed-to-eggs ratio of Silky fowl, egg mass and egg production of White Leghorn and Silky fowl, EST of the Dwarf Layer and White Leghorn, EYW of Dwarf Layer, and AW of Shouguang chicken. In Trial 2, no significant effects of dietary FSO were observed on performance and egg quality parameters in White Leghorn or Dwarf layer breeds. This lack of significant effects may be attributed to differences in chicken breeds, ages, sources of n-3 PUFA, and varying dosages of supplementation used across studies.
In our study, supplementing the diet of hens with FSO led to a significant increase in the ALA, EPA, DHA, and total n-3 PUFA contents in eggs (
p < 0.05), consistent with previous findings [
40,
41,
42]. However, Lee et al. (2021) reported that in their study, only DHA and n-3 PUFA contents were significantly increased after feeding Hy-Line Brown laying hens with FSO at varying doses (0.2%, 0.4%, 0.6%, and 0.8%) for 4 weeks, while there was no significant difference in the ALA (0.23 vs. 0.20) or EPA (0.01 vs. 0.01) content [
22]. This discrepancy may have been due to differences in the doses of FSO added between the studies. Additionally, in the present study, FSO (2.0%) supplementation resulted in the content of SFA in the egg yolk lipids, as was observed by a significant reduction in myristic (0.69 vs. 0.52) and palmitic acid (27.53 vs. 25.70) percentages (
p < 0.05), consistent with the results of the study by Souza et al.’s research (2008) [
43]. In addition, our study demonstrated that the supplementation of FSO in hen diets substantially augmented the n-3 PUFA content in eggs, which corresponded to a significant reduction in the n-6 PUFA-to-n-3 PUFA ratio (
p < 0.05), akin to the effects of other dietary sources of n-3 PUFA [
20,
22,
31,
32,
40].
Supplementation of SEY significantly increased the amount of selenium in eggs (
p < 0.05), which is consistent with other findings [
6,
7,
8,
44]. There were no notable impacts observed on ESS, AH, YC, or HU as a result of dietary SEY. This is in agreement with previous reports, which have also observed no significant effects of SEY on these particular parameters [
5,
7,
9]. However, in this study, the EW of the Dwarf Layer breed was significantly altered after supplementation with SEY (
p < 0.05). When it comes to selenium deposits, there was no significant difference observed in selenium deposits among various chicken breeds (
p > 0.05).
The study showed that no significant effects of dietary MFE were observed on ESS, AH, HU, or EW, which aligns with the results of previous research by Grcevic et al. (2019) [
4] and Wen et al. (2021) [
45]. However, feeding hens with MFE did significantly enhance yolk color, which is in line with earlier studies conducted by Grcevic et al. (2019) (0 vs. 1 g/kg of marigold extract, 9.63 vs. 12.77; 0 vs. 2 g/kg of marigold extract, 9.63 vs. 13.5) [
4], Islam et al., (2017) [
46], and Wen et al., (2021) [
45] (
p < 0.05). Interestingly, the color of the egg yolk also significantly improved in Dwarf Layer (8.3 vs. 9.3) and White Leghorn (8.2 vs. 9.1). There are certain differences from previous research results, which may be caused by factors such as the source of added lutein and dosage. Feeding with MFE led to a meaningful increase in lutein levels found in eggs (166.8–174.6 μg/egg to 238.7–268.8 μg/egg) (
p < 0.05), which aligns with the results reported by Wen et al. (2021) [
45]. However, lutein deposits did not vary significantly among the chicken breeds (Dwarf Layer vs. White Leghorn, 268.8 μg/egg vs. 238.7 μg/egg) (
p > 0.05).
It is worth mentioning that our study’s results should be compared to prior research with care. Although there were no notable contrasts in ALA, EPA, DHA, or total n-3 PUFA levels of eggs from different breeds’ CON groups, we detected a substantial difference in the eggs from the FSO groups (
p < 0.05). To be specific, our study observed that in the FSO groups, the levels of ALA, DHA, and n-3 PUFA in the eggs of Dwarf Layer were significantly amplified compared to the FSO groups for White Leghorn and other three breeds (
p < 0.05). Conversely, the EPA levels in the eggs of the FSO group for the Dwarf Layer were significantly lower than those in the FSO group of the White Leghorn breed (
p < 0.05). There are likely two reasons for this difference. First, the White Leghorn may be more efficient at converting ALA to EPA than the Dwarf Layer. Second, the Dwarf Layer may convert more EPA to DHA for deposition in eggs. This may be related to the breed’s lipid metabolism, which has been linked to sex-linked dwarfism in chickens caused by mutations in the growth hormone receptor gene on the Z chromosome [
47]. Additionally, Dwarf Layers have been found to have the breed-specific characteristics of conjugated linoleic acid isomers. As such, their yolk lipids tend to be more enriched in fatty acids in response to dietary conjugated linoleic acid than those of White Leghorn hens [
48]. Finally, sex-linked dwarf chickens have been found to have a greater deposition of abdominal fat and larger adipocytes than normal Xinghua chickens [
47], which may also have an effect on the lipid metabolism and fatty acid composition of their eggs.
Chickens are unable to synthesize ALA and other n-3 PUFA on their own [
49,
50]. However, it should be noted that by inserting a double bond at the 3rd and 6th carbon positions (counted from the CH
3 end location), hens are capable of adding further double bonds [
49]. The yolk lipids are produced in the liver of the hen and then transported to the yolk using triacylglycerol-enriched very low-density lipoprotein (VLDL) and phospholipid-rich very high-density lipoprotein vitellogenin via serum [
51]. In laying hens, a distinct form of VLDL that is exclusively present in the yolk, VLDLy, exists, which is almost half the size of ordinary VLDL. VLDLy associates with apolipoprotein B100 and apovitellenin-1, preventing the action of lipoprotein lipase and allowing for triglyceride deposition in the oocyte in their intact form [
52]. Dietary fatty acid composition has a greater influence on the yolk lipid precursor’s fatty acid composition in Dwarf Layer hens than in regular hens. It is plausible that the dwarfing gene could reduce the hepatic synthesis of de novo fatty acids or that dwarf hens may assimilate more dietary lipids into the yolk relative to normal hens [
53]. Efficient nutrient absorption by laying hens depends on the condition of the intestinal absorption surface, which is regulated by the morphology of the intestine, specifically the length and recess depth of the intestinal villus. The villi in the small intestine are responsible for nutrient absorption, while the crypts are responsible for the regeneration of villous mucosal cells [
54,
55]. Moreover, the macroscopic and microstructural integrity of the intestine plays a crucial role in nutrient absorption and growth efficiency of laying hens [
56]. An increased nutrient absorption ability in efficient birds can be attributed to their larger duodenal surface area and a greater ratio of villus height to crypt depth than those of non-efficient birds [
33]. The difference in n-3 PUFA deposition between breeds may be related to the conversion of fatty acids in the liver and intestinal absorption. Although differences in n-3 PUFA deposition among breeds were compared in this experiment, the reasons for this difference still need further research.
To better understand the mechanisms for the greater deposition of n-3 PUFA in eggs from the Dwarf Layer breed, further research is required. Future studies should aim to investigate the deposition pattern of n-3 PUFA from the perspective of intestinal absorption, liver lipid synthesis, and liver uptake of dietary lipids. Additionally, candidate genes involved in the efficiency of n-3 PUFA deposition should be characterized, including their mutations. This will aid in the development of specialized strains to improve the production efficiency of n-3 PUFA-enriched eggs.