Effects of Hesperidin and Naringin Dietary Supplementation on Egg Yolk Colour and Lipid Composition in Laying Hens

Abstract

This research examines the effect of a 9-week feeding intervention on laying hens with flavonoid-enriched rations to modulate the colour and lipid quality of egg yolks. Triglycerides and phosphatidylcholine were the principal neutral and polar classes, respectively, as determined by TLC-FID. Oleic, palmitic, and linoleic were the major fatty acids (FAs) as determined by GC-FID/MS, while essential DHA predominated omega-3 FAs. Flavonoid supplementation did not markedly modify the total fat, lipid classes, or FA profile of yolks, whilst FAs were mostly affected by basal diet ingredients. However, higher-dose hesperidin and naringin improved the MUFA/PUFA ratio of yolk fatty acids. Egg yolks were not differentiated by colour or total carotenoid content irrespective of the feeding intervention. Study time, on the other hand, contributed to observed differences. Remarkably, a 1-week adaptation to a standardised regime proved adequate for the production of yolks with a homogenous colour profile, while incremental changes in the total carotenoids boosted red/yellow colouration. By completing the intervention, significant nutritional improvements emerged for all groups: (i) phosphatidylcholine levels increased; (ii) n-6/n-3 shifted to lower values; and (iii) peroxidisability indices diminished. The aforementioned findings, along with low atherogenic/thrombogenic indices and combined with the oxidative stability of yolk lipophilic constituents, feature eggs with consistent yolk colour and enhance lipid-rich yolks’ contribution towards a healthy diet.

1. Introduction

Hen eggs are classified as one of nature’s functional foods used for the preparation of a wide range of meals and food products. Eggs play a key part in human diet thanks to a plethora of constituents found in the yolk or the albumen with nutritional value or bearing biological activities [1]. In this regard, nowadays, health-conscious consumers show a preference towards nutritionally labelled eggs, such as omega-3 eggs [2]. To tap further into this market, manufacturers and poultry farmers have taken steps to enhance the nutritional value of eggs using natural methods and ingredients. The incorporation of plant-derived agro-industrial by-products, including citrus peel meals, fruit pulp residues, and polyphenol-rich plant extracts, as functional feed additives in laying hen diets represents a major focus of current sustainable poultry nutrition research [3,4,5]. This has given rise to an entire industry dedicated to improve the egg quality by modifying its composition, with proactive ideas and the fast commercialisation of research findings through new products in the market place.

Egg is indeed an excellent candidate for man-made functional food because of the highly achievable transfer and/or conversion of nutrients from a hen’s diet to the egg [2]. The chemical composition of eggs can thus be easily manipulated through dietary interventions that are feasible in all types of poultry enterprises. In particular, the high fat content of egg yolk serves as an excellent medium for the straightforward manipulation of yolk lipophilic compounds through the diet. This stands as a great opportunity to favourably modulate the lipid profile in yolk, providing exceptional functionality and a manifold composition of its constituents [6]. Yolk lipids, proteins, and lipoproteins possess biochemical and supra-molecular traits, while compared with egg white, yolk is used for large-scale production of commodities with important economic value [7]. Furthermore, many of the yolk-derived compounds seem promising for future applications in the design of innovative products beyond the food industry, such as the production of biodegradable and biocompatible packaging materials [8,9], or cosmetic formulations [10]. Egg yolk could also replace soybean and other seed oils as a plentiful source to recover phospholipids for utilisation by nutraceutical, cosmetic, food, and liposome industries [11,12].

Nutrition can markedly alter yolk composition, although a number of factors may also define its functional traits and nutritional profile, such as a hen’s breed/hybrid and age [13,14]. In particular, several studies have shown that the carotenoid profile of egg yolk can be directly modulated by the carotenoid composition of a hen’s diet [15,16,17]. Carotenoids, mainly lutein and zeaxanthin, are responsible not just for the yellow–orange–red colour of the yolk but also its intensity and homogeneity [18,19]. Yolk colour is the benchmark for consumers, and it is important in the manufacture of yolk-based foodstuffs, like pasta, noodles, and cakes [20]. Either natural or synthetic carotenoids are commonly used as feed additives to achieve a desirable yolk colour according to market trends [21]. Moreover, several studies have established the connection between the fatty acid composition in a hen’s diet and the corresponding profile in yolk [22,23,24].

Apart from favourably altering nutritional traits of egg yolk, a main focus that has received attention more recently is to avoid possible deteriorations of yolk lipid constituents caused by oxidation phenomena [25]. To decelerate oxidation, synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, and tert-butylhydroquinone are regularly added to diets [19]. Nevertheless, the demand for natural antioxidants instead of synthetic ones has currently increased because of the negative perceptions of consumers about the long-term impacts on human health regarding possible toxicity [26,27]. In this context, the supplementation of poultry diets with natural antioxidants, e.g., vitamin E, phenolic compounds, or Se, may strengthen the endogenous defence mechanisms of yolk against lipid peroxidation [28,29,30,31,32,33]. Among these, citrus-derived and broader fruit-waste-derived materials have received increasing attention as effective and economically viable natural antioxidant sources for poultry nutrition, with recent reviews demonstrating their positive impact on egg quality characteristics [34] and broader One Health benefits across animal production systems, as promoted by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) [35].

Among natural antioxidants, signature flavonoids, namely hesperidin and naringin, have been evaluated for their potent beneficial effects as feed additives in animal products, poultry included [36,37]. Both flavonoids accumulate to substantial levels in citrus pulp, which is the inexpensive residue of peel fragments, membranous fruit tissue, and whole culled fruit left over once their juice has been extracted [38]. Their antioxidant behaviours, characteristic of flavonoids bearing aromatic hydroxyl groups, extends in hesperidin and naringin to a wider set of health-relevant activities, including anti-inflammatory actions and the scavenging of reactive oxygen species [38]. In particular, their use as supplements in agro-feeds positively influenced the antioxidant properties of poultry meat without negative implications on growth performance or meat quality [38,39]. Parallel beneficial outcomes have been documented in laying hen trials involving dietary dried orange pulp, where egg yolk fatty acid composition, antioxidant capacity, and oxidative stability were all significantly improved [40]. Similarly, dietary supplementation with dried lemon citrus peel in spent laying hens enhanced egg production and improved internal and external egg quality traits [41]. The use of fermented orange pulp in broiler diets has likewise demonstrated significant improvements in meat oxidative stability and serum lipid profile [42].

Numerous animal studies have indicated that the oral administration of hesperidin, naringin, or their aglycones clearly decreases plasma cholesterol and/or triglycerides levels while also reducing hepatic lipid content [43,44,45,46]. Investigations into the effects of supplemental levels of hesperidin inclusion in hens’ diet for 31 days revealed that 1 or 3 g flavonoid per kg feed significantly improved yolk oxidative stability in fresh and stored eggs, with no side effects on egg production or weight [33]. Hens offered diets fortified with hesperidin or naringin for 8 weeks displayed improved antioxidant markers in serum, although only hesperidin managed to lower serum cholesterol concentration [47]. Moreover, the inclusion of either flavonoid at 2 g per kg feed for 10 weeks produced eggs with a lower yolk cholesterol content and significantly enhanced antioxidant activity [48]. A recent comparative study further demonstrated that, while isolated hesperidin and naringin supplementation at levels equivalent to those naturally present in dried orange pulp exerted limited effects on egg elemental composition, the inclusion of the whole dried orange pulp matrix significantly altered the deposition of multiple elements across the yolk, albumen, and eggshell, suggesting that the intact citrus by-product matrix mediates compositional effects beyond those of its isolated flavonoid constituents [49].

Within this context, in the present work, we aim to investigate the potential of natural flavonoids for amending egg yolk’s nutritional value as provided by the main yolk lipophilic species, as well as the consistency of the yolk colour as a major contributor of sensory properties and product acceptability. Hesperidin and naringin were chosen over other citrus flavonoids for their low cost and ready availability as citrus-processing residues, together with earlier evidence from a laying hen trial by some of the present co-authors showing that dietary hesperidin improved yolk oxidative stability without compromising egg output [33]. To this end, the two flavonoids were utilised as feed supplements for a nine-week dietary intervention to laying hens in order to evaluate their effects at different time points on the lipid and colour quality of produced eggs. A control group, together with a positive control group with vitamin E acetate, was used for comparison reasons. Different analytical methodologies were implemented for the assessment of neutral and polar lipid classes, the profile of fatty acids, total carotenoid content, and colour evaluation, including chromatographic/spectrometric (thin-layer chromatography–flame ionisation detection, TLC-FID; gas chromatography–flame ionisation detection, GC-FID; and gas chromatography–mass spectrometry, GC-MS), spectrophotometric (UV-Vis), and chromatometric techniques.

2. Materials and Methods

2.1. Reagents, Solvents and Standards

Standard phenolic compounds used were of high-performance liquid chromatography (HPLC) grade; hesperidin [>90.0% (w/w)] was supplied by TSI Europe NV (Zwijndrecht, Belgium), and naringin hydrate [98% (w/w)] from Alfa Aesar (Karlsruhe, Germany). DL-a-tocopheryl acetate [≥96% (w/w)] was purchased from DSM Nutritional Products Hellas (Oinofyta, Greece). Standard lipid compounds used were cholesterol (Chol), cholesteryl oleate, octadecyl hexadecanoate, squalene, tristearoyl-glycerol, stearic acid, oleic acid, 1,3-distearoyl-glycerol, 1,2-distearoyl-glycerol, 1-monostearoylrac-glycerol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PtdIns), phosphatidylserine (PS), and sphingomyelin (Sphm) from Sigma Chemical Co (Sigma-Aldrich Company, Dorset, UK and St. Louis, MO, USA). Commercially available authentic standard mixtures of fatty acid methyl esters with chain lengths from C4 to C24 Supelco 37 Component FAME Mix C4–C24 and Supelco PUFA No. 1 (marine source, analytical standard), as well as palmitic acid M-E, stearic acid M-E, oleic acid (cis-9) M-E, linoleic acid (cis-9,12) M-E, and cis-5,8,11,14,17-eicosapentaenoic acid ME, were bought from Sigma Aldrich Co. (Gillingham, UK) and MIDI Inc. (Newark, DE, USA). Lutein [≥95% (w/w)] and zeaxanthin [≥98% (w/w)] were bought from Extrasynthese (Genay, France), and trans-canthaxanthin [≥95% (w/w)] was supplied by Sigma-Aldrich (Steinheim, Germany; Postfach, Switzerland; St. Louis, MO, USA). Solvents and reagents were of analytical, HPLC, GC, or MS grade, and they were acquired from Merck (Darmstadt, Germany), Riedel-de Haën AG (Seelze, Germany), LGC Standards (Wesel, Germany), Chem-Lab NV (Zedelgem, Belgium), Sigma Aldrich Co. (Gillingham, UK), Carlo Erba Reagents (Val de Reuil, France), Fluka Analytical (Steinheim, Germany), Fisher Scientific (Loughborough, UK), and Scharlab S.L. (Barcelona, Spain).

2.2. Animal Dietary Intervention, Sampling and Sample Preparation

The animal study was set up in the Laboratory of Animal Breeding and Husbandry in the Department of Animal Science of the Agricultural University of Athens. The experimental project included laying hens of the subspecies Gallus gallus domesticus L. (Phasianidae Family), which were assigned to different dietary groups. For the dietary intervention with antioxidants, i.e., hesperidin, naringin, and acetate of vitamin E, 72 Lohmann Brown-Classic laying hens (54 weeks old) were randomly allocated into six dietary treatment groups, each consisting of 12 hens, and the experiment was conducted over a period of nine weeks. Microclimate conditions in the poultry house, namely the temperature and relative humidity, ranged from 19 to 26 °C and from 50 to 63%, respectively, throughout the experimental period. The hens were individually caged, and each hen was considered as an experimental unit. The experimental design and hens’ performance results were presented in detail in the study by Goliomytis et al. [50]. One of the groups served as a control (C) and was offered a commercial basal diet, whereas the other five groups were offered the same diet further supplemented with hesperidin at 0.75 g·kg⁻¹ (HD1) or 1.5 g·kg⁻¹ of feed (HD2), naringin at 0.75 g·kg⁻¹ (NG1) or 1.5 g·kg⁻¹ of feed (NG2), or a-tocopheryl acetate, a synthetic form of vitamin E, at 0.20 g·kg⁻¹ feed (VE), to serve as a positive control. A sample size of 192 eggs from all treatment groups (N = 8 egg samples per group, randomly selected) were collected at 4 sampling time points: on the first day (D0) of the experiment and at the end of the first (W1), fifth (W5), and ninth (W9) week. At each of the four sampling time points, eggs were independently and randomly drawn from among those available on the collection day rather than tracked from a fixed subset of hens. Therefore, the four time points do not constitute repeated measurements on the same experimental units. Table 1 presents the ingredients and composition of the basal diet as it is provided by the feed manufacturer. For the clarity of presentation herein, the coding and supplement dosing of the different sample groups are presented in Table 2.

Table 1. Composition of the basal diet offered to the laying hens of the experimental set.

Composition	g·kg−1 Feed
Maize	540
Wheat	50
Wheat bran	46
Soybean meal [crude protein 47% (w/w)]	246
Soybean oil	12
Limestone	89
Monocalcium phosphate	9
Sodium chloride	2.40
Sodium bicarbonate	2.7
Methionine 99%	1.1
Choline chloride 70%	0.6
Phytase, Natuphos®	0.06
Antioxidant (butylated hydroxyanisole)	0.11
Pigment (Carophyl® Red)	0.04
Vitamin and mineral premix 1	1.00
Crude protein	175
Fibre	25
Fat	38
Ash	124
Lysine 2	9
Methionine + cystine 2	6.8
Calcium 2	37.5
Available phosphorus 2	3.6
Metabolisable energy (MJ·kg−1 feed) 2	11.48

¹ The vitamin and mineral premix provided per kg of diet: 3.00 mg of vitamin A (retinol), 62.5 μg of vitamin D₃, 30 mg of vitamin E (a-tocopherol), 5 mg of vitamin K₃, 1 mg of vitamin B₁, 5 mg of vitamin B₂, 3 mg of vitamin B₆, 20 μg of vitamin B₁₂, 30 mg of nicotinic acid, 10 mg of pantothenic acid, 0.8 mg of folic acid, 100 μg of biotin, 10 mg of vitamin C, 450 mg of choline chloride, 0.2 mg of Co, 0.5 mg of I, 0.3 mg of Se, 25 mg of Fe, 120 mg of Mn, 10 mg of Cu, and 100 mg of Zn. ² Calculated.

Table 2. Sample set and coding of the dietary groups of laying hens for the experimental study.

Dietary Group	Coding	Supplement (g·kg−1 Feed)
Control	C	–
Hesperidin	HD1	0.750
	HD2	1.50
Naringin	NG1	0.750
	NG2	1.50
Vitamin E acetate	VE	0.20

The feed of each group was replenished every 2 days. Feed and water were provided ad libitum, and a photoperiod programme of 16 h of continuous light per day was established throughout the experimental period.

After immediate collection at each time point, the eggs were weighed and the yolks were separated from the egg white per manus, without removing the vitelline membrane, which were also then weighed. Egg yolks were freeze-dried and homogenised to a fine powder prior to extraction, not only to preserve the samples and avoid possible degradation of the constituents or microbial growth but also to remove water that would cause a restriction for the following extraction procedure. The lyophilised samples were placed in aluminium-foil-wrapped containers, which were labelled and kept air-sealed at −80 °C for further use during the study period.

2.3. Measurement of Colour Parameters

The CIE L*a*b* (CIE, Commission Internationale de l’Éclairage) coordinates (L*, a*, b*, C*, hue) were determined directly for lyophilised yolks using a chroma-meter (Chroma Meter CR-400, Konica Minolta Sensing, Tokyo, Japan). Measurements of the five CIE Lab values were obtained from 3 different points of each sample and in triplicate. A white calibration plate with the specifications of Y = 93.5, x = 0.3137, and y = 0.3197 was used to standardise the chroma-meter prior to sample measurements.

2.4. Extraction Methodology

A multi-staged methodology was elaborated for the extraction of polar and non-polar constituents of the egg yolk samples [51]. The methodology was based on the classical co-solvent Folch method [52] with some modifications. Extraction was performed in a fume hood for as much of the procedure as possible, under a flow of nitrogen to minimise oxygen exposure. To finalise the extraction protocol, a set of preliminary tests was conducted to assure repeatability and maximum yield, based on gravimetric measurements of total fat. The lipophilic extract was yielded for each sample. All procedures were carried out under dim light as much as possible. Amber glassware was used throughout the extraction procedure to further minimise light exposure.

At the first step, the extraction was performed with a 15:1 (v/w) solvent/material ratio. Approximately 0.5 g of lyophilised yolk was homogenised with 3.75 mL of 2:1 chloroform–methanol (v/v), then the mixture was vortexed thoroughly, and it was allowed to extract for 20 min before the supernatant was collected after centrifugation at 3500 rpm for 5 min. The remaining sample was re-extracted twice with 1.88 mL of the Folch solvent mixture following the procedure as described above. The three recovered extracts were then combined, and 1.5 mL of water (0.2 volume to the total volume of chloroform–methanol) was added to form a two-phase solvent system, which was left to equilibrate overnight at 7 °C. The lower phase of chloroform and the upper polar phase were separated, while the film of cell debris and protein, which was formed between the two phases, was rejected. The chloroformic extract was collected, its volume was measured for each sample using a graduated pipette, and the extracts were kept in sealed amber glass vials at −20 °C until further use.

2.5. Total Fat Determination

The total fat yield for each yolk or feed sample was determined gravimetrically after evaporating 1 mL of the chloroform extract to dryness under a gentle stream of nitrogen till a constant weight. Each measurement was carried out in triplicate. The concentration of total fat was expressed as mg per g of the dry yolk sample.

2.6. TLC-FID Analysis of Lipid Classes

An Iatroscan analyser (Model MK-6 TLC/FID—FPD Analyser Iatron Laboratories, Tokyo, Japan), equipped with a thin-layer chromatograph (TLC) and a flame ionisation detector (FID), was used for the separation, identification, and quantification of the polar and neutral classes of total lipids of the lipophilic extracts of yolks, according to the method described by Sinanoglou et al. [53]. The separation of lipids was performed on silicic-acid-coated quartz rods and chromarods (silica-gel-coated quartz rod, Type SIII, particle size 5 μm, Mitsubishi Kagaku Iatron, Tokyo, Japan). Approximately 0.5 μL of each extract or lipid standard solution was spotted on each rod with an 1 μL Hamilton syringe (Hamilton Co., Reno, NV, USA). The rods were developed in a lined chromatography chamber with a two-step development sequence. The polar solvent system was a mixture of chloroform:methanol:water 50:20:2 (v/v/v), and the neutral solvent system mixture consisted of n-hexane:diethyl ether:formic acid 60:15:1.5 (v/v/v). Each chamber was prepared 30 min before development with a filter paper wetted with the solvent system and placed along one side of the chamber in order to achieve a good saturation of vapours. The room temperature was stable at 22 °C during development. Before FID analysis, each chromarod set was carefully dried in a desiccator, with FID operation conditions set to a hydrogen flow rate of 160 mL·min⁻¹, an air flow rate of 2 L·min⁻¹, and a scan speed of 30 s per rod scan. In each of the two development steps, the solvent front was allowed to migrate a distance of approximately 9.5 cm from the base of the chromarods. All measurements were held at least in triplicate. Individual lipid classes were evaluated as described previously [53]. The detection and quantitation limits were 0.02 μg·μL⁻¹ and 0.05 μg·μL⁻¹, respectively, for each lipid standard. The processing of the chromatograms for the calculation of retention times and peak areas was performed with Chromstar 6.3 Software.

2.7. GC-FID Analysis of Fatty Acid Methyl Esters

Analysis of fatty acid methyl esters (FAMEs) of total lipids in the chloroformic extracts of lyophilised yolks was performed on an Agilent 6890 Series gas chromatograph equipped with a flame ionisation detector (Agilent Technologies, Palo Alto, CA, USA). Fatty acid methyl esters (FAMEs) were prepared by cold trans-esterification. An aliquot of the lipophilic extract, corresponding to approximately 20–50 mg of lipids, was evaporated to dryness, and the dry residue was reconstituted in 0.75 mL of n-hexane. Trans-esterification was induced by the addition of 0.1 mL of 2 N methanolic potassium hydroxide; the mixture was vortexed for 2 min and then allowed to stand for 25 min at room temperature to complete the reaction. The resulting mixture was dried over anhydrous sodium sulphate to remove residual moisture and, following phase separation, the upper n-hexane layer containing the derivatised FAMEs was then collected and injected directly into the gas chromatograph. Hewlett-Packard ChemStation Software (Agilent Technologies, Inc., Wilmington, DE, USA) was used to calculate the retention times and peak areas for the qualitative and quantitative results. Each analyte was identified by comparing the retention time with those of the authentic standards. The relative content of fatty acids in the sample was determined according to a previously described method [53].

2.8. GC-MS Analysis of Fatty Acid Methyl Esters

A qualitative analysis of FAMEs of total lipids of lipophilic extracts of yolks to confirm the presence of the analytes was performed using a gas chromatograph–mass spectrometer (GC-MS) QP2010 Series (Shimadzu USA Manufacturing, Inc., Kyoto, Japan). A column of medium polarity was used (DB-5 MS, 30 m, 0.25 μm i.d. and 0.25 μm film thickness; Agilent Technologies, Palo Alto, CA, USA) with a stationary phase of phenyl arylene polymer virtually equivalent to 5% (w/w) phenylmethylpolysiloxane, and helium was used as the carrier gas under a constant column flow rate of 1 mL·min⁻¹.

The temperature of the injector was set at 265 °C, while the interface was set to 270 °C and the ion source to 240 °C. The GC oven temperature programme is provided in the Supplementary Materials (Table S1). Specifically, the oven temperature was set to 100 °C and then increased to 120 °C at a rate of 15 °C per minute. It then increased to 140 °C at a rate of 7 °C per minute, and then to 180 °C at a rate of 13 °C per minute. The temperature was held constant at 180 °C for 2 min, then increased to 185 °C at a rate of 1 °C per minute and held for 3 min. It then increased to 190 °C at a rate of 1 °C per minute and was held at this temperature for a further 7 min. Finally, the temperature increased to 195 °C at a rate of 2 °C per minute and was held for 1 min. It rose to 200 °C at a rate of 1 °C per minute and remained at this temperature for 2 min. This was followed by an increase to 240 °C at a rate of 14 °C per minute, which was held for 2 min. Then, it increased to 250 °C at a rate of 13 °C per minute and was held for 2 min. Finally, it rose from 250 °C to 260 °C at a rate of 13 °C per minute and was held for a further 2 min. The total analysis time was 50 min. The split ratio was 10.0 and the injection volume was 1.0 μL. Blank samples were injected after every fourth analysis.

For the MS analysis, electron impact (EI) ionisation was produced by accelerating electrons from a filament through a potential difference of 70 eV. The scan mode was operated at the m/z (mass-to-charge ratio) range of 40.00 to 700.00.

The chromatograms and mass spectral data were processed with the GCMS Solution software (version 2.71, Shimadzu Corporation, Analytical Instruments Division, Kyoto, Japan). The identification of the FAMEs in sample solutions was achieved through similarity searches in mass spectral libraries (NIST05, NIST05s, NIST08, NIST08s, NIST21, NIST107, WILEY7, PMW_TOX2, SZTERP).

2.9. Determination of Total Carotenoid Content

The total carotenoid content in the lipophilic extracts of egg yolks was estimated spectrophotometrically using a double-beam UV–Vis spectrophotometer Hitachi U-3210 (Hitachi Ltd., Tokyo, Japan). Three standard carotenoids, namely trans-canthaxanthin, lutein, and zeaxanthin, were each used to construct an independent calibration curve at its own wavelength of maximum absorbance [54], and the total carotenoid content of samples was accordingly estimated in parallel as trans-canthaxanthin, lutein, and zeaxanthin equivalents. Acetone–n-hexane 1:1 (v/v) was used as the solvent mixture for samples, standard solutions and blanks. A matrix-matched blank was not used, since carotenoids are endogenous to egg yolk and a genuinely carotenoid-free yolk matrix could not be obtained; a solvent blank was used instead. In short, 3 mL of lipohilic extract was dried under nitrogen stream, the dry residue was reconstituted with an even volume of the acetone–n-hexane mixture, and then the solution was transferred into a glass cuvette. The absorbance value was recorded at the wavelength of maximum absorbance observed for the three standard compounds: 475.0 nm for trans-canthaxanthin, 445.9 nm for lutein, and 451.5 nm for zeaxanthin. Triplicate spectrophotometric measurements were conducted for all determinations.

2.10. Statistical Analysis

The results are expressed as mean values ± standard deviation (SD). Normality tests for data distribution were performed with the Shapiro–Wilk test at a significance level of 0.05. Data were analysed on a significance level of 0.05 with one-way analysis of variance (ANOVA) and post hoc test analysis comprising multiple pairwise comparisons conducted with Tukey’s Honestly Significant Difference (HSD) test. Probabilities lower than 0.05 were considered statistically significant (p < 0.05). All statistical calculations were performed using the Origin Pro 8 SR4 (v8.0951, B951, 2008; OriginLab Corporation, Northampton, MA, USA) statistical software package.

3. Results and Discussion

3.1. Total Fat, Distribution of Neutral and Polar Lipids and Lipid Classes

At first, we examined the impact of hesperidin, naringin, or vitamin E on total lipids, neutral and polar lipid classes of total lipids in egg yolk samples. The correlation between antioxidant administration to laying hens at four sampling time points and yolk lipid constituents was tested in comparison with the control group (C) offered only the commercial basal diet (Table 1). Neutral and polar lipids and their totals were determined in egg yolk samples and are shown in Table 3.

Table 3. Total fat of lipophilic extracts of dry yolk samples expressed as mg per g of dry yolk (DY), and total neutral (NL) and polar lipids (PL) expressed as weight percent of total lipids (TL) and as mg per g of dry yolk. Results are presented as mean ± SD (standard deviation) (measurement repeats, N = 3).

Sampling	Treatment Group 1	Total Fat (mg·g−1 DY) 2	% NL of TL 2	% PL of TL 2	NL (mg·g−1 DY) 2	PL (mg·g−1 DY) 2
D0	C	700 ± 42 a,b,A	67.0 ± 1.1 a,A	33.0 ± 1.1 a,B	469 ± 33 a,A	231 ± 11 a,A
	HD1	745 ± 44 a,A	67.7 ± 1.5 a,A	32.3 ± 1.5 a,B	505 ± 37 a,A	240 ± 13 a,A
	HD2	702 ± 36 a,b,A	66.6 ± 2.3 a,A	33.4 ± 2.3 a,C	468 ± 37 a,A	234 ± 13 a,B
	NG1	719 ± 39 a,b,A	67.6 ± 1.7 a,A	32.4 ± 1.7 a,B	486 ± 35 a,A	233 ± 11 a,B
	NG2	672 ± 35 b,A	66.82 ± 0.91 a,A	33.18 ± 0.91 a,B	449 ± 26 a,A	223 ± 12 a,B
	VE	717 ± 70 a,b,A	66.7 ± 1.4 a,A	33.3 ± 1.4 a,B	478 ± 43 a,A	238 ± 29 a,A
W1	C	670 ± 13 a,A	61.9 ± 3.3 a,B	38.1 ± 3.3 a,A	415 ± 26 a,A,B	255 ± 20 a,A
	HD1	671 ± 13 a,B	61.0 ± 1.7 a,B	39.0 ± 1.7 a,A	409 ± 15 a,B	262 ± 12 a,A
	HD2	683 ± 23 a,A	60.7 ± 3.0 a,B,C	39.3 ± 3.0 a,A,B	415 ± 27 a,A,B	268 ± 20 a,A,B
	NG1	668 ± 17 a,A	62.0 ± 3.5 a,B	38.0 ± 3.5 a,A	414 ± 27 a,B	254 ± 22 a,A,B
	NG2	679 ± 15 a,A	62.4 ± 3.4 a,A,B	37.6 ± 3.4 a,A,B	424 ± 27 a,A	255 ± 22 a,A,B
	VE	669 ± 19 a,A	60.8 ± 3.6 a,B	39.2 ± 3.6 a,A	407 ± 30 a,B	262 ± 24 a,A
W5	C	682 ± 16 a,A	63.9 ± 1.8 a,A,B	36.1 ± 1.8 a,A,B	436 ± 18 a,A,B	246 ± 12 a,A
	HD1	684 ± 19 a,A,B	63.4 ± 1.6 a,A,B	36.6 ± 1.6 a,A,B	434 ± 18 a,B	250 ± 12 a,A
	HD2	686 ± 14 a,A	64.34 ± 0.53 a,A,B	35.66 ± 0.53 a,B,C	441 ± 10 a,A,B	244.6 ± 6.3 a,A,B
	NG1	683.8 ± 8.9 a,A	62.7 ± 1.5 a,B	37.3 ± 1.5 a,A,B	429 ± 11 a,B	255 ± 11 a,A,B
	NG2	684 ± 19 a,A	64.0 ± 1.2 a,A,B	36.0 ± 1.2 a,A,B	438 ± 17 a,A	245.7 ± 8.5 a,A,B
	VE	684.3 ± 7.9 a,A	63.7 ± 1.4 a,A,B	36.3 ± 1.4 a,A,B	436 ± 12 a,A,B	248.6 ± 8.4 a,A
W9	C	662 ± 50 a,A	61.3 ± 3.8 a,B	38.7 ± 3.8 a,A	405 ± 39 a,B	256 ± 33 a,A
	HD1	674 ± 28 a,B	61.9 ± 2.8 a,B	38.1 ± 2.8 a,A	417 ± 26 a,B	257 ± 21 a,A
	HD2	678 ± 15 a,A	59.4 ± 2.4 a,C	40.6 ± 2.4 a,A	403 ± 21 a,B	275 ± 16 a,B
	NG1	688 ± 46 a,A	60.4 ± 2.3 a,B	39.6 ± 2.3 a,A	415 ± 28 a,B	273 ± 27 a,B
	NG2	677 ± 23 a,A	61.4 ± 3.9 a,B	38.6 ± 3.9 a,A	416 ± 34 a,A	261 ± 23 a,B
	VE	687 ± 13 a,A	60.7 ± 3.3 a,B	39.3 ± 3.3 a,A	417 ± 26 a,B	270 ± 22 a,A

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

According to Table 3, the total fat content of yolks collected at the start of the experiment (D0) varied from 672(±35) to 745(±44) mg·g⁻¹ dry yolk. The differences between the dietary groups on D0 reflect the inhomogeneity of the previously offered feed to the hens before setting the dietary intervention. However, introducing the hens to a standardised regime from the first day of the study balanced out the level of total fat for all groups on each of the following three subsequent sampling points, including the NG2 group that initially presented the lowest values (p < 0.05) for yolk total fat on D0. As a result, regarding each sampling, the total fat content in the yolks did not differ between groups (p ≥ 0.05). It is noteworthy that only the treatment with 0.75 g hesperidin per kg feed (HD1) for one week seemed to alleviate (p < 0.05) the total fat content in the yolks of the corresponding group. Comparing the present findings with those of other studies confirms the stability of the total fat levels in egg yolks. Generally, there are limited reports on significant changes in the total fat content of the egg yolk as a result of different dietary treatments [13,55].

The reduction in the total fat content observed in the HD1 group after one week of dietary intervention warrants further consideration. Given that egg lipids are deposited in the yolk as lipoprotein forms synthesised in the hen’s liver, a possible mechanistic explanation may be sought for the influence of hesperidin on hepatic lipoprotein assembly, since exposure of cultured human liver cells to hesperetin, an aglycone of hesperidin, has been shown to lower apolipoprotein B-100 secretion [43]. In vivo, dietary hesperidin has likewise been reported to lower plasma and hepatic triglyceride and cholesterol content in a mouse model of type-2 diabetes, an effect linked to the reduced hepatic activity of lipogenic and cholesterogenic enzymes [44]. Similarly, in mice fed a high-fat diet, dietary hesperidin has been shown to activate hepatic genes involved in fatty acid oxidation while downregulating lipogenic gene expression, accompanied by a reduced liver weight and an improved serum lipid profile [45]. A similar, transient suppression of hepatic lipoprotein output may plausibly account for the reduction observed in the HD1 group, although this remains a tentative hypothesis that would require direct confirmation in laying hens.

Furthermore, the total fat content of the studied yolks did not seem to be influenced by the age of the laying hens, as depicted by the different time samplings. A similar outcome was reported in the study by Peng et al. [56] in which no significant differences were observed concerning the total fat content in egg yolks from younger versus older hens. However, other research has stated that the fat content of egg yolks is age-dependent, such that older hens lay eggs had a higher fat content than that of eggs laid by younger ones. The fat content of yolks may also fluctuate towards higher or lower values with age [13,57]. Cherian [58] showed that egg yolks from 42-week-old hens possessed a higher total fat content than eggs laid by hens of other ages (26, 34, 50, 58, and 62 weeks old).

Regarding the results in Table 3, none of the antioxidants significantly modified (p ≥ 0.05) the total neutral lipids of total yolk lipids either between treatments or between samplings from different time points. Instead, it was the effect of changing the basal ration fed to all hens when the study commenced that led to a significant decline (p < 0.05) of neutral lipids during the nine weeks of the study. In particular, yolks collected on W9 presented the lowest proportion (p < 0.05) of neutral lipids for all samples, ranging from 59.4(±2.4) to 61.9(±2.8)% (w/w) and corresponding to 403(±21) and 417(±26) mg·g⁻¹ dry yolk. Regarding the NL content, only the NG2 group did not follow the same trend shown for the other treatment groups, where the concentration of total neutral lipids in the yolks of this group was maintained at the same levels (p ≥ 0.05) till the ninth week of the study. The proportion of total polar lipids was elevated (p < 0.05) after the first week of the study, while the higher proportions were observed at the end of the final week of the experimental period (W9). However, different treatments with natural antioxidants had no effect (p ≥ 0.05) compared with the control group at each time point. Polar lipids represented 32.3(±1.5) and up to 40.6(±2.4)% (w/w) of total lipids in the studied egg yolk samples.

Furthermore, the individual neutral and polar lipid classes of the lipophilic extracts of dry yolk samples were investigated via TLC-FID analysis of the different treatment groups for the four time points. It is apparent from Table 4 that neutral lipids classes were not altered significantly (p ≥ 0.05) between the different treatments at each time point. The most obvious finding to emerge from the analysis was that the levels of the individual classes fluctuated (p < 0.05) during the study regardless of antioxidant supplementation in the hen’s diet. Concerning the proportion of triglycerides (TGs), egg yolks presented generally higher values at D0 compared to W1, W5, and W9, apart from the values for the C and NG2 groups that remained stable (p ≥ 0.05) throughout the nine weeks.

Table 4. Distribution of neutral and polar lipid classes of lipophilic extracts of dry yolk samples, expressed as weight percent of total lipids (% of TL). Results are presented as mean ± SD (standard deviation) (measurement repeats, N = 3).

		% (w/w) Neutral Lipid Classes of Total Lipids					% (w/w) Polar Lipid Classes of Total Lipids
Sampling	Treatment Group 1	TG 2,3	FFA 2,3	Sterols 2,3	DG 2,3	MG 2,3	PE 2,3	PtdIns 2,3	PC 2,3	Sphm 2,3	LPC 2,3
D0	C	61.7 ± 1.3 a,A	0.63 ± 0.22 a,A	4.20 ± 0.35 a,A	0.29 ± 0.10 a,A	0.17 ± 0.07 a,A	5.39 ± 0.66 a,A	0.16 ± 0.19 a,A	26.94 ± 0.82 a,B	0.29 ± 0.20 a,A	0.18 ± 0.13 a,A
	HD1	62.2 ± 1.5 a,A	0.74 ± 0.32 a,A	4.20 ± 0.40 a,A	0.46 ± 0.31 a,B	0.14 ± 0.02 a,A	5.39 ± 0.68 a,B	0.09 ± 0.14 a,A	26.27 ± 0.83 a,B	0.42 ± 0.28 a,A	0.12 ± 0.13 a,B
	HD2	61.5 ± 1.8 a,A	0.61 ± 0.18 a,A	4.03 ± 0.56 a,A	0.30 ± 0.13 a,A	0.16 ± 0.03 a,A	5.93 ± 0.65 a,A	0.01 ± 0.03 a,A	26.9 ± 1.8 a,C	0.40 ± 0.18 a,A	0.22 ± 0.14 a,A
	NG1	62.7 ± 1.2 a,A	0.56 ± 0.09 a,A	3.87 ± 0.47 a,A	0.29 ± 0.07 a,A	0.14 ± 0.03 a,A	5.79 ± 0.80 a,A	0.19 ± 0.27 a,A	26.0 ± 1.1 a,B	0.37 ± 0.14 a,A	0.13 ± 0.13 a,A
	NG2	61.70 ± 0.98 a,A	0.57 ± 0.12 a,A	4.12 ± 0.11 a,A	0.32 ± 0.09 a,A	0.12 ± 0.02 a,A	5.77 ± 0.43 a,A	0.14 ± 0.31 a,A	26.62 ± 0.74 a,B	0.37 ± 0.16 a,A	0.27 ± 0.11 a,A
	VE	61.5 ± 1.3 a,A	0.56 ± 0.10 a,A	4.22 ± 0.21 a,A	0.29 ± 0.11 a,A	0.14 ± 0.02 a,A	5.34 ± 0.44 a,A	0.19 ± 0.26 a,A	27.3 ± 1.4 a,B	0.40 ± 0.25 a,A	0.12 ± 0.17 a,A
W1	C	57.9 ± 3.4 a,A	0.49 ± 0.17 a,A,B	3.18 ± 0.45 a,B	0.21 ± 0.11 a,A,B	0.14 ± 0.05 a,A,B	6.47 ± 0.70 a,A	0.11 ± 0.11 a,A	30.9 ± 2.8 a,A	0.34 ± 0.05 a,A	0.29 ± 0.04 a,A
	HD1	56.7 ± 1.6 a,B	0.48 ± 0.13 a,A,B	3.43 ± 0.46 a,B	0.21 ± 0.10 a,A	0.13 ± 0.04 a,A,B	6.90 ± 0.81 a,A	0.05 ± 0.07 a,A	31.4 ± 1.2 a,A	0.35 ± 0.04 a,A	0.29 ± 0.08 a,A
	HD2	56.9 ± 2.6 a,B,C	0.46 ± 0.25 a,A,B	3.08 ± 0.42 a,B	0.20 ± 0.10 a,A	0.13 ± 0.09 a,A	6.92 ± 0.94 a,A	0.04 ± 0.08 a,A	31.7 ± 2.1 a,A,B	0.35 ± 0.07 a,A	0.28 ± 0.11 a,A
	NG1	58.0 ± 3.4 a,B	0.39 ± 0.14 a,A	3.24 ± 0.30 a,A,B	0.17 ± 0.09 a,A	0.11 ± 0.05 a,A	6.77 ± 0.85 a,A	0.04 ± 0.07 a,A	30.6 ± 2.6 a,A	0.37 ± 0.10 a,A	0.28 ± 0.05 a,A
	NG2	58.1 ± 3.4 a,A	0.58 ± 0.15 a,A	3.33 ± 0.27 a,B	0.27 ± 0.11 a,A	0.16 ± 0.05 a,A	6.35 ± 0.79 a,A	0.13 ± 0.28 a,A	30.5 ± 2.9 a,A,B	0.37 ± 0.08 a,A	0.24 ± 0.06 a,A
	VE	56.9 ± 3.3 a,B	0.40 ± 0.19 a,A	3.15 ± 0.41 a,B	0.21 ± 0.12 a,A	0.09 ± 0.07 a,A	6.15 ± 0.82 a,A	0.07 ± 0.12 a,A	32.4 ± 3.2 a,A	0.34 ± 0.05 a,A	0.29 ± 0.08 a,A
W5	C	60.0 ± 1.6 a,A	0.21 ± 0.15 a,B	3.54 ± 0.43 a,A,B	0.08 ± 0.07 a,B	0.07 ± 0.06 a,B	6.74 ± 0.54 a,b,A	0.04 ± 0.07 a,A	28.9 ± 1.5 a,A,B	0.25 ± 0.06 a,A	0.25 ± 0.08 a,A
	HD1	59.8 ± 1.7 a,A,B	0.15 ± 0.10 a,C	3.31 ± 0.31 a,B	0.08 ± 0.07 a,A	0.04 ± 0.05 a,B	6.67 ± 0.88 a,b,A,B	0.04 ± 0.06 a,A	29.4 ± 1.6 a,A,B	0.25 ± 0.06 a,A	0.24 ± 0.08 a,A,B
	HD2	60.61 ± 0.35 a,A,B	0.24 ± 0.14 a,B	3.33 ± 0.36 a,B	0.09 ± 0.08 a,A	0.07 ± 0.06 a,A	6.49 ± 0.56 a,b,A	0.05 ± 0.07 a,A	28.64 ± 0.65 a,B,C	0.23 ± 0.07 a,A	0.25 ± 0.06 a,A
	NG1	58.9 ± 1.5 a,A,B	0.28 ± 0.16 a,A	3.29 ± 0.30 a,A,B	0.12 ± 0.10 a,A	0.08 ± 0.06 a,A	7.16 ± 0.39 a,A	0.05 ± 0.09 a,A	29.6 ± 1.3 a,A,B	0.22 ± 0.07 a,A	0.26 ± 0.07 a,A
	NG2	60.0 ± 1.2 a,A	0.35 ± 0.13 a,A	3.44 ± 0.25 a,A,B	0.14 ± 0.11 a,A,B	0.11 ± 0.02 a,A	6.49 ± 0.71 a,b,A	0.02 ± 0.06 a,A	28.97 ± 0.71 a,A,B	0.22 ± 0.04 a,A	0.25 ± 0.05 a,A
	VE	59.7 ± 1.2 a,A,B	0.27 ± 0.15 a,A	3.52 ± 0.26 a,B	0.09 ± 0.10 a,A	0.09 ± 0.04 a,A	6.12 ± 0.55 b,A	0.12 ± 0.15 a,A	29.6 ± 1.2 a,A,B	0.25 ± 0.08 a,A	0.26 ± 0.05 a,A
W9	C	57.6 ± 3.3 a,A	0.31 ± 0.11 a,B	3.18 ± 0.37 a,B	0.09 ± 0.07 a,B	0.11 ± 0.05 a,A,B	6.46 ± 0.93 a,A	0.07 ± 0.08 a,A	31.6 ± 3.0 a,A	0.30 ± 0.07 a,A	0.27 ± 0.08 a,A
	HD1	58.1 ± 2.5 a,A,B	0.29 ± 0.18 a,B,C	3.25 ± 0.21 a,B	0.08 ± 0.06 a,A	0.10 ± 0.10 a,A,B	7.04 ± 0.53 a,B	0.08 ± 0.14 a,A	30.5 ± 2.6 a,A	0.27 ± 0.07 a,A	0.21 ± 0.04 a,A,B
	HD2	55.8 ± 2.2 a,C	0.36 ± 0.09 a,A,B	2.98 ± 0.28 a,B	0.09 ± 0.06 a,A	0.13 ± 0.03 a,A	6.77 ± 0.89 a,A	0.12 ± 0.11 a,A	33.3 ± 1.9 a,A	0.25 ± 0.08 a,A	0.23 ± 0.05 a,A
	NG1	56.9 ± 2.1 a,B	0.32 ± 0.12 a,A	2.92 ± 0.20 a,B	0.12 ± 0.04 a,A	0.10 ± 0.05 a,A	6.96 ± 0.84 a,A	0.04 ± 0.07 a,A	32.1 ± 2.1 a,A	0.28 ± 0.14 a,A	0.26 ± 0.09 a,A
	NG2	57.6 ± 3.3 a,A	0.31 ± 0.18 a,A	3.27 ± 0.45 a,B	0.05 ± 0.05 a,B	0.13 ± 0.09 a,A	6.36 ± 0.87 a,A	0.03 ± 0.08 a,A	31.7 ± 3.3 a,A	0.28 ± 0.05 a,A	0.21 ± 0.06 a,A
	VE	57.1 ± 3.0 a,B	0.31 ± 0.13 a,A	3.07 ± 0.29 a,B	0.09 ± 0.06 a,A	0.11 ± 0.06 a,A	6.34 ± 0.58 a,A	0.07 ± 0.11 a,A	32.3 ± 2.9 a,A	0.31 ± 0.07 a,A	0.26 ± 0.08 a,A

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² TG: triglyceride; FFA: free fatty acid; DG: diglyceride; MG: monoglyceride; PE: phosphatidylethanolamine; PtdIns: phosphatidylinositol; PC: phosphatidylcholine; Sphm: sphingomyelin; LPC: lysophosphatidylcholine. ³ Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

Moreover, the proportion of sterols in egg yolks was significantly lower (p < 0.05) at W9 than at the beginning of the study. Specifically, a decline from 20.0 to 27.2% was observed in the values of total sterols between D0 and W9. Likewise, the proportions of free fatty acids (FFAs), diglycerides (DGs), and monoglycerides (MGs) measured for all treatment groups were lower after the first week (W1), possibly implying a reduction in lipolysis from the hydrolytic cleavage of ester bonds in TGs [51]. On the fifth week of the experiment (W5), the levels of FFA proportions were significantly lower (p < 0.05) compared to D0 for the C, HD1, and HD2 groups. On the other hand, di- and mono-glycerides presented lower values on both W5 and W9, although they were not significantly different (p ≥ 0.05) for most cases.

Concerning polar lipid assessments in yolk extracts, phosphatidylcholine (PC) predominated among the polar lipid classes, followed by phosphatidylethanolamine (PE) for all the studied egg samples. Based on the overall results of Table 4, there were no notable modifications in the profile of the main lipid classes as expected. Egg lipids are deposited in the yolk in lipoprotein forms synthesised in the hen’s liver, and are then transported to the ovary as vitellogenins and very-low-density lipoproteins (VLDLs). These lipid precursors are finally transferred to egg yolk without any further catabolism modifications. Thus, it is not feasible to change from scratch the main lipid profile of yolk by modifying the (lipid) content in the diet. Instead, it is possible to achieve short modifications in non-structural yolk lipids [59]. Notwithstanding this, a more important finding in our study concerns the PC proportion, which showed a significant (p < 0.05) increase by W9. Phosphatidylcholines are a class of phospholipids that incorporate a polar part of choline, which has important functions in brain development and liver performance [60,61]. As choline is a well-known important nutrient, it should be noted that the experimental diet, irrespective of the antioxidant addition, led to an optimised dietary polar lipid profile of egg yolks.

3.2. Profile of Fatty Acids and Lipid Quality Indices

The fatty acid composition of total lipids in the lipophilic extracts of egg yolks was determined using GC-FID and standard compounds. In parallel, the screening of FAMEs was conducted by GC-MS, with the support of GC-FID analysis, to confirm the identity of individual fatty acids due to the complexity of yolk fatty acid composition. A cost-effective dual analysis was achieved by using the same derivatised lypophilic extract of each yolk sample, which was first injected into GC-FID and then sequentially into GC-MS. Characteristic GC-MS chromatograms in an overlay view, corresponding to a representative sample from all six different treatment groups on the ninth week of the experimental study, along with the labelled peaks of several major fatty acids, are presented in Figure S1 (Supplementary Materials). The identification of FAMEs by GC-MS was accomplished through similarity searches in mass spectral libraries. Figure S2 (Supplementary Materials) illustrates the supervised identification for the mass profile matched to the methyl ester of palmitoleic acid. The qualitative investigation for each analyte was based on similarity scores in reference to the mass spectra of standard compounds from the libraries, as well as on the different fragmentation patterns (m/z values, intensities and ratios) recorded in the mass spectra of tested samples.

The percentage compositions of the major fatty acids per total fatty acids in total lipids of lipophilic extracts of dry yolk samples are shown in Table 5. Palmitic (C16:0) and stearic (C18:0) acids were the predominant saturated fatty acids (SFAs), and they accounted for more than 35% of total fatty acids in the studied egg yolks from all treatment groups throughout the 9 weeks. Oleic (C18:1 n-9 cis), palmitoleic (C16:1 n-7 cis), and vaccenic (C18:1 n-7 cis) acids contributed the most to the total concentration of monounsaturated fatty acids (MUFAs). Linoleic acid (C18:2 n-6 cis) was dominant among n-6 polyunsaturated fatty acids (PUFAs), with percentages ranging from 12.3(±1.6) to 18.79(±0.87)% for all studied samples. Alpha-linolenic acid (ALA, C18:3 n-3 cis), clupanodonic acid (C22:5 n-3 cis), and docosahexaenoic acid (DHA, C22:6 n-3 cis) were the main n-3 PUFAs identified in yolk samples (Table 5).

Table 5. Composition of the major fatty acids expressed as percentages (w/w) of total fatty acids in total lipids of lipophilic extracts of dry yolk samples. Results are presented as mean ± SD (standard deviation) (measurement repeats, N = 3).

Sampling	Treatment Group 1	C18:1 n-9 (cis) 2	C16:0 2	C18:2 n-6 (cis) 2	C18:0 2	C16:1 n-7 (cis) 2	C20:3 n-3 (cis) 2	C18:1 n-7 (cis) 2	Iso-C16:0 2	C22:6 n-3 (cis) 2	18:3 n-3 (cis) 2	C22:5 n-3 (cis) 2	C17:0 2
D0	C	36.6 ± 1.9 a,A,B	27.2 ± 2.6 a,A	18.1 ± 1.4 a,A	7.68 ± 0.73 a,A	2.76 ± 0.61 a,C	1.63 ± 0.24 a,A	1.33 ± 0.21 a,B	0.96 ± 0.07 a,A	0.58 ± 0.13 a,A	0.51 ± 0.06 a,A	0.44 ± 0.10 a,A	0.26 ± 0.03 a,A,B
	HD1	37.4 ± 2.0 a,A,B	27.2 ± 2.2 a,A	17.3 ± 1.1 a,A	7.72 ± 0.65 a,A	2.67 ± 0.39 a,B	1.56 ± 0.22 a,A	1.43 ± 0.27 a,A	1.05 ± 0.20 a,A,B	0.58 ± 0.12 a,A	0.46 ± 0.06 a,A	0.45 ± 0.12 a,A	0.25 ± 0.04 a,A,B
	HD2	37.28 ± 0.72 a,A	25.6 ± 1.3 a,B	18.79 ± 0.87 a,A	8.43 ± 0.99 a,A	2.10 ± 0.30 a,C	1.59 ± 0.15 a,A	1.28 ± 0.36 a,B	1.16 ± 0.21 a,A,B	0.62 ± 0.09 a,A	0.49 ± 0.02 a,A	0.51 ± 0.16 a,A	0.29 ± 0.03 a,A
	NG1	36.3 ± 1.9 a,A,B	27.2 ± 3.2 a,B	18.6 ± 1.9 a,A	7.9 ± 1.1 a,A	2.45 ± 0.57 a,B	1.59 ± 0.42 a,A	1.25 ± 0.17 a,B	1.03 ± 0.20 a,A,B	0.54 ± 0.15 a,A	0.50 ± 0.07 a,A	0.47 ± 0.20 a,A,B	0.27 ± 0.04 a,A
	NG2	35.4 ± 1.6 a,A	28.1 ± 2.9 a,A	18.7 ± 1.2 a,A	7.72 ± 0.50 a,A	2.77 ± 0.70 a,C	1.52 ± 0.22 a,A	1.24 ± 0.19 a,B	0.90 ± 0.17 a,A,B	0.50 ± 0.16 a,A	0.52 ± 0.07 a,A	0.45 ± 0.21 a,A	0.28 ± 0.06 a,A,B
	VE	36.5 ± 2.0 a,A,B	28.3 ± 1.4 a,A	16.9 ± 1.5 a,A	7.79 ± 0.79 a,A	3.02 ± 0.65 a,A	1.61 ± 0.11 a,A	1.39 ± 0.21 a,A	0.97 ± 0.18 a,A,B	0.51 ± 0.07 a,A	0.45 ± 0.04 a,A	0.44 ± 0.06 a,A	0.26 ± 0.06 a,A,B
W1	C	34.7 ± 1.3 a,B	30.3 ± 1.7 a,A	17.6 ± 2.1 a,A	7.24 ± 0.70 a,A	3.08 ± 0.99 a,B,C	1.43 ± 0.13 a,A	1.44 ± 0.26 a,A,B	1.10 ± 0.14 a,A	0.39 ± 0.08 a,B	0.38 ± 0.05 a,B	0.31 ± 0.05 a,A	0.30 ± 0.06 a,A
	HD1	34.9 ± 1.8 a,B	30.8 ± 2.7 a,A	17.0 ± 2.1 a,A	7.11 ± 0.93 a,A	3.1 ± 1.0 a,A,B	1.39 ± 0.21 a,A	1.43 ± 0.15 a,A	1.22 ± 0.24 a,A	0.36 ± 0.08 a,B	0.34 ± 0.04 a,B	0.29 ± 0.10 a,A	0.31 ± 0.06 a,A
	HD2	35.6 ± 1.6 a,A	30.0 ± 1.6 a,A	17.1 ± 1.7 a,A	7.38 ± 0.47 a,A	2.77 ± 0.85 a,B,C	1.37 ± 0.10 a,A	1.41 ± 0.15 a,A,B	1.30 ± 0.32 a,B	0.32 ± 0.04 a,B	0.34 ± 0.06 a,B	0.32 ± 0.07 a,A	0.32 ± 0.06 a,A
	NG1	34.0 ± 1.4 a,B	31.2 ± 1.5 a,A	17.6 ± 1.3 a,A	6.87 ± 0.41 a,A	3.33 ± 0.77 a,A,B	1.33 ± 0.11 a,A	1.43 ± 0.19 a,A,B	1.14 ± 0.22 a,B	0.32 ± 0.04 a,B	0.38 ± 0.03 a,B	0.30 ± 0.05 a,B	0.29 ± 0.05 a,A
	NG2	35.1 ± 2.1 a,A	30.4 ± 2.3 a,A	17.1 ± 1.3 a,A	7.28 ± 0.42 a,A	2.96 ± 0.80 a,B,C	1.40 ± 0.21 a,A	1.41 ± 0.13 a,A,B	1.22 ± 0.24 a,A	0.35 ± 0.08 a,A	0.35 ± 0.07 a,B	0.31 ± 0.08 a,A	0.29 ± 0.03 a,A
	VE	34.2 ± 1.9 a,B	31.2 ± 3.2 a,A	16.9 ± 1.7 a,A	6.77 ± 0.72 a,A	3.6 ± 1.3 a,A	1.38 ± 0.28 a,A	1.52 ± 0.17 a,A	1.26 ± 0.24 a,A	0.33 ± 0.08 a,A	0.36 ± 0.04 a,B	0.32 ± 0.13 a,A	0.28 ± 0.06 a,A
W5	C	38.7 ± 1.8 a,A	28.4 ± 1.3 a,A	13.7 ± 1.9 a,B	7.34 ± 0.51 a,A	4.43 ± 0.95 a,A,B	1.67 ± 0.20 a,A	1.80 ± 0.31 a,A	0.81 ± 0.16 a,A	0.46 ± 0.10 a,A,B	0.31 ± 0.06 a,B	0.39 ± 0.07 a,A	0.20 ± 0.05 a,B
	HD1	38.7 ± 2.0 a,A	28.0 ± 1.4 a,A	14.0 ± 1.6 a,B	7.51 ± 0.55 a,A	4.30 ± 0.38 a,A	1.67 ± 0.14 a,A	1.80 ± 0.28 a,A	0.71 ± 0.15 a,B	0.48 ± 0.11 a,A,B	0.30 ± 0.04 a,B	0.47 ± 0.12 a,A	0.19 ± 0.03 a,B
	HD2	38.0 ± 3.2 a,A	27.9 ± 1.6 a,A,B	14.2 ± 1.1 a,B	8.6 ± 2.6 a,A	4.03 ± 0.66 a,A,B	1.60 ± 0.11 a,A	1.60 ± 0.14 a,A,B	0.82 ± 0.04 a,B	0.43 ± 0.09 a,B	0.32 ± 0.04 a,B	0.47 ± 0.15 a,A	0.20 ± 0.02 a,B
	NG1	37.2 ± 3.8 a,A,B	27.9 ± 1.4 a,A,B	14.5 ± 1.1 a,B	7.5 ± 4.0 a,A	4.00 ± 0.76 a,A	1.69 ± 0.13 a,A	1.56 ± 0.10 a,A,B	0.76 ± 0.13 a,B	0.50 ± 0.10 a,A	0.33 ± 0.03 a,B	0.52 ± 0.11 a,A	0.19 ± 0.02 a,B
	NG2	38.9 ± 2.2 a,A	28.5 ± 1.3 a,A	13.3 ± 1.4 a,B	7.49 ± 0.39 a,A	4.38 ± 0.95 a,A,B	1.57 ± 0.16 a,A	1.73 ± 0.13 a,A	0.80 ± 0.20 a,B	0.42 ± 0.09 a,A	0.30 ± 0.05 a,B	0.49 ± 0.12 a,A	0.18 ± 0.02 a,B
	VE	38.6 ± 1.6 a,A	28.43 ± 0.65 a,A	13.5 ± 1.5 a,B	7.44 ± 0.55 a,A	4.44 ± 0.74 a,A	1.69 ± 0.19 a,A	1.77 ± 0.21 a,A	0.83 ± 0.19 a,B	0.44 ± 0.08 a,A	0.29 ± 0.04 a,B	0.44 ± 0.15 a,A	0.19 ± 0.04 a,B
W9	C	37.8 ± 1.9 a,A,B	30.2 ± 2.5 a,A	12.94 ± 0.93 a,B	7.2 ± 1.2 a,A	4.7 ± 1.2 a,A	1.46 ± 0.25 a,A	1.84 ± 0.42 a,A	0.84 ± 0.09 a,A	0.34 ± 0.11 a,B	0.29 ± 0.02 a,B	0.32 ± 0.10 a,A	0.19 ± 0.03 a,B
	HD1	38.3 ± 1.5 a,A,B	30.1 ± 1.5 a,A	13.1 ± 1.3 a,B	7.37 ± 0.54 a,A	4.04 ± 0.76 a,A,B	1.52 ± 0.17 a,A	1.66 ± 0.27 a,A	0.92 ± 0.09 a,A,B	0.32 ± 0.07 a,B	0.27 ± 0.03 a,B	0.31 ± 0.06 a,A	0.22 ± 0.03 a,B
	HD2	38.8 ± 2.0 a,A	30.1 ± 1.6 a,A	12.3 ± 1.6 a,B	7.20 ± 0.76 a,A	4.53 ± 0.94 a,A	1.41 ± 0.17 a,A	1.78 ± 0.26 a,A	0.90 ± 0.17 a,B	0.32 ± 0.05 a,B	0.26 ± 0.04 a,B	0.35 ± 0.10 a,A	0.19 ± 0.02 a,B
	NG1	38.0 ± 1.4 a,A	30.6 ± 1.4 a,A,B	12.9 ± 1.9 a,B	6.85 ± 0.38 a,A	4.62 ± 0.94 a,A	1.34 ± 0.15 a,A	1.78 ± 0.23 a,A	0.93 ± 0.17 a,A,B	0.29 ± 0.07 a,B	0.27 ± 0.05 a,B	0.31 ± 0.09 a,A,B	0.20 ± 0.03 a,B
	NG2	37.4 ± 2.1 a,A	30.8 ± 1.5 a,A	12.83 ± 0.84 a,B	7.20 ± 0.80 a,A	4.66 ± 0.78 a,A	1.40 ± 0.20 a,A	1.80 ± 0.16 a,A	0.85 ± 0.18 a,B	0.32 ± 0.07 a,A	0.28 ± 0.03 a,B	0.34 ± 0.10 a,A	0.20 ± 0.02 a,B
	VE	40.4 ± 3.7 a,A	27.4 ± 4.7 a,A	13.1 ± 2.4 a,B	7.56 ± 0.82 a,A	4.11 ± 0.72 a,A	1.61 ± 0.24 a,A	1.68 ± 0.27 a,A	0.99 ± 0.12 a,A,B	0.37 ± 0.08 a,A	0.27 ± 0.06 a,B	0.37 ± 0.13 a,A	0.20 ± 0.06 a,B

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

As indicated in Table 5, the monounsaturated fatty acids C16:1 n-7 cis and C18:1 n-7 cis showed an increasing tendency (p < 0.05) for all supplement groups, which stabilised by W5 of the dietary intervention period. On the other hand, margaric acid (C17:0) presented a statistically significant reduction (p < 0.05) by W5, but it was generally not affected by the supplements in general. Moreover, the polyunsaturated C18:2 n-6 cis and C18:3 n-3 cis decreased by W9, especially for the cases of the HD2, NG1, and NG2 groups. For dihomo-linolenic acid (C20:3 n-3 cis), neither the diet with different antioxidants nor the period of dietary intervention led to percentage alterations for all tested yolks. The iso-C16:0 concentration was mostly influenced by the week of the experiment rather than the dietary group. Furthermore, according to our study, C20:5 n-3 (cis) (eicosapentaenoic acid, EPA) was found in negligible amounts and below the limit of quantification (<LOQ, LOQ = 0.05 μg·μL⁻¹) in the lipophilic extracts of all egg yolks samples; meanwhile, the percentage proportion of the essential DHA showed significant (p < 0.05) alterations during the experiment and was in agreement with other researchers [62,63], whereas DHA was strongly affected by the combined effects of the hen’s age and strain, together with the dietary treatment. A similar observation was reported for egg yolks produced from younger hens, which presented higher percentages of arachidonic acid and DHA [64,65].

As a general remark and illustrated by Table 5, fluctuations are marked along the different time samplings for the proportion of some major fatty acids. Specifically, oleic acid, palmitic acid, and DHA presented both upward and downward trends during the experimental period of nine weeks. Lešić et al. [13], who evaluated the effect of hen age on the fatty acid content of yolks produced from Lohman Brown hens from 21 to 55 weeks old, also revealed significant ageing-based variations in fatty acid composition, in particular in the profile of linoleic, alpha-linolenic, and arachidonic acids. It is important to acknowledge that several yolk parameters varied over the course of the experimental period across all dietary groups in our study; since hen age, duration of dietary exposure, and adaptation to the regime occurred concomitantly, the present design does not allow their underlying cause to be disentangled.

Moreover, the proportion of the major fatty acids on total fatty acids, the proportion of the SFAs, MUFAs, and PUFAs of the total fatty acids contained in each of the total lipid fractions studied, as well as the lipid quality indices, are presented in Table 6. Most of the fatty acid proportions underwent significant variations during the experimental period, and they were mostly affected by the duration of feeding irrespective of the feed type provided. MUFAs increased significantly (p < 0.05) from day 0 till the end of the ninth week, whereas total PUFAs decreased (p < 0.05). The total SFAs did not significantly differ (p ≥ 0.05) when comparing the content of egg yolks among samplings or treatment groups. Concerning the fatty acid ratios of egg yolk fat, the n-6/n-3 ratio decreased on the fifth week of feeding, revealing a favourable change considering the nutritional value of tested yolks, which remained constant (p < 0.05) until the ninth week. Furthermore, MUFAs and n-6 PUFAs showed a contradictory trend during the experimental period via a significant increase in the MUFA proportion and a parallel decrease in the n-6 PUFA proportion. Moreover, according to the results shown in Table 6, the MUFA/PUFA ratio showed a significant increase for the cases of HD2, NG1, and NG2 during the experimental period, especially by W9. Therefore, the addition of flavonoids seems to enhance this fatty acid ratio. Supporting these findings, Hussein et al. reported that dietary supplementation with dried orange pulp in laying hens significantly increased the proportion of n-3 polyunsaturated fatty acids while also reducing the saturated fatty acid content and n-6/n-3 ratio in egg yolk [40].

Table 6. Sums % (w/w) and ratios of fatty acid groups of total fatty acids in total lipids, as well as lipid quality indices of lipophilic extracts, of dry yolk samples, as expressed on the basis of the dry yolk weight. Results are presented as mean ± SD (standard deviation, N = 3).

Sampling	Treatment Group 1	SFA 2,3	MUFA 2,3	PUFA 2,3	MUFA/SFA 2,3	PUFA/SFA 2,3	MUFA/PUFA 2,3	n-6/n-3 2,3	AI 2,3	TI 2,3	PI 2,3	H 2,3	H 2,3	h/H 2,3
D0	C	37.0 ± 2.3 a,A	41.1 ± 1.7 a,B,C	21.9 ± 1.7 a,A	1.12 ± 0.10 a,A,B	0.60 ± 0.08 a,A	1.89 ± 0.18 a,A	5.98 ± 0.91 a,A,B	0.46 ± 0.06 a,A	0.90 ± 0.11 a,A	32.6 ± 3.0 a,A	56.4 ± 2.5 a,A	27.7 ± 2.7 a,A	2.06 ± 0.28 a,A
	HD1	37.1 ± 1.9 a,A	41.8 ± 2.3 a,A,B	21.1 ± 1.0 a,A	1.13 ± 0.12 a,A,B	0.57 ± 0.04 a,A	1.99 ± 0.17 a,A	6.0 ± 1.0 a,A,B	0.46 ± 0.05 a,A	0.90 ± 0.09 a,A	31.6 ± 1.9 a,A	56.2 ± 1.5 a,A	27.6 ± 2.3 a,A	2.05 ± 0.21 a,A
	HD2	36.3 ± 1.3 a,A	40.99 ± 0.95 a,B,C	22.7 ± 1.1 a,A	1.13 ± 0.06 a,A	0.63 ± 0.05 a,A	1.81 ± 0.11 a,B	6.10 ± 0.61 a,A,B	0.42 ± 0.03 a,B	0.86 ± 0.06 a,A	33.9 ± 2.4 a,A	57.8 ± 1.1 a,A	26.0 ± 1.3 a,B	2.23 ± 0.15 a,A
	NG1	37.2 ± 2.5 a,A	40.4 ± 1.8 a,B,C	22.4 ± 2.1 a,A	1.09 ± 0.10 a,A	0.61 ± 0.09 a,A	1.82 ± 0.21 a,C	6.5 ± 1.5 a,A,B	0.46 ± 0.08 a,B	0.91 ± 0.13 a,A	32.8 ± 4.1 a,A	56.6 ± 2.6 a,A	27.6 ± 3.3 a,B	2.09 ± 0.33 a,A
	NG2	37.8 ± 2.7 a,A	39.8 ± 1.9 a,B,C	22.4 ± 1.8 a,A	1.06 ± 0.11 a,A,B	0.60 ± 0.08 a,A	1.79 ± 0.15 a,A	6.7 ± 1.3 a,A,B	0.48 ± 0.07 a,A	0.94 ± 0.15 a,A	32.3 ± 4.3 a,A	55.7 ± 2.4 a,A	28.5 ± 2.9 a,A	1.98 ± 0.27 a,A
	VE	38.08 ± 0.87 a,A	41.3 ± 2.0 a,B,C	20.6 ± 1.5 a,A	1.09 ± 0.07 a,B,C	0.54 ± 0.04 a,A	2.02 ± 0.24 a,A	5.80 ± 0.56 a,A,B	0.48 ± 0.03 a,A	0.95 ± 0.04 a,A	30.6 ± 1.8 a,A	55.0 ± 1.3 a,A	28.7 ± 1.5 a,A	1.92 ± 0.14 a,A
W1	C	39.8 ± 1.3 a,A	39.5 ± 1.8 a,C	20.7 ± 2.3 a,A	1.00 ± 0.05 a,B	0.52 ± 0.07 a,A,B	1.94 ± 0.30 a,A	7.21 ± 0.41 a,A	0.54 ± 0.05 a,A	1.04 ± 0.07 a,A	28.8 ± 2.8 a,A,B	53.5 ± 2.2 a,A,B	30.8 ± 1.8 a,A	1.75 ± 0.17 a,A
	HD1	40.3 ± 2.2 a,A	39.8 ± 1.7 a,B	19.9 ± 2.4 a,A,B	0.99 ± 0.08 a,B	0.50 ± 0.08 a,A,B	2.03 ± 0.30 a,A	7.38 ± 0.93 a,A	0.55 ± 0.08 a,A	1.07 ± 0.12 a,A	27.6 ± 3.3 a,A,B	53.0 ± 2.9 a,A	31.3 ± 2.8 a,A	1.71 ± 0.22 a,A
	HD2	39.9 ± 1.4 a,A	40.1 ± 1.5 a,C	20.0 ± 1.7 a,A,B	1.01 ± 0.06 a,A	0.50 ± 0.05 a,B	2.02 ± 0.23 a,B,C	7.54 ± 0.89 a,A	0.53 ± 0.04 a,A	1.05 ± 0.07 a,A	27.5 ± 1.6 a,B	53.8 ± 2.0 a,A,B	30.5 ± 1.7 a,A	1.77 ± 0.16 a,A,B
	NG1	40.4 ± 1.5 a,A	39.1 ± 1.8 a,C	20.5 ± 1.4 a,A,B	0.97 ± 0.07 a,A	0.51 ± 0.04 a,A,B	1.92 ± 0.20 a,B	7.77 ± 0.79 a,A	0.56 ± 0.04 a,A	1.08 ± 0.07 a,A	27.9 ± 1.6 a,B,C	52.7 ± 1.7 a,A,B	31.8 ± 1.6 a,A	1.67 ± 0.12 a,A
	NG2	40.0 ± 2.0 a,A	39.8 ± 1.3 a,C	20.2 ± 1.4 a,A	1.00 ± 0.08 a,B	0.51 ± 0.06 a,A,B	1.98 ± 0.16 a,A	7.46 ± 0.83 a,A	0.54 ± 0.06 a,A	1.06 ± 0.11 a,A	27.9 ± 2.3 a,A,B	53.4 ± 2.4 a,A	30.9 ± 2.3 a,A	1.75 ± 0.22 a,A
	VE	40.5 ± 2.6 a,A	39.7 ± 1.2 a,C	19.9 ± 2.2 a,A,B	0.98 ± 0.08 a,C	0.49 ± 0.08 a,A	2.02 ± 0.19 a,A	7.35 ± 0.80 a,A	0.56 ± 0.09 a,A	1.08 ± 0.15 a,A	27.6 ± 3.5 a,A,B	52.2 ± 3.4 a,A	31.8 ± 3.3 a,A	1.67 ± 0.28 a,A
W5	C	37.5 ± 1.5 a,A	45.3 ± 2.4 a,A	17.2 ± 2.1 a,B	1.21 ± 0.09 a,A	0.46 ± 0.06 a,B	2.68 ± 0.43 a,B	5.02 ± 0.68 a,B	0.48 ± 0.03 a,A	0.94 ± 0.07 a,A	26.6 ± 2.7 a,B	53.6 ± 1.4 a,A,B	28.8 ± 1.4 a,A	1.86 ± 0.13 a,A
	HD1	37.2 ± 1.5 a,A	45.2 ± 2.2 a,A	17.6 ± 1.6 a,B,C	1.22 ± 0.10 a,A	0.47 ± 0.05 a,A,B	2.60 ± 0.33 a,B	5.02 ± 0.87 a,B	0.47 ± 0.04 a,A	0.92 ± 0.07 a,A	27.6 ± 1.7 a,A,B	54.1 ± 1.4 a,A	28.4 ± 1.5 a,A	1.91 ± 0.15 a,A
	HD2	38.3 ± 2.5 a,A	44.0 ± 2.8 a,A,B	17.6 ± 1.1 a,B,C	1.16 ± 0.14 a,A	0.46 ± 0.04 a,B	2.51 ± 0.26 a,A,B	5.21 ± 0.64 a,B	0.48 ± 0.04 a,A,B	0.97 ± 0.11 a,A	27.1 ± 2.0 a,B	53.5 ± 2.8 a,A,B	28.3 ± 1.7 a,A,B	1.90 ± 0.19 a,A,B
	NG1	38.6 ± 3.6 a,A	43.2 ± 3.5 a,A,B	18.2 ± 1.3 a,B,C	1.13 ± 0.17 a,A	0.48 ± 0.06 a,B	2.38 ± 0.26 a,A,B	4.94 ± 0.51 a,B	0.48 ± 0.04 a,A,B	0.97 ± 0.15 a,A	28.7 ± 2.4 a,A,B	53.1 ± 4.1 a,A,B	28.3 ± 1.4 a,A,B	1.88 ± 0.18 a,A
	NG2	37.8 ± 1.1 a,A	45.5 ± 1.5 a,A,B	16.7 ± 1.5 a,B	1.21 ± 0.06 a,A	0.44 ± 0.05 a,B	2.74 ± 0.35 a,B	4.99 ± 0.71 a,C	0.49 ± 0.03 a,A	0.95 ± 0.06 a,A	26.3 ± 2.5 a,B	53.6 ± 1.8 a,A	29.0 ± 1.3 a,A	1.85 ± 0.15 a,A
	VE	37.67 ± 0.49 a,A	45.3 ± 1.9 a,A,B	17.1 ± 2.0 a,B,C	1.20 ± 0.05 a,A,B	0.45 ± 0.05 a,A	2.69 ± 0.39 a,B	4.92 ± 0.37 a,B	0.48 ± 0.02 a,A	0.94 ± 0.04 a,A	26.7 ± 3.4 a,A,B	53.42 ± 0.86 a,A	28.87 ± 0.68 a,A	1.85 ± 0.07 a,A
W9	C	39.3 ± 1.7 a,A	44.7 ± 1.3 a,A,B	16.0 ± 1.2 a,B	1.14 ± 0.08 a,A,B	0.41 ± 0.04 a,B	2.81 ± 0.24 a,B	5.72 ± 0.91 a,A,B	0.53 ± 0.06 a,A	1.04 ± 0.11 a,A	24.0 ± 2.6 a,B	51.8 ± 2.6 a,B	30.7 ± 2.7 a,A	1.70 ± 0.22 a,A
	HD1	39.4 ± 1.2 a,A	44.4 ± 1.1 a,A	16.2 ± 1.3 a,C	1.13 ± 0.05 a,A,B	0.41 ± 0.04 a,B	2.76 ± 0.28 a,B	5.67 ± 0.67 a,B	0.53 ± 0.04 a,A	1.04 ± 0.07 a,A	23.9 ± 1.7 a,B	52.5 ± 1.9 a,A	30.6 ± 1.6 a,A	1.72 ± 0.15 a,A
	HD2	39.2 ± 1.3 a,A	45.5 ± 1.5 a,A	15.2 ± 1.7 a,C	1.16 ± 0.06 a,A	0.39 ± 0.05 a,B	3.03 ± 0.41 a,A	5.51 ± 0.89 a,B	0.53 ± 0.04 a,A,B	1.04 ± 0.07 a,A	23.0 ± 2.2 a,B	52.2 ± 2.0 a,B	30.6 ± 1.7 a,A	1.71 ± 0.16 a,B
	NG1	39.4 ± 1.5 a,A	44.8 ± 2.2 a,A	15.7 ± 2.1 a,C	1.14 ± 0.08 a,A	0.40 ± 0.06 a,B	2.92 ± 0.55 a,A	6.05 ± 0.94 a,B	0.54 ± 0.04 a,A,B	1.06 ± 0.08 a,A	23.0 ± 2.9 a,C	51.9 ± 1.7 a,B	31.1 ± 1.4 a,A,B	1.67 ± 0.12 a,A
	NG2	39.9 ± 1.2 a,A	44.3 ± 1.5 a,A	15.8 ± 1.0 a,B	1.11 ± 0.07 a,A,B	0.40 ± 0.03 a,B	2.82 ± 0.24 a,B	5.78 ± 0.86 a,B,C	0.55 ± 0.04 a,A	1.07 ± 0.07 a,A	23.5 ± 2.0 a,B	51.4 ± 1.6 a,A	31.3 ± 1.6 a,A	1.65 ± 0.13 a,A
	VE	37.0 ± 4.0 a,A	46.6 ± 3.9 a,A	16.4 ± 2.7 a,C	1.29 ± 0.29 a,A	0.45 ± 0.11 a,A	2.91 ± 0.58 a,B	5.25 ± 0.87 a,B	0.47 ± 0.09 a,A	0.93 ± 0.15 a,A	25.1 ± 3.7 a,B	54.7 ± 4.0 a,A	27.9 ± 4.7 a,A	2.07 ± 0.74 a,A

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; n-6: omega-6 fatty acids; n-3: omega-3 fatty acids; AI: atherogenic index; TI: thrombogenic index; PI: peroxidability index; h: hypocholesterolaemic fatty acids index; H: hypercholesterolaemic fatty acids index; h/H = hypocholesterolaemic/hypercholesterolaemic fatty acids ratio. ³ Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

Health-aggravating lipid quality indices—the atherogenic index (AI), thrombogenic index (TI), and peroxidisability index (PI)—and hypocholesterolaemic (h) and hypercholesterolaemic (H) fatty acids were calculated according to Sinanoglou et al. [66]. The corresponding equations for lipid quality indices are cited in Equations (S1)–(S6) (Supplementary Materials). The AI indicates the relationship between SFAs and the main classes of unsaturated fatty acids. The clotting activity of platelets and their aggregation to a thrombus is closely related to the intake of long-chain SFAs. Omega-6 PUFAs are anti-atherogenic, whereas n-3 PUFAs are antithrombogenic. Furthermore, the TI indicates the tendency for the formation of clots in the blood vessels. The h/H ratio is related to the functional activity of fatty acids in the metabolism of lipoproteins regarding plasma cholesterol transport and the risk of cardiovascular disease. The proportions of other minor fatty acids of the total fatty acids used for the evaluation of the indices are also provided in Supplementary Materials (Table S2). The corresponding values for all indices are set out in Table 6. AI, TI, and H showed similar variations throughout the weeks, with the higher values being recorded after the first week of feeding (W1). There were no significant differences (p ≥ 0.05) among the values of health-related lipid quality indices, PI and H, throughout the feeding period after the first week of adaptation to the new feeding regime. According to Table 6, only the peroxidisability index was shown to decrease (p < 0.05), especially by W9 and for all cases of treatment. This is an important finding, as a lower PI renders the lipid-rich egg yolk less susceptible to lipid peroxidation, thus presenting a greater protective potential for pathological conditions, such as ageing, atherosclerosis, and coronary artery disease [67]. Recent studies have demonstrated that plant-extract supplementation reduces lipid peroxidation markers in egg yolks: dietary olive pomace extract significantly decreased yolk malondialdehyde levels during storage while concurrently increasing total n-3 PUFA proportions in laying hens [68], and supplementation with either oyster mushroom waste [69] or dried orange pulp [40] similarly produced markedly lower lipid peroxidation rates in both fresh and stored egg yolks. AI and TI, after nine weeks of dietary intervention, were within 0.47(±0.09)–0.55(±0.04) and 0.93(±0.15)–1.07(±0.07), respectively. Such low values of AI and TI are recommended for a healthy diet, while AI and TI were found to be lower or quite similar when compared with edible yolks from different avian species [70].

3.3. Colour Analysis of Egg Yolks

The colour of egg yolks can be accurately determined using spectrocolourimeters with predefined parameters following the international CIE colour system. In particular, the CIE L*a*b* system is regarded as the most suitable for determining the colour of chicken egg yolks [71]. In the present study, the colour analysis of the powdered lyophilised yolk samples was executed accordingly. The values of the coordinates L*, a*, b*, C*, and hue were determined. Parameter L* symbolises luminosity or lightness corresponding to the equivalence of each colour to a member of the greyscale; that is, between black and white. Positive values for the parameter a* stand for reddish colours and negative values for greenish ones, whereas yellowish and bluish colours are presented by positive and negative values of parameter b*, respectively. Moreover, the chroma (C*) and hue values were determined, where chroma is a quantitative characteristic of colour saturation, so that the higher the chroma value, the higher is the colour intensity of a sample. The hue angle (h*) is a qualitative colour parameter, and it is related to the absorbance at different wavelengths of the visible spectra, such that colours are labelled as yellowish, bluish, etc., and it is used to define the difference in a certain colour with reference to the grey colour of the same L* value. The hue angle is depicted on a 360° grid where the red colour is set to 0°, 90° for yellow, 180° for green, and 270° for blue [72,73].

The results of the colour analysis for the lyophilised yolk samples are presented in Table 7. It is apparent that the colour profile of egg yolks obtained from hens fed with either of the three antioxidants, namely hesperidin, naringin, and vitamin E, did not differ significantly (p ≥ 0.05) from the control group for all samplings regardless of the supplemented dose. The analytical assessment of carotenoid-driven yolk pigmentation by a liquid chromatography–tandem mass spectrometry (LC-MS/MS) workflow, enabling simultaneous quantification of the eleven most abundant egg yolk carotenoids, confirmed that carotenoid profiles are strongly diet- and farming-dependent [74]. The absence of colour modulation by hesperidin and naringin contrasts with reported effects of other phytogenic compounds; curcumin supplementation at 200 mg/kg, for example, has been shown to significantly improve the egg yolk colour score in laying hens [75], and a range of plant extracts including thyme, anise, and black cumin has similarly been reported to significantly affect yolk colour parameters in laying hens [76]. As presented herein, pronounced colour changes, in terms of lightness, redness, yellowness, chroma, and hue, were recorded with respect to the feeding duration.

Table 7. Values of CIE L*a*b* colour parameters for lyophilised yolk samples from the different treatment groups and samplings. Results are presented as mean ± SD (standard deviation) (measurement repeats, N = 9).

Sampling	Treatment Group 1	Luminosity, L* 2,3	Redness, a* 2,3	Yellowness, b* 2,3	Chroma, C* 2,3	Hue (°) 3
D0	C	74.7 ± 6.8 a,B	2.48 ± 0.89 a,B	38.0 ± 4.1 a,B	38.1 ± 4.1 a,B	86.4 ± 1.1 a,A
	HD1	75.8 ± 5.2 a,B	3.1 ± 1.2 a,B	40.2 ± 4.1 a,B	40.3 ± 4.1 a,B	85.7 ± 1.5 a,A
	HD2	75.9 ± 7.0 a,B	2.18 ± 0.67 a,B	39.1 ± 2.9 a,B	39.1 ± 2.9 a,B	86.85 ± 0.73 a,A
	NG1	72.4 ± 5.0 a,B	2.98 ± 0.77 a,B	39.6 ± 2.8 a,B	40.8 ± 3.6 a,B	85.71 ± 0.89 a,A
	NG2	71.1 ± 6.3 a,B	2.4 ± 1.0 a,B	37.5 ± 2.6 a,B	37.5 ± 2.6 a,B	86.4 ± 1.3 a,A
	VE	75.9 ± 6.1 a,B	2.95 ± 0.69 a,B	40.1 ± 3.5 a,B	40.2 ± 3.5 a,B	85.83 ± 0.62 a,A
W1	C	81.4 ± 2.1 a,A,B	9.9 ± 1.6 a,A	48.9 ± 3.4 a,A	49.9 ± 3.6 a,A	78.6 ± 1.4 a,B
	HD1	81.8 ± 2.3 a,A,B	8.7 ± 1.9 a,A	48.4 ± 5.2 a,A	49.2 ± 5.4 a,A	79.9 ± 1.3 a,B
	HD2	80.8 ± 2.3 a,A,B	10.2 ± 2.4 a,A	51.1 ± 3.7 a,A	52.1 ± 4.0 a,A	78.8 ± 2.1 a,B
	NG1	80.1 ± 3.3 a,A	9.8 ± 1.8 a,A	50.0 ± 2.5 a,A	51.0 ± 2.6 a,A	79.0 ± 1.8 a,B
	NG2	81.7 ± 1.4 a,A	8.9 ± 1.9 a,A	50.3 ± 4.3 a,A	51.1 ± 4.4 a,A	80.0 ± 1.8 a,B
	VE	80.2 ± 2.3 a,A,B	10.4 ± 1.5 a,A	51.4 ± 5.1 a,A	52.5 ± 5.3 a,A	78.6 ± 1.2 a,B
W5	C	84.5 ± 1.5 a,A	8.7 ± 1.1 a,A	52.8 ± 2.7 a,A	53.5 ± 2.8 a,A	80.66 ± 0.83 a,B
	HD1	84.4 ± 1.5 a,A	8.43 ± 0.99 a,A	47.7 ± 2.8 a,A	48.5 ± 2.8 a,A	80.0 ± 1.3 a,B
	HD2	83.5 ± 1.6 a,A	8.7 ± 1.9 a,A	50.1 ± 3.5 a,A	50.9 ± 3.7 a,A	80.3 ± 1.7 a,B
	NG1	85.5 ± 1.1 a,A	7.81 ± 0.81 a,A	48.6 ± 1.9 a,A	49.2 ± 2.0 a,A	80.85 ± 0.63 a,B
	NG2	84.9 ± 1.5 a,A	7.96 ± 0.73 a,A	50.9 ± 1.7 a,A	51.5 ± 1.7 a,A	81.11 ± 0.81 a,B
	VE	83.8 ± 1.8 a,A	9.6 ± 1.5 a,A	52.1 ± 4.6 a,A	53.0 ± 4.6 a,A	79.6 ± 1.3 a,B
W9	C	75.9 ± 3.3 a,B	8.8 ± 1.4 a,A	51.9 ± 1.9 a,A	52.7 ± 2.0 a,A	80.4 ± 1.4 a,B
	HD1	77.0 ± 2.0 a,B	8.4 ± 1.3 a,A	47.5 ± 3.6 a,A	48.2 ± 3.5 a,A	80.0 ± 1.3 a,B
	HD2	77.9 ± 2.2 a,A,B	8.1 ± 1.8 a,A	47.9 ± 2.8 a,A	48.6 ± 3.0 a,A	80.4 ± 1.6 a,B
	NG1	73.5 ± 3.6 a,B	7.8 ± 1.4 a,A	49.3 ± 3.3 a,A	49.9 ± 3.5 a,A	81.1 ± 1.1 a,B
	NG2	76.4 ± 2.1 a,B	9.26 ± 0.96 a,A	50.9 ± 2.9 a,A	51.7 ± 2.9 a,A	79.7 ± 1.0 a,B
	VE	75.2 ± 5.2 a,B	10.0 ± 1.1 a,A	51.6 ± 3.4 a,A	52.6 ± 3.4 a,A	79.0 ± 1.1 a,B

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² Unitless values. ³ Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

In detail, as shown in Table 7, L* ranged from 71.1(±6.3) to 75.9(±7.0) (p ≥ 0.05) for samples collected on the first day of the experiment (D0), and it shifted to higher values when measured after one week (W1) of treatment for all dietary groups. Luminosity for the C, HD1, HD2, and VE groups did not differ significantly (p ≥ 0.05) between D0 and W1 samplings, whereas naringin-supplemented hens laid eggs by W1 with significantly higher luminosity (p < 0.05), at 80.1(±3.3) for NG1 and 81.7(±1.4) for NG2, compared to the eggs from the corresponding groups on D0. However, no differences (p ≥ 0.05) were found for L* values of yolks among the treatment groups concerning the samplings on W1, W5, or W9. Among all samplings, the highest (p < 0.05) values for L*, signifying yolks of a lighter colour, were reported for the samples collected on W1 and W5. On the ninth week of the study, egg yolks from all the treatment groups shifted to a darker colour (p < 0.05), compared to yolks from W1 and W5, at about the same level of lightness as in D0, precisely from 73.5(±3.6) to 77.9(±2.2). A possible explanation for the fluctuations observed in yolk luminosity are the differences in the deposition rates of various carotenoid pigments, which are negatively correlated with L* [77].

With regard to the coordinate for redness (a*), a clear trend dependent on the treatment period was observed for yolk samples. The a* values increased remarkably (p < 0.05) within the first week of the experimental period for the egg yolks derived from all treatment groups (Table 7). Specifically, the intensity of red colour was 3.6-fold higher (p < 0.05) in the yolks collected on W1 compared with the yolks of D0, and remained unaltered (p ≥ 0.05) till the ninth week (W9) of the experiment. On W1, W5, and W9, hens fed vitamin E produced eggs with a slightly more intense (p ≥ 0.05) red yolk colour compared to the control or HD and NG groups. Even though the influence of vitamin E on colour is not statistically different when compared with the other treatments, dietary vitamin E has been proven effective in the prevention of lipid oxidation, including carotenoid pigments, in egg yolks [78].

In a similar way to yolk redness, the yellowness of yolks from W1 presented statistically significantly higher values (p < 0.05) than the yolks from D0 (Table 7). The yolk colour of eggs collected from all treatment groups by D0, ranging from 37.5(±2.6) to 40.2(±2.1), shifted to a more intense (p < 0.05) yellow colour, with b* values levelled at approximately 50.0. Yellowness is the most essential colour factor to determine egg quality. Dietary lutein and zeaxanthin enhance the yellowness of dried egg yolks, thus elevating the value of the b* colour parameter [79]. Elevated values for component b* render the product more desirable, as the yellow intensity is a very important criterion in the consumer’s choice because it connects colour with nutritional value. Moreover, eggs with intense yellow yolks are advantageous for the production of egg derivatives and processed foods, since the yolk colour will contribute to the sensory quality of the product [80]. Furthermore, the a*/b* ratios of the W1, W5, and W9 samples are about three-fold higher than the corresponding one at D0. This finding could be attributed to the antioxidant phytochemicals of maize and wheat rather than the addition of the flavonoids or vitamin E.

For context, the present study constitutes a follow-up analysis of egg yolk samples collected during the same feeding trial first reported by Goliomytis et al. [50]; the present analyses and outcomes were not included in this earlier publication. In the original experiment, Goliomytis et al. [50] estimated egg yolk colour using the DSM Yolk Colour Fan, a visual evaluation method based on comparison with a standard colour scale ranging from very light yellow to red. The researchers investigated the effect of a dietary intervention on laying hens with hesperidin-, naringin-, or vitamin-E-enriched feed for approximately 9.5 weeks (67 d). The results of their study revealed a relationship between the supplementation to laying hens and yolk colour. Specifically, a linear shift (P-linear < 0.05) from a yellow to red colour was observed with increasing doses of both phenolic compounds. Considering that none of the supplements are pigments, the observations were attributed to the higher conservation and deposition of carotenoids in egg yolk as a result of the protective mechanisms against oxidation of the three antioxidants under study. This hypothesis is not supported by our results, and might be attributed to the different techniques used to measure yolk colour in the two studies.

Furthermore, according to Table 7, an increase was observed for the yolk chroma (C*) (p < 0.05) after one week of dietary treatment, indicating an enhancement of colour saturation. Both b* and C* remained stable (p ≥ 0.05) for the rest of the studied period and for all dietary groups. Hue measurements on the yolks from all subjects at the beginning of the experiment, as shown in Table 7, approached 90°, which indicates a yellow pigmentation of the yolks. Hue depends on the carotenoid composition of yolk, specifically from lutein and zeaxanthin [20]. However, all yolk samples from the samplings of W1, W5, and W9 presented lower hue values compared to the yolks from D0, which were perceived as having a slight shift towards a redder colouration of yolks. Changes for hue values of the yolks are in accordance with the corresponding changes recorded for a* and b* values, indicating an inverse relationship of the colour coordinates.

As discussed above, the variation in egg yolk colour was primarily associated with sampling time rather than with the different dietary treatments. Differences reported after one week of dietary treatment can therefore be attributed to the adaptation of hens’ diets to the regulated regime with the new commercially available basal feed reported in Table 1. The colour profile of the eggs collected on the first day of the experimental period (D0) reflects the regime implemented to the hen groups prior to the beginning of the experiment.

As listed in Table 1, the main ingredients of the standard diet offered to all subjects from the different groups during the whole period of the experiment were maize, wheat, and soybean. Maize (Zea mays L.) was the principal component of the poultry diet, accounting for 54% per weight. Yellow corn grains are a commonly used as a dietary base for laying hens. The contribution of maize carotenoids to yolk colour is often underestimated by zootechnicians, although there are corn hybrids with high carotenoid contents that can affect yolk colour. Corn grains in hens’ diets, without added pigments, can adequately supply carotenoids for an acceptable egg yolk colour by increasing a* values of yolks [81]. Moreover, green chlorophyll pigments, a and b, have been found in wheat and soybean seeds [82,83]. It was reported that the yolk colour can also be affected by the consumption of chlorophyll a and b when present in the feed of laying hens [84]. The potential of a chlorophyll extract to increase the yellowness, redness, and chroma while decreasing the hue of egg yolks was demonstrated when supplemented to laying hens [72]. Nevertheless, it was pointed out that the colouring effect might also be associated with other compounds contained in the extract, such as xanthophylls of a yellow and orange colour which are known to be readily transported into the yolk [20].

Overall, with respect to the results of the colour analysis for the yolk samples of the dietary groups collected on the four time points of the dietary treatment, it was not possible to discriminate the yolks laid by hens that followed diets including different supplements on a colour basis. The addition of either hesperidin, naringin, or vitamin E in hens’ feeds did not affect the profile of the yolk colour based on the CIE L*a*b* colorimetric determination. On the other hand, the yolk colour was markedly influenced by the feeding duration with the basal ration rather than the inclusion of antioxidants in the hens’ regime. These findings suggest that the adaptation period in which the hens can adjust to a newly introduced basal diet can be narrowed to one week rather than the time frame of two-to-four weeks, which is usually allowed for the production of eggs with a stable yolk colour [85,86,87,88,89]. From an industrial and scientific standpoint, this finding bears considerable practical significance. Extended acclimation periods represent a principal contributor to the overall cost and duration of animal-based nutrition trials, as hens must be maintained under standardised housing and feeding conditions before any treatment effect can be reliably assessed against a stable baseline. Narrowing this requirement to one week would therefore allow for a substantial reduction in feed, labour, and housing costs per trial, all while shortening the overall experimental timeline and enabling a faster turnover between successive studies. This is of particular relevance to the poultry feed industry, where the efficacy of novel feed additives is routinely evaluated through comparative trials of considerable duration; a shortened adaptation phase would allow a greater number of formulations to be screened within a given research budget, thereby accelerating the pace of product development.

3.4. Total Carotenoid Content

The total carotenoid content in the lipophilic extracts of egg yolks was estimated spectrophotometrically. Trans-canthaxanthin, lutein, and zeaxanthin were used as standards to estimate the equivalent concentration of total carotenoids in yolks. The analytical figures of merit for the photometric determination of total carotenoids are illustrated in Table 8.

Table 8. Concentration ranges, constants of the linear regression equations, determination coefficients (R²), limits of detection (LODs), and limits of quantitation (LOQs), as determined photometrically, for the standard carotenoids.

	Trans-Canthaxanthin	Lutein	Zeaxanthin
Concentration range (μg·mL−1)	1.0–10	0.70–8.0	0.60–6.0
Slope, a 1,2	0.1521(±0.0016)	0.2002(±0.0075)	0.3104(±0.068)
Intercept, b 1,2	0.0053(±0.037)	0.009(±0.021)	0.017(±0.017)
R2	0.996	0.993	0.999
LOD (μg·mL−1)	0.37	0.22	0.19
LOQ (μg·mL−1)	1.1	0.67	0.57

¹ Values are presented as mean (±standard deviation). ² Measurement repeats, N = 3.

The results of the spectrophotometric determination for total carotenoids in the lipophilic extracts of yolk samples, expressed as equivalents of trans-canthaxanthin, lutein, or zeaxanthin, are presented in Table 9.

Table 9. Total carotenoid content of yolk samples from the different treatment groups and samplings, expressed as trans-canthaxanthin, lutein, and zeaxanthin equivalents, in μg per g total lipids (TL) of yolk and as μg per g dry yolk (DY). Results are presented as mean ± standard deviation (measurement repeats, N = 3).

		Trans-Canthaxanthin		Lutein		Zeaxanthin
Sampling	Treatment Group 1	(μg·g−1 TL) 2	(μg·g−1 DY) 2	(μg·g−1 TL) 2	(μg·g−1 DY) 2	(μg·g−1 TL) 2	(μg·g−1 DY) 2
D0	C	67.2 ± 4.1 a,b,A	46.9 ± 4.8 a,b,c,A	54.3 ± 4.4 a,b,A	37.8 ± 4.0 a,A	43.1 ± 2.1 a,A	30.0 ± 2.4 a,A
	HD1	55.8 ± 9.1 b,A	41.2 ± 5.7 b,c,A	46.1 ± 7.9 b,c,A	34.0 ± 7.4 a,b,A	35.1 ± 6.8 a,A	26.2 ± 4.5 a,A
	HD2	52.2 ± 9.7 b,A,B	36.8 ± 5.5 c,A,B	37.9 ± 8.4 c,A,B	26.8 ± 5.8 b,A	35.7 ± 9.3 a,A	24.7 ± 6.8 a,A
	NG1	73.2 ± 8.3 a,A	52.9 ± 3.8 a,A	50.0 ± 2.6 a,c,A	36.4 ± 3.3 a,b,A	35.1 ± 3.8 a,A	25.6 ± 4.3 a,A
	NG2	76 ± 10 a,A	50.0 ± 9.1 a,b,A	62.8 ± 8.1 a,A	42.5 ± 6.9 a,A	47.8 ± 8.2 a,A	32.4 ± 5.1 a,A
	VE	75.5 ± 8.3 a,A	54.9 ± 7.3 a,A	55.8 ± 7.8 a,b,A	42.9 ± 7.4 a,A	44.4 ± 9.8 a,A	30.6 ± 7.8 a,A
W1	C	65.9 ± 5.4 a,b,A	44.1 ± 3.4 a,A	42.6 ± 5.6 a,A,B	28.5 ± 3.4 a,b,A,B	33.2 ± 6.4 a,A,B	22.2 ± 4.0 a,A,B,C
	HD1	58.0 ± 7.6 a,b,A	38.7 ± 4.7 a,A,B	40.2 ± 8.3 a,A,B	26.7 ± 5.0 a,b,A,B	32.0 ± 6.6 a,A,B	21.3 ± 4.0 a,A,B
	HD2	61.8 ± 9.2 a,b,A	45.4 ± 8.4 a,A	43.0 ± 9.2 a,A	32.4 ± 7.1 a,b,A,B	33.9 ± 7.9 a,A,B	25.4 ± 3.0 a,A
	NG1	51.3 ± 7.2 b,B	35.8 ± 3.3 a,B	35.6 ± 3.7 a,A,B	23.9 ± 2.2 a,b,B	29.3 ± 3.6 a,A	20.2 ± 2.2 a,A,B
	NG2	70.2 ± 4.8 a,A,B	47.4 ± 3.9 a,A,B	49.6 ± 2.9 a,A,B	33.5 ± 2.1 a,A	39.8 ± 4.9 a,A,B	27.1 ± 3.0 a,A,B
	VE	53.0 ± 4.9 a,b,B	35.3 ± 2.3 a,B	31.5 ± 5.2 a,B	21.0 ± 2.5 b,B	26.9 ± 4.6 a,B	17.9 ± 2.8 a,B
W5	C	44.5 ± 1.9 a,B	30.5 ± 1.9 a,B	34.0 ± 5.4 a,B	23.4 ± 4.2 a,B	25.9 ± 3.2 a,B	17.8 ± 2.6 a,A,C
	HD1	36.6 ± 4.1 a,B	26.3 ± 4.2 a,B	26.1 ± 1.9 a,B	17.7 ± 1.3 a,B	20.6 ± 1.7 a,B	14.0 ± 1.2 a,B
	HD2	38.4 ± 2.5 a,B	28.0 ± 3.4 a,B	28.0 ± 3.3 a,B	19.2 ± 2.5 a,B	23.1 ± 2.4 a,B	15.8 ± 1.9 a,B
	NG1	36.0 ± 5.2 a,C	24.7 ± 3.6 a,C	27.4 ± 4.5 a,B	18.9 ± 3.2 a,B	23.4 ± 4.7 a,A	16.1 ± 3.4 a,B
	NG2	38.4 ± 4.0 a,B,C	26.2 ± 2.3 a,C	27.9 ± 3.1 a,C	19.1 ± 1.8 a,B	22.4 ± 2.4 a,C	15.4 ± 1.8 a,C
	VE	38.7 ± 3.6 a,B	26.5 ± 2.4 a,B	27.4 ± 2.5 a,B	18.8 ± 1.8 a,B	22.5 ± 2.0 a,B	15.4 ± 1.4 a,B
W9	C	47.3 ± 3.3 a,B	31.9 ± 4.8 a,B	33.4 ± 2.7 a,B	23.8 ± 2.0 a,B	27.9 ± 5.3 a,B	18.4 ± 4.3 a,B,C
	HD1	56.7 ± 2.5 a,A	39.0 ± 1.3 a,A	40.8 ± 1.5 a,A	28.1 ± 1.0 a,A	32.4 ± 3.4 a,A	22.1 ± 2.5 a,A
	HD2	62.2 ± 1.0 a,A	42.5 ± 1.9 a,A	47.8 ± 5.1 a,A	32.7 ± 4.3 a,A	31.1 ± 1.1 a,A,B	20.8 ± 1.4 a,A,B
	NG1	49.7 ± 7.3 a,B,C	34.0 ± 5.6 a,B,C	36.0 ± 4.4 a,A,B	24.6 ± 3.4 a,B	30.9 ± 1.2 a,A	21.2 ± 1.0 a,A,B
	NG2	52.5 ± 7.7 a,B	36.0 ± 6.3 a,B,C	37.7 ± 1.7 a,B,C	25.7 ± 1.9 a,B	30.2 ± 2.1 a,B,C	20.6 ± 1.4 a,B,C
	VE	48.2 ± 5.5 a,B	33.2 ± 4.2 a,B	34.0 ± 4.5 a,B	23.4 ± 3.6 a,B	27.8 ± 4.7 a,B	19.2 ± 3.6 a,B

¹ C: control group offered a commercial basal diet; HD1: group offered the basal diet supplemented with 0.75 g hesperidin per kg feed; HD2: group offered the basal diet supplemented with 1.5 g hesperidin per kg feed; NG1: group offered the basal diet supplemented with 0.75 g naringin per kg feed; NG2: group offered the basal diet supplemented with 1.5 g naringin per kg feed; VE: group offered the basal diet supplemented with 0.20 g a-tocopheryl acetate per kg feed. ² Lowercase superscript letters indicate comparisons between treatment groups within the same sampling time, whereas uppercase superscript letters indicate comparisons between sampling times within the same treatment group. Means sharing at least one common letter are not significantly different according to Tukey’s HSD test at the 5% significance level.

When evaluating the results presented in Table 9, no strong evidence was found to associate the intervention of either antioxidant with the total carotenoid content in egg yolk samples during the experimental period. In line with the results depicted from the yolk colour assessment (Section 3.3), neither the low nor high doses of added flavonoids or vitamin E acetate seemed to markedly affect total carotenoids per g total lipids or g dry yolk. The only exception that stood out was the case of the naringin dose of 1.5 g·kg⁻¹ feed (NG2), for which the levels of trans-canthaxanthin and lutein equivalents were higher on the first week of the study; lutein equivalents expressed per g dry yolk were significantly higher (p < 0.05) in NG2 than in VE, and trans-canthaxanthin equivalents expressed per g total lipids were significantly higher (p < 0.05) in NG2 than in NG1, although not significantly different (p ≥ 0.05) from the remaining groups.

In contrast, the amounts of carotenoids in the yolk samples from all treatment groups were reduced compared to D0, presenting at a minimum on W5, but were not statistically significant (p ≥ 0.05) irrespective of the supplementation. However, after four more weeks (W9) of the hens’ regime, the total carotenoid content was again elevated, especially when expressed as zeaxanthin equivalents, which reached the levels of W1. The lower total carotenoid concentration for yolks collected on W5 correlates with the highest (p < 0.05) L* values observed for all yolks of this time sampling, pointing out the reversed interplay between the carotenoid levels and yolk colour lightness. Likewise, the elevated carotenoid content in W1 samples comports with the trend followed by redness (a*) and yellowness (b*) for the same feeding period (Table 7), indicating the role of dietary carotenoids in the development of yolk colour. Gałazka-Czarnecka et al. [90] demonstrated that the red and yellow colour intensity of egg yolks increased accordingly to the content of lutein in the layer feed. Lutein affected yolk colour towards a yellow–orange pigmentation. They also confirmed that the colour concerning a* and b* parameters, but not L*, was significantly influenced by the ratio of lutein to zeaxanthin. Similarly, the supplementation of a lutein-rich marigold extract to laying hens increased the degree of redness and yellowness of egg yolks while significantly decreasing lightness [91].

Concerning canthaxanthin, 40 mg Carophyll^®Red were incorporated per kg of feed that was offered to all dietary groups for 9 weeks, as listed in Table 1. Carophyll^®Red is a common additive pigment in poultry feeds of synthetic origin, including canthaxanthin as its main ingredient, and it serves as a means for the pigmentation of egg yolks [92,93]. Supplemented canthaxanthin, either naturally isolated or industrially produced, effectively enhances the reddish yolk colour [94] and promotes the antioxidant status with efficient canthaxanthin transfer to the yolk [93,95]. Interestingly, the fact that our results did not provide evidence for an increase in canthaxanthin equivalents during supplementation of its synthetic form included in the basal ration possibly indicates the low transferability to egg yolk at the corresponding initial concentration added in the hens’ basal ration.

3.5. Study Limitations

When interpreting and generalising the reported findings, it is important to consider the experimental limitations of the present study. In particular, the study was conducted exclusively with Lohmann Brown-Classic laying hens at a specific age (54 weeks at the onset), and caution should therefore be exercised when attempting to extrapolate the present findings to other commercial breeds, hybrid lines, or different stages of laying hen production. Individual housing of the hens, whilst ensuring precise dietary intake control per experimental unit, differs from the conditions of standard commercial egg production, where hens are typically maintained in group or cage systems. It is also noteworthy that inherent inter-individual differences in the daily feed intake and metabolic rate among hens may introduce a degree of biological variability that cannot be fully eliminated; accordingly, the actual daily exposure to the supplemented compounds may not have been entirely uniform across experimental units within each treatment group. Furthermore, supplementation was evaluated at only two dose levels per flavonoid (0.75 and 1.50 g·kg⁻¹ feed), which precludes a formal dose–response analysis across a wider range of dietary concentrations.

Another limitation concerns the composition of the basal diet, which already contained the antioxidants butylated hydroxyanisole and vitamin E, as well as the carotenoid pigment Carophyll^®Red. This background presence of antioxidants may have produced a ceiling effect, potentially masking differences in oxidative susceptibility, carotenoid content, or colour parameters between treatment groups. It should also be noted that the dietary administration of isolated hesperidin and naringin as pure supplemented compounds does not replicate a realistic feeding scenario, where hens are offered a ration naturally incorporating citrus agro-industrial by-products, such as dried orange or lemon pulp, which contain these flavonoids as integral components of a complex phytochemical matrix; accordingly, the potential contribution of the intact by-product matrix and its associated bioactive constituents to yolk quality may differ from the outcomes observed with isolated flavonoid supplementation. Finally, the nine-week experimental period is relatively short for a comprehensive long-term assessment; however, this duration was selected to ensure that all subjects remained within the same defined production phase throughout the study (54–63 weeks of age), thereby minimising age-related confounding effects on yolk composition, while the persistence of the reported improvements in yolk lipid quality characteristics beyond this timeframe remains to be confirmed.

Additionally, two further limitations warrant mention regarding aspects of storage-related egg quality that were not addressed in the present study. First, the oxidative stability of yolk lipids during storage was not assessed. Whilst the calculated peroxidisability index (PI) provides an indirect estimate of the yolks’ susceptibility to lipid oxidation, it does not substitute for direct oxidative stability assays, such as malondialdehyde or peroxide value determinations, performed on eggs held under refrigerated or ambient storage conditions. Future studies should therefore incorporate such storage trials to establish whether the compositional differences observed at collection translate into an actual improvement in oxidative stability over time. Second, no broader shelf-life quality data were collected, such as changes in the Haugh unit, yolk index, albumen pH, or weight loss during storage, which are the parameters conventionally used to define commercial egg shelf-life. Future research may include a comprehensive shelf-life evaluation of eggs from flavonoid-supplemented hens under commercially relevant storage conditions to establish whether the reported improvements in yolk lipid quality confer a practical benefit for the food market.

4. Conclusions

This research investigated the potential of natural flavonoids, i.e., hesperidin and naringin, incorporated in a basal layers’ feeds, with the intention to favourably modulate the colour and lipid profile of egg yolks. The effect of dietary intervention on the lipid quality of the produced yolks was monitored throughout a 9-week period with four consequent time points.

Triglycerides were the dominant neutral lipid class, and phosphatidylcholine the main polar lipid class in the lipid fractions of the studied yolks. A significant finding emerged from the present work was that introducing the hens to a standardised regime for merely one week can alleviate possible inhomogeneity concerning the total fat concentration in yolks. On the other hand, the treatment for 9 weeks with natural flavonoids did not significantly alter the amount of total fat or total neutral and total polar lipids of egg yolks, except from the treatment with 0.75 g hesperidin per kg feed (HD1) for one week, which seemed to alleviate (p < 0.05) the total fat content in yolk. An increase (p < 0.05) in phosphatidylcholine was observed at the end of the intervention for all sample groups, that signified a nutritional improvement of yolk lipid profile.

Regarding yolk fatty acids, palmitic and stearic acids were the predominant SFAs, while oleic, palmitoleic, and vaccenic acids contributed the most to the total MUFAs. Linoleic acid was dominant among n-6 PUFAs, as well as a-linolenic and clupanodonic (DPA) acids and DHA among n-3 PUFAs. The fatty acid composition was mainly affected by the major ingredients of the basal ration, based on maize, wheat, and soybean, and not by the supplemented antioxidants. Of note, the oleic, palmitic, and DHA fatty acids presented both upward and downward trends, which could be perceived as layer-ageing-based fluctuations. Moreover, the inclusion of either hesperidin or naringin in hens’ diets for nine weeks seemed to enhance the MUFA/PUFA ratio of total fatty acids in total lipids of yolks. From a nutritional point of view, the shift in the n-6/n-3 ratio to lower values and the significantly decreased peroxidisability index at the end of dietary intervention for all cases of treatment, along with low values for atherogenic and thrombogenic indices and combined with the oxidative stability of yolk lipophilic constituents, enhance the potential of lipid-rich yolks to contribute to a healthy diet. Collectively, these findings substantiate the potential of dietary hesperidin and naringin as natural, sustainable functional feed additives for the production of eggs with improved yolk lipid nutritional quality.

Concerning yolk colour, contingent upon the total carotenoid content, the adaptation period in which the hens can adjust to a newly introduced standard diet can be narrowed to one week rather than the time frame of two-to-four weeks, which is usually allowed for the production of eggs with a stable yolk colour. Neither the low nor high dose of added hesperidin or naringin in hens’ diets for nine weeks seemed to affect the amount of yolk total carotenoids in the examined samples. Overall, yolks laid from hens that were offered different supplements or the control feed were not distinguished on a colour basis. On the other hand, colour was markedly influenced by the feeding duration with the basal ration rather than the inclusion of antioxidants in the hens’ regime. Moreover, the lower total carotenoid concentration in yolks was negatively correlated with a lighter colour. In contrast, the incremental change in total carotenoids seemed to enhance the development of a red and yellow yolk colour. Interestingly, as a side inference made from our results, the incorporation of 40 mg Carophyll^®Red per kg of a basal ration offered to layers for a nine-week duration seemed to be ineffective in raising the corresponding total carotenoid content of canthaxanthin equivalents in egg yolks. Of particular practical relevance is the finding that a single week of adaptation to a standardised dietary regime proved sufficient to homogenise both yolk’s total fat content and colour profile, a result that may bear meaningful implications for the design and interpretation of future dietary intervention studies in laying hens.

Future research efforts should focus on extending the present analytical framework to include LC-MS/MS-based individual carotenoid profiling in freshly collected and refrigerated-stored egg yolks in order to provide a more comprehensive evaluation of the antioxidant efficacy of the studied flavonoids. Studies employing whole-citrus agro-industrial by-products, such as dried orange or lemon pulp and peel, rather than isolated compounds would allow for the investigation of potential matrix effects and synergistic interactions between phytochemical constituents that may not be captured when pure flavonoids are supplemented alone. Additionally, expanding the experimental model to encompass different hen breeds, wider age ranges, and longer supplementation periods, complemented by in vivo assessments of flavonoid bioavailability and metabolic fate, would contribute to a more mechanistic and generalisable understanding of the dose-dependent effects of citrus flavonoid-enriched diets on the nutritional quality of eggs.