Fatty Acid Profile and Oxidative Stability of Layers’ Egg Yolk as Affected by Dietary Supplementation with Fresh Purslane and Addition of Aromatic Plant Essential Oils to Drinking Water

Abstract

The objective of this study was to investigate the possibility of producing eggs enriched in omega-3 polyunsaturated fatty acids (PUFA) while also increasing the oxidative stability of egg yolk. Here, 432 68-week-old Isa Brown layers were split into two groups of 216, consisting of three subgroups of 72 each. Group C was fed a standard corn–soybean meal diet, while Group P received the same basic diet with an additional 24 g of fresh purslane. In the drinking water of hens of the three subgroups of group C and the three subgroups of group P, either no essential oil (C-0, P-0) or 100 ppm of oregano essential oil (C-ORE, P-ORE) or 100 ppm of a blend of oregano, sage, and fennel essential oils (C-BLEND, P-BLEND) was administered. The purslane supplementation resulted in increased egg weight, improved yolk color, higher levels of α-linolenic and linoleic acids, and an improved omega-6/omega-3 nutritional index. The addition of essential oils resulted in a significant increase in the oxidative stability of the egg yolk, with the BLEND being the most effective. In conclusion, the combined administration of fresh purslane and essential oils of aromatic plants could be suggested for the production of eggs enriched in omega-3 PUFA, protected with natural antioxidants of plant origin.

1. Introduction

It is well documented that enriching the human diet with high levels of polyunsaturated fatty acids (PUFA) is associated with a reduced risk of early cardiovascular disease [1]. Investigating the effect of PUFA on cholesterol fractions, it has been found that these fatty acids cause a decrease in low-density lipoprotein-cholesterol (LDL-C) and an increase in high-density lipoprotein-cholesterol (HDL-C) [2]. Dietary supplementation with PUFA leads to an increased proportion of these fatty acids in plasma lipoproteins, cell and tissue lipids [3]. It has been suggested that an adequate intake level for total omega-3 fatty acids of 1.1–1.6 g/day for humans can potentially reduce the risk of diseases, such as cardiovascular disease [4].

Atherosclerosis might be reduced or slowed by long-chain omega-3 PUFA by modifying the risk factor profile [5]. Daily supplementation of healthy people with omega-3 PUFA may have protective and antioxidant effects on LDL and is supportive of the global recommendations for long-chain omega-3 PUFA for the primary prevention of coronary disease [6]. Yang et al. [7] demonstrated that a low omega-6/omega-3 PUFA ratio (1:1 up to 5:1) had a beneficial effect on cardiovascular risk factors by enhancing favorable lipid profiles, having anti-inflammatory and anti-oxidative stress effects, and improving endothelial function.

Omega-3 α-linolenic acid (ALA) is an essential fatty acid that cannot be produced by humans and, therefore, must be ingested. This fatty acid plays a crucial role in human growth, development, and disease prevention [8]. Additionally, it is the precursor to longer-chain omega-3 fatty acids such as eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), which are generally found in marine organisms [9].

Nowadays, there is an increased interest and demand for functional foods for human consumption that provide various benefits and help prevent nutrition-related diseases. This is achieved by changing the composition of foods to include certain ingredients that are beneficial to health [10,11].

In this sense, eggs, as a conventional food, can be enriched with certain nutrients through dietary manipulation to be promoted as functional foods. Egg production is also an economically sustainable activity because it offers a moderate calorie source and a high-quality protein product at a low economic cost [12]. Functional eggs are primarily enriched with omega-3 PUFA or low-cholesterol content through modification of the layer hen’s diet.

Consumers have become increasingly concerned about the amount and type of animal-based foods in recent years due to their high saturated fatty acid (SFA) content and low polyunsaturated fatty acid content, which have been linked to health problems. Increasing the functional and nutritional value of egg lipid fractions may have positive effects on human health [13]. However, modifying animal diets to increase the degree of unsaturation of tissue fatty acids to meet dietary guidelines increases the risk of oxidative deterioration.

Enriching animal products such as eggs with omega-3 PUFA can be achieved with fish by-products such as fish oil and fishmeal. However, this is not a sustainable solution for conserving the Earth’s precious and finite resources [14]. Additionally, the addition of fish meal or fish oil to the feed may have a negative effect on the sensory quality of eggs, especially with regard to consumer acceptance [15].

In recent years, efforts have been made in egg-laying poultry to increase the omega-3 PUFA content of eggs through alternative and sustainable plant-based raw materials. These production practices for functional foods enriched with fatty acids should also take care of their oxidative stability during storage by utilizing additional antioxidants from sustainable sources.

Purslane (Portulaca oleracea L.) is one of the most widespread herbaceous plants in the world and has a long history of use for human food, animal feed, and medical purposes. A nutritional characterization of purslane accessions conducted by Ezekwe et al. [16] revealed that, despite its genetic diversity, purslane remains one of the most abundant terrestrial sources of omega-3 fatty acids, which are potentially beneficial for humans and animals. These benefits include the prevention and treatment of cardiovascular disease, some autoimmune diseases, diabetes, certain types of cancer, and their significant role in neuronal development. According to Simopoulos et al. [17] and Simopoulos [18], 100 g of fresh purslane leaves contain approximately 0.3–0.4 g ALA, 1.0 mg EPA, 0.0122 g α-tocopherol (vitamin E), 0.0266 g ascorbic acid (vitamin C), 0.0019 g β-carotene (a precursor of vitamin A), and 0.0148 g glutathione. This is a remarkable amount of EPA for land-based vegetable sources. Purslane also contains DHA and DPA [19], vitamins (primarily vitamins A and C, and lesser amounts of vitamin B and carotenoids), as well as dietary minerals such as magnesium, calcium, potassium, and iron [20]. As ALA can be converted into DHA and EPA, the inclusion of ALA in feed and the metabolic efficiency of hens in bioconversion of ALA plays an important role in long-chain (EPA, DPA, DHA) fatty acid enrichment in poultry tissues and eggs [21]. Additionally, purslane contains less cholesterol than fish oils.

Essential oils are important secondary metabolites of aromatic plants [22]. Oregano (Origanum vulgare) and sage (Salvia officinalis) are aromatic plants with ornamental, culinary, and phytotherapeutic uses all over the world. In Europe, they are traditionally used in the southern countries, particularly in the Mediterranean area [23].

The essential oils of aromatic plants have been widely used in animal nutrition as additives to replace antibiotic growth promoters. Within the Labiatae plant family, thyme (Thymus vulgaris), oregano, and sage are the most popular members, as they contain phenolic compounds such as carvacrol, eugenol, thymol, p-cymene, and γ-terpinene, which possess strong antibacterial and antioxidant properties [24]. The essential oil derived from oregano is known to possess antimicrobial, antifungal, insecticidal, and antioxidant properties [25]. Regarding antibacterial properties, cultivated sage had a 2.37% extract concentration, with basic components being cis-thujone (33.80%) and trans-thujone (6.97%) [26].

Fennel (Foeniculum vulgare) is also a plant whose essential oils are exploited as strong antioxidants and antimicrobials due to the presence of compounds, including anethole and polyphenols [27].

Dietary herbal additives can manipulate serum cholesterol due to their total phenolic ingredients [28]. Functional foods can be made by transferring the components of active herbal ingredients to egg yolks and enhancing them with phytonutrients [29]. Lipid oxidation accelerates quality deterioration in muscle-based foods (fish, red meat, and poultry), resulting in off-odors/flavors, color problems, texture defects, and safety concerns [30]. Malondialdehyde (MDA) is one of the most widely studied degradation products of lipid hydroperoxides and serves as a marker of lipid peroxidation [31]. The antioxidant components of these active plant compounds are thought to protect lipids from oxidation, thereby preventing lipid peroxidation and improving the oxidative stability of fat and egg yolk, as well as their storage quality [32].

Many studies have previously examined the antioxidant properties of aromatic plants and their effect on the oxidative stability of egg yolks in feed [26,33,34,35]. However, limited studies have been conducted on essential oils of aromatic plants supplemented in water for their antioxidant activity.

The objective of this study was to investigate the possibility of producing eggs enriched in omega-3 PUFA by adding fresh leaves and stems of the plant species purslane (Portulaca oleracea L.) while also increasing the oxidative stability of egg yolk by adding essential oils of aromatic plants derived from Origanum vulgare (oregano), Salvia officinalis (sage), and Foeniculum vulgare (fennel seeds) to the drinking water of laying hens.

2. Materials and Methods

2.1. Housing

The experiment was conducted at the poultry shed of the Aristotle University of Thessaloniki Farm located in Thermi, Thessaloniki (Latitude: 40.540238861182964° N, Longitude: 22.99927815920721° E, WGS 84). The poultry shed’s ventilation system, consisting of two side vents and two roof vents, is automated and facilitates effective airflow regulation. This system ensures a constant supply of fresh air, removes stale air and odors, and promotes a healthy and comfortable environment for the laying hens, enhancing their well-being, productivity, and overall conditions. The vent openings are automatically adjusted by motorized actuators controlled by a centralized system, which takes into account temperature, humidity, and air quality, thereby maintaining stable environmental conditions and optimizing airflow. The poultry shed is equipped with enriched cages housing six hens each, which meet European Union welfare standards. The dimensions of each cage are 90 cm (width) × 60 cm (depth) × 50 cm (height), which adequately ensures the minimum requirement of 750 cm² surface area per hen. Each cage is equipped with a linear feeder, a nipple-type drinker, a private nest area for laying eggs, a litter pad for pecking and scratching, and perches for birds to rest on.

The experiment lasted 30 days. The lighting of the building during the experiment amounted to 16 h per day. The indoor temperature averaged 24.5 ± 2.4 °C, while the relative humidity ranged between 55 and 58%.

2.2. Birds

Four hundred and thirty-two (432) 68-week-old Isa Brown layers were used in the experiment and were divided into 6 treatments of 72 hens each. Each treatment had 12 replicates (cages) of 6 hens each.

The hens were weighed at the beginning and at the end of the experiment, with the cage of 6 hens constituting the experimental unit. The egg production rate and daily mass of eggs produced per hen (egg production rate × egg weight) were determined at the end of the experiment.

During the last three days of the experiment, 144 eggs were collected each day (2 eggs from each cage). These eggs were used to determine egg quality characteristics, the fatty acid profile of the egg yolk, and the oxidative stability of the egg yolk through the MDA method.

2.3. Diets

During the 30 days of the experiment, the hens were fed a standard corn–soybean meal diet containing 2750 kcal ME, 160 g crude protein, and 44 g ether extract per kg (

Table 1. Ingredients and nutrient content of the experimental control diet.

	Control Diet
Ingredients (%)
Corn	63.25
Soybean meal (45% crude protein)	23.90
Soybean oil	1.70
Limestone (calcium carbonate)	9.50
Monocalcium phosphate	0.80
Salt	0.30
Sodium carbonate	0.30
DL-methionine	0.15
Vitamin and mineral premix 1	0.10
Total	100.00
Nutrient content
Metabolizable energy (kcal/kg)	2750.00
Dry matter (%)	89.02
Crude protein (%)	16.00
Digestible protein (%)	13.32
Crude fat (%)	4.40
Crude fiber (%)	2.83
Total lysine (%)	0.82
Total methionine (%)	0.41
Ash (%)	12.73
Calcium (%)	3.92
Total phosphorus (%)	0.51
Available phosphorus (%)	0.38

¹ Supplying per kg of feed: 10,000 IU vitamin A; 3000 IU vitamin D3; 30 mg vitamin E; 2.5 mg vitamin K3; 3 mg thiamine; 5 mg riboflavin; 5.4 mg nicotinamide; 4 mg pyridoxine; 20 mcg vitamin B12; 50 mg pantothenic acid; 0.5 mg folic acid; 150 mcg biotin; 1045 mg choline; 30 mg Fe; 10 mg Cu; 30 mg Zn; 50 mg Mn; 2 mg I; 0.2 mg Se; 1.5 mg Mo, and phytase 500 FTU.

). The 432 hens were split into 2 groups of 216, consisting of 3 subgroups of 72 each. The hens of the first group (group C) were fed daily with an average amount of ration of 120 g/day, an amount which, based on the laying rate and weight of the hens, is considered sufficient to support the normal nutritional requirements of the hens for maintenance and laying. The hens of the second group (group P) were given the same basic control diet and an additional 24 g of finely chopped fresh leaves and stems of the plant species purslane (Portulaca oleraceae) collected daily from the surrounding area of the poultry house. In the drinking water of hens of the 3 subgroups of group C and the 3 subgroups of group P, either no essential oil (C-0, P-0, respectively) or 100 ppm of oregano essential oil (C-ORE, P-ORE, respectively) or 100 ppm of a blend of oregano, sage, and fennel essential oils (C-BLEND, P-BLEND, respectively) was administered. Thus, 6 nutritional treatments emerged. The oregano essential oil used is marketed as a commercial formulation by the company “Ecopharm Hellas” under the trade name “Ecodiar Liquid”. It is natural oregano essential oil in liquid form (5% v/v concentration) intended for livestock use and administered through drinking water. The blend of essential oils is released as a non-patented commercial formulation by the company “Dioscurides”. It is a mixture of natural essential oils of oregano, sage, and fennel in a ratio of 4:1:1, respectively (6% v/v concentration). Like the previous formulation, it is administered to the animals through drinking water. The formulations mentioned above, produced by the two companies operating in Northern Greece, contain soy lecithin. Soy lecithin is used as an emulsifier to enhance the solubility of essential oils and facilitate their absorption through the intestinal tract.

Fresh and clean, healthy water was supplied from three independent tanks with a float, where essential oil was mixed daily with a proportionate amount of water to reach a concentration of 100 ppm. Each hen consumed approximately 0.5 L of water daily, which is approximately four times the amount of dry matter consumed through their diet.

2.4. Egg Collection and Determinations

2.4.1. Egg Quality Traits

On the 28th day of the experiment, a total of 144 eggs were collected, i.e., 6 treatments × 12 replicates (cages)/treatment × 2 eggs/cage, which were used to determine their quality characteristics.

Quality traits measured/estimated were egg weight; longitudinal and transverse axes; specific gravity; Haugh units; yolk weight, diameter, height, pH, and color; albumen weight, height, and pH; and shell weight, thickness, and hardness.

2.4.2. Materials and Reagents

The reagents used in the analyses for fatty acid profile and oxidative stability of egg yolk included: n-hexane pesticide grade (ChemLab, Zedelgem, Belgium), sodium hydroxide p.a. (Honeywell Fluka, München, Germany), methanol (VWR chemicals, Radnor, PA, USA), sodium bisulfate pure (Fluca München, Germany), 2-thiobarbituric acid (Sigma-Aldrich, Darmstadt, Germany), butylated hydroxytoluene (Sigma Aldrich, Darmstadt, Germany), trichloroacetic acid (Merck, Darmstadt, Germany), ferrous sulfate p.a. (Merck Darmstadt, Germany), sodium sulfate p.a. (Honeywell Fluka, München, Germany), chloroform (Merck Darmstadt, Germany), ascorbic acid p.a. (Panreac, Barcelona, Spain) and 1,1,3,3 tetraethoxypropane (Sigma Aldrich, Darmstadt, Germany). A 37-component mixture of fatty acids, methyl esters, and FAME mix C₆–C₂₄ (SIGMA 18919-1AMP, certified reference material) was used as the reference standard.

2.4.3. Fatty Acid Profile of Egg Yolk

On the 29th day, an additional 144 eggs were collected using the same method as described above. These eggs were then placed in a freezer at −20 °C to be subsequently used for the determination of their yolk fatty acid profile. The eggs were then thawed at room temperature, broken, the yolks were separated, and the adhering albumen was removed by rolling on a paper towel. Yolk pools were prepared from 2 eggs from each cage and mixed with a wire whisk, resulting in 72 samples.

Lipid extraction of egg yolk was performed using Folch procedure [36]. Fatty acid methyl esters (FAMEs) were prepared and determined by gas chromatography (GC). Identification of FAMEs involved comparing their retention times with the retention times of the reference standards, and the results were expressed as a percentage (%) of the total fatty acids present in the sample.

2.4.4. Oxidative Stability of Egg Yolk

On the 30th day, another batch of 144 eggs was collected using the same method as previously described. These eggs were stored in a freezer at −20 °C for the subsequent determination of the oxidative stability of the egg yolk. The same procedure as described for the fatty acid profile determination was followed to generate 72 samples for analysis.

Lipid oxidation and iron-induced lipid oxidation of egg yolks were determined on the basis of the formation of MDA using a selective third-order derivative spectrophotometric method [37].

Susceptibility of eggs to iron-induced lipid oxidation was evaluated according to a slightly modified method [38]. In brief, yolk samples were homogenized, and four 1 g sub-samples from each yolk sample were weighed into 50 mL centrifuge tubes. Then, 1.5 mL of a solution containing 1.138 mM ferrous sulfate and 0.368 mM ascorbic acid was added to three of the sub-samples, and they were incubated at 37 °C for either 50, 100, or 150 min. Following incubation, all three iron-induced sub-samples, along with the 4th non-induced sub-sample, were immediately submitted to MDA assay for assessing the extent of lipid oxidation.

2.4.5. Determination of MDA

Determination of MDA, the compound used as an index of lipid peroxidation, was carried out by a third-order derivative method slightly modified to suit egg yolk analysis [37,39]. In brief, yolk samples were homogenized (Polytron homogenizer, PCU, Malters, Switzerland) in the presence of 8 mL of aqueous trichloroacetic acid (5% w/v) and 5 mL of butylated hydroxytoluene in hexane (0.8% w/v) and the mixture was centrifuged. The top layer was discarded, and the bottom aqueous layer was transferred to a volumetric flask (10 mL). Diluted to 10 mL with an aqueous solution of trichloroacetic acid (5% w/v), a 2.5 mL aliquot from this solution was mixed with 1.5 mL of aqueous 2-thiobarbituric acid (0.8% w/v). The mixture was further incubated at 70 °C for 30 min. After incubation, the mixture was cooled in a cold-water bath and submitted to conventional spectrophotometry. Lipid oxidation was expressed as nanograms of MDA per gram of yolk sample.

2.5. Statistical Analysis

Data were subjected to analysis of variance using the SPSS 20 statistical package [40]. A significance level of p < 0.05 was used. Differences between means were tested with Duncan’s test.

The quality characteristics of the eggs and the fatty acid profiles were analyzed using a factorial experimental design (2 × 3) that included two factors: the type of diet (C or P) and the presence or absence of essential oils in the water (0, ORE, or BLEND). The main effects of both the diet and essential oils were determined, as well as their interaction.

The data related to the oxidative stability, as determined by the MDA method, were processed using a 2 × 3 × 4 factorial experimental design that included three factors: the type of diet (C or P), the presence or absence of essential oils in the water (0, ORE, or BLEND), and the duration of the incubation period (0, 50, 100, or 150 min). The main effects of the diet, essential oils, and time were determined, as well as their two-way and three-way interactions.

3. Results

3.1. Egg Production Rate and Egg Quality Traits

No mortality was observed throughout the experiment. Furthermore, no significant difference was observed between the treatments in terms of the change in the mean body weight of the hens between initial and final weighing (1948 g vs. 1934 g, respectively).

Table 2. Effect of purslane supplementation and essential oil addition on egg production rate and egg quality traits.

Variable	Egg Production Rate	Egg Weight	Egg Mass Production	Longitudinal Axis	Transverse Axis	Specific Gravity
	(%)	(g)	(g/hen/day)	(mm)	(mm)	(g/cm3)
Diet (D)
C	68.5	68.5 b	46.8 b	59.1 b	48.7 b	1.077
P	68.4	70.7 a	48.4 a	62.5 a	50.0 a	1.078
SEM	0.253	0.494	0.374	0.827	0.297	0.004
Essential Oils (EO)
0	68.4	69.7	47.8	60.6	49.4	1.080
ORE	68.2	69.8	47.5	60.9	49.2	1.075
BLEND	68.9	69.4	47.7	60.9	49.6	1.076
SEM	0.208	0.120	0.088	0.100	0.115	0.002
D × EO
C-0	68.5	68.2 b	46.7 b	58.9 b	49.0 b	1.080
C-ORE	68.2	68.8 b	46.8 b	59.2 b	48.4 b	1.074
C-BLEND	68.8	68.6 b	47.0 b	59.1 b	48.8 b	1.077
P-0	68.3	71.1 a	48.8 a	62.3 a	49.8 a	1.079
P-ORE	68.1	70.7 a	48.1 a	62.6 a	49.9 a	1.076
P-BLEND	68.9	70.2 a	48.4 a	62.7 a	50.3 a	1.075
SEM	0.203	0.327	0.261	0.759	0.192	0.002
p-value
D	0.372	0.023	0.028	0.039	0.018	0.817
EO	0.277	0.090	0.240	0.428	0.317	0.587
D × EO	0.130	0.038	0.042	0.032	0.036	0.256
Variable	Haugh Units	Yolk Weight	Yolk Diameter	Yolk Height	Yolk pH	Yolk Color
		(g)	(mm)	(mm)		(DSM Scale)
Diet (D)
C	62.70	19.8	42.6	18.7	6.26	9.1 b
P	64.04	20.3	43.3	18.3	6.28	10.1 a
SEM	0.451	0.162	0.256	0.134	0.024	0.231
Essential Oils (EO)
0	63.33	20.0	43.6	18.6	6.27	9.5
ORE	62.18	20.2	42.7	18.9	6.31	9.6
BLEND	64.60	19.9	42.7	18.1	6.24	9.7
SEM	0.699	0.088	0.300	0.233	0.020	0.058
D × EO
C-0	62.45	19.4	42.7	19.1	6.25	9.2 b
C-ORE	60.73	20.0	41.4	18.8	6.32	9.0 b
C-BLEND	64.91	19.9	43.7	18.1	6.21	9.1 b
P-0	64.21	20.5	44.4	18.0	6.29	9.8 a
P-ORE	63.62	20.4	43.9	18.9	6.29	10.1 a
P-BLEND	64.29	19.9	41.6	18.0	6.26	10.3 a
SEM	1.187	0.362	0.852	0.306	0.031	0.137
p-value
D	0.412	0.068	0.317	0.343	0.849	0.035
EO	0.435	0.121	0.695	0.258	0.712	0.329
D × EO	0.347	0.062	0.512	0.480	0.743	0.027
Variable	Albumen Weight	Albumen Height	Albumen pH	Shell Weight	Shell Thickness	Shell Hardness
	(g)	(mm)		(g)	(mm)	(N)
Diet (D)
C	41.9	7.5	8.75	6.7	0.45	37.6
P	43.3	7.4	8.74	6.9	0.43	36.3
SEM	0.433	0.040	0.032	0.063	0.016	0.434
Essential Oils (EO)
0	42.7	7.5	8.69	6.9	0.44	37.3
ORE	42.4	7.6	8.83	7.0	0.45	38.3
BLEND	42.8	7.3	8.75	6.5	0.44	35.4
SEM	0.120	0.088	0.041	0.153	0.006	0.850
D × EO
C-0	42.0	7.5	8.66	6.6	0.45	38.3
C-ORE	41.7	7.7	8.84	6.9	0.45	39.2
C-BLEND	42.0	7.3	8.76	6.5	0.44	35.4
P-0	43.3	7.5	8.71	7.1	0.42	36.3
P-ORE	43.1	7.5	8.81	7.0	0.44	37.3
P-BLEND	43.6	7.2	8.74	6.5	0.43	35.3
SEM	0.516	0.142	0.052	0.199	0.009	1.247
p-value
D	0.072	0.856	0.912	0.574	0.833	0.517
EO	0.133	0.647	0.737	0.483	0.819	0.344
D × EO	0.057	0.494	0.549	0.639	0.754	0.612

C: control diet; P: diet containing leaves and stems of purslane; C-0, P-0: no addition of essential oils in drinking water; C-ORE, P-ORE: addition of 100 ppm of oregano essential oil in drinking water; C-BLEND, P-BLEND: addition of 100 ppm of blend of oregano, sage, and fennel essential oils in drinking water; SEM: standard error of the means; ^a,b means within a column with different superscripts inside each factor differ significantly at p < 0.05.

shows the effect of purslane supplementation and essential oil addition on egg production rate and egg quality traits. According to the table, the addition of purslane to the ration or essential oils to the drinking water of hens did not significantly affect the rate of egg production.

Treatments supplemented with fresh, finely chopped purslane leaves and stems showed a significant (p < 0.05) increase in egg weight compared to those without supplementation. The addition of essential oils did not have a significant effect on egg weight. Egg mass production per hen per day significantly (p < 0.05) increased in the purslane treatments, similar to the observed increase in egg weight. Additionally, the length of the longitudinal and transverse axes of the eggs of hens treated with purslane significantly (p < 0.05) increased compared to those of hens that did not receive purslane. In contrast, the addition of essential oils did not have a statistically significant effect on these parameters. Dietary supplementation with purslane also resulted in a significant (p < 0.05) improvement in yolk color, whereas providing essential oils through water did not produce a similar outcome.

The supplementation of purslane and the addition of essential oils to the hens’ drinking water did not have a significant impact on the other egg quality characteristics, including specific gravity; Haugh units; yolk weight, diameter, height, and pH; albumen weight, height, and pH; and shell weight, thickness, and hardness.

3.2. Fatty Acid Profile of Egg Yolk

From

Table 3. Fatty acid (FA) profile of egg yolk.

FA (% of Total FA)	C14:0	C16:0	C16:1n-7	C18:0	C18:1n-9	C18:1n-7
	(Myristic)	(Palmitic)	(Palmitoleic)	(Stearic)	(Oleic)	(Vaccenic)
Diet (D)
C	0.40	28.29	3.55	9.32 a	39.47	3.66
P	0.35	27.76	3.10	7.26 b	39.14	3.79
SEM	0.014	0.184	0.130	0.395	0.173	0.040
Essential Oils (EO)
0	0.39	27.87	3.38	8.18	39.58	3.58
ORE	0.34	27.61	3.19	8.44	39.67	3.70
BLEND	0.39	28.60	3.41	8.26	38.67	3.89
SEM	0.017	0.296	0.069	0.077	0.319	0.090
D × EO
C-0	0.43	28.17	3.63	9.18 a	39.74	3.56
C-ORE	0.34	27.94	3.34	9.46 a	39.83	3.54
C-BLEND	0.42	28.75	3.67	9.33 a	38.84	3.88
P-0	0.35	27.57	3.12	7.18 b	39.41	3.60
P-ORE	0.33	27.28	3.04	7.41 b	39.51	3.86
P-BLEND	0.36	28.44	3.15	7.18 b	38.49	3.90
SEM	0.029	0.383	0.190	0.405	0.356	0.121
p-value
D	0.612	0.654	0.411	0.007	0.797	0.487
EO	0.593	0.599	0.519	0.378	0.676	0.313
D × EO	0.585	0.613	0.427	0.008	0.387	0.421
FA (% of Total FA)	C18:2n-6	C18:3n-3	C20:1n-9	C20:4n-6	C22:4n-6	C22:6n-3
	(Linoleic, LA)	(α-Linolenic, ALA)	(Gondoic)	(Arachidonic)	(Adrenic)	(Docosahexaenoic, DHA)
Diet (D)
C	10.69 b	0.18 b	0.18	2.64	0.27	1.64
P	12.74 a	0.85 a	0.19	2.84	0.29	1.65
SEM	0.463	0.123	0.004	0.059	0.009	0.006
Essential Oils (EO)
0	11.79	0.48	0.18	2.75	0.29	1.63
ORE	11.97	0.54	0.19	2.67	0.27	1.60
BLEND	11.39	0.50	0.21	2.82	0.30	1.69
SEM	0.171	0.027	0.009	0.043	0.009	0.026
D × EO
C-0	10.60 b	0.18 b	0.18	2.57	0.27	1.62
C-ORE	11.06 b	0.17 b	0.19	2.52	0.26	1.59
C-BLEND	10.41 b	0.18 b	0.20	2.84	0.29	1.70
P-0	12.97 a	0.77 a	0.17	2.92	0.31	1.63
P-ORE	12.88 a	0.96 a	0.19	2.81	0.27	1.61
P-BLEND	12.36 a	0.81 a	0.21	2.79	0.30	1.68
SEM	0.303	0.116	0.013	0.131	0.015	0.034
p-value
D	0.008	<0.001	0.873	0.085	0.713	0.878
EO	0.135	0.221	0.855	0.150	0.694	0.139
D × EO	0.007	<0.001	0.843	0.079	0.633	0.216

C: control diet; P: diet containing leaves and stems of purslane; C-0, P-0: no addition of essential oils in drinking water; C-ORE, P-ORE: addition of 100 ppm of oregano essential oil in drinking water; C-BLEND, P-BLEND: addition of 100 ppm of blend of oregano, sage, and fennel essential oils in drinking water; SEM: standard error of the means; ^a,b means within a column with different superscripts inside each factor differ significantly at p < 0.05.

, it can be concluded that dietary supplementation with purslane significantly modified the content of saturated and polyunsaturated fatty acids in egg yolk. Specifically, treatments receiving purslane had significantly (p < 0.05) higher levels of polyunsaturated omega-3 fatty acid α-linolenic (ALA), as well as omega-6 fatty acid linoleic (LA), compared to those not receiving purslane. Correspondingly, the content of stearic acid in the egg yolk decreased significantly (p < 0.05) in the purslane groups. The administration of oregano essential oil (ORE) or the blend of oregano, sage, and fennel essential oils (BLEND) in the drinking water of the layers did not result in significant differences. Furthermore, statistical analysis showed no significant differences among the treatments regarding the remaining fatty acids.

Table 4. Grouped fatty acids (%) in egg yolk.

Grouped FA (%)	SFA	MUFA	PUFA	Omega-3 FA	Omega-6 FA
Diet (D)
C	37.8 a	47.0	15.3 b	1.8 b	13.5 b
P	35.2 b	46.4	18.3 a	2.5 a	15.8 a
SEM	0.622	0.244	0.568	0.152	0.516
Essential Oils (EO)
0	36.5	46.7	16.9	2.1	14.8
ORE	36.2	46.8	17.1	2.2	14.9
BLEND	36.9	46.5	16.6	2.2	14.4
SEM	0.203	0.088	0.145	0.033	0.265
D × EO
C-0	37.8 a	47.1	15.1 b	1.8 b	13.3 b
C-ORE	37.4 a	47.0	15.6 b	1.8 b	13.8 b
C-BLEND	38.2 a	46.8	15.3 b	1.9 b	13.4 b
P-0	35.1 b	46.3	18.6 a	2.4 a	16.2 a
P-ORE	34.9 b	46.6	18.5 a	2.6 a	15.9 a
P-BLEND	35.5 b	46.2	17.9 a	2.5 a	15.4 a
SEM	0.453	0.250	0.531	0.112	0.356
p-value
D	0.037	0.156	<0.001	0.007	0.005
EO	0.364	0.413	0.271	0.815	0.402
D × EO	0.042	0.274	<0.001	0.008	0.007

C: control diet; P: diet containing leaves and stems of purslane; C-0, P-0: no addition of essential oils in drinking water; C-ORE, P-ORE: addition of 100 ppm of oregano essential oil in drinking water; C-BLEND, P-BLEND: addition of 100 ppm of blend of oregano, sage, and fennel essential oils in drinking water; SEM: standard error of the means; ^a,b means within a column with different superscripts inside each factor differ significantly at p < 0.05.

presents the cumulative concentrations of saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA), omega-3, and omega-6 fatty acids. From the table, it can be observed that the inclusion of purslane in the layers’ diet resulted in a significant (p < 0.05) decrease in SFA. This reduction was accompanied by a significant (p < 0.05) increase in PUFA, omega-3, and omega-6 fatty acids. The concentration of MUFA appeared to remain unchanged with the addition of purslane to the diet.

Table 5. Nutritional indices of fatty acids in egg yolk.

Nutritional Indices of FA	MUFA/SFA	PUFA/SFA	UFA/SFA	Omega-6 FA/Omega-3 FA
Diet (D)
C	1.24 b	0.40 b	1.65 b	7.56 a
P	1.32 a	0.52 a	1.84 a	6.34 b
SEM	0.020	0.026	0.047	0.299
Essential Oils (EO)
0	1.29	0.46	1.75	7.06
ORE	1.29	0.48	1.77	6.99
BLEND	1.27	0.46	1.72	6.81
SEM	0.007	0.007	0.015	0.074
D × EO
C-0	1.25 b	0.39 b	1.65 b	7.58 a
C-ORE	1.24 b	0.42 b	1.67 b	7.76 a
C-BLEND	1.23 b	0.40 b	1.63 b	7.35 a
P-0	1.32 a	0.53 a	1.85 a	6.54 b
P-ORE	1.34 a	0.53 a	1.87 a	6.21 b
P-BLEND	1.30 a	0.51 a	1.80 a	6.26 b
SEM	0.012	0.021	0.028	0.193
p-value
D	0.041	0.006	0.043	0.030
EO	0.673	0.483	0.224	0.184
D × EO	0.038	0.006	0.041	0.033

C: control diet; P: diet containing leaves and stems of purslane; C-0, P-0: no addition of essential oils in drinking water; C-ORE, P-ORE: addition of 100 ppm of oregano essential oil in drinking water; C-BLEND, P-BLEND: addition of 100 ppm of blend of oregano, sage, and fennel essential oils in drinking water; SEM: standard error of the means; ^a,b means within a column with different superscripts inside each factor differ significantly at p < 0.05.

shows the relationships between the aforementioned concentrations. According to the table, the inclusion of purslane led to a significant (p < 0.05) increase in MUFA/SFA, PUFA/SFA, and UFA/SFA ratios. Moreover, the nutritional index omega-6 FA/omega-3 FA was significantly (p < 0.05) improved. On the other hand, the addition of ORE or BLEND to the hens’ drinking water did not seem to significantly affect the concentrations of fatty acids and the relationships between them.

3.3. Oxidative Stability of Egg Yolk

The degree of oxidation was measured by determining the concentration of malondialdehyde (MDA), which is one of the final products of peroxidation of polyunsaturated fatty acids. Oxidative rancidity was measured at four incubation periods: 0, 50, 100, and 150 min. The main effects of diet (D), essential oils (EO), and time of incubation (T), as well as their interactions on MDA concentration in egg yolk, are presented in

Table 6. Main effects of diet, essential oils, and time and their interactions on MDA concentration in egg yolk.

	p-Value
Main effects
D	0.482
EO	<0.001
T	<0.001
Interactions
D × EO	<0.001
D × T	<0.001
EO × T	<0.001
D × EO × T	<0.001

MDA: concentration of malondialdehyde expressed in ng per g of egg yolk; D: diet; EO: essential oils; T: time of incubation; values with p < 0.05 are statistically significant.

and Figure 1 and Figure 2.

Based on the results, supplementation of purslane in the diet did not significantly affect MDA concentration. However, the addition of essential oils led to a significant (p < 0.05) reduction in MDA concentration, with BLEND being more effective than ORE. The length of incubation time had a significant (p < 0.05) increasing effect on MDA concentration.

All two-way interactions were significant (p < 0.05) on MDA concentration. Specifically, the addition of essential oils significantly (p < 0.05) reduced MDA concentration in both the no-purslane and purslane treatments, with BLEND being more effective. Increasing the length of incubation time significantly (p < 0.05) increased the concentration of MDA in treatments without purslane and in those with purslane. The length of incubation time exerted an increasing effect on MDA concentration, which was, however, significantly (p < 0.05) limited by the addition of essential oils, with BLEND having a greater limiting effect compared to ORE.

The D × EO × T triple interaction was significant (p < 0.05). While the addition of purslane and incubation time positively interacted to increase MDA concentration, the addition of essential oils dramatically limited this increase, resulting in ORE and BLEND treatments having significantly (p < 0.05) lower MDA concentrations than treatments without essential oils.

In summary, the results indicate that the addition of essential oils contributed to a significant (p < 0.05) increase in the oxidative stability of egg yolk at all time points, independent of the administration of purslane, with the blend of oregano, sage, and fennel essential oils (BLEND) being the most effective.

4. Discussion

In the present study, it was found that the addition of purslane resulted in a significant increase in egg weight and egg mass production. This increase may be attributed to the hens’ increased feed and nutrient intake in the rations, which contained 24 g of fresh chopped leaves and stems of purslane per day per hen. Notably, there are no reports in the literature on the use of fresh purslane in the diets of laying hens. Previous research has primarily focused on the use of dried purslane and reported conflicting results regarding its effect on egg production rate, egg weight, and egg mass production.

The findings of the present research are consistent with the results reported by Aydin and Dogan [8] and Evaris et al. [41]. These studies found that the addition of dried purslane at rates of 10 or 20 g/kg and 100 or 200 g/kg, respectively, resulted in positive effects on egg weight and egg mass production. In the latter study, an increase in egg production was also observed. Conversely, other studies have reported positive effects only on egg weight [42] or only on egg mass production [43]. Moazedian and Saemi [9] did not find any effect on egg production or egg characteristics. On the other hand, Dalle Zotte and Pranzo [14] reported lower egg weight and total egg production in hens fed rations containing 20% dried purslane. This may be due to the reduced feed intake of the hens.

Yolk color was a trait that significantly improved in the purslane treatments, which is consistent with the findings of Kartikasari et al. [10]. An increased intensity of yolk color can be achieved by incorporating purslane, which is rich in xanthophylls and β-carotene, into the diets of laying hens [20].

The present research found that the addition of purslane significantly increased the concentration of the polyunsaturated fatty acids linoleic (LA) and α-linolenic (ALA) in the egg yolk. Similarly, Moazedian and Saemi [9] reported a significant increase in omega-3 fatty acids ALA and docosahexaenoic (DHA) in the egg yolk, which became even greater with an increase in the participation of purslane in the ration. Evaris et al. [41] also reported similar results. It is worth noting that the conversion efficiency of ALA to DHA by the hen’s body is only 0–9%. However, a diet rich in ALA can still lead to an increase in yolk DHA content [10,44]. In the present study, the concentration of DHA was not significantly increased by the addition of purslane, possibly because the concentration of ALA in purslane leaves and stems was not high enough. It is also noteworthy that the concentration of LA was significantly increased in the purslane treatments, which is consistent with the findings of Moazedian and Saemi [9] and Dalle Zotte and Pranzo [14]. On the other hand, the decrease in the concentration of stearic acid observed in our study in the purslane treatments may be related to the low concentration (0.048 mg/g) of purslane leaves in stearic acid [17].

The percentage of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in the leaves of purslane are 16.4, 4.8, and 78.7%, respectively, while in the stems, it is 24.6, 3.9, and 71.5%, respectively [45]. Supplementing the diet of laying hens with feeds containing high levels of unsaturated fatty acids (UFA), such as purslane, can help reduce cholesterol in the egg yolk. This hypothesis is confirmed by Shehata and El-Krim [43], who found that Portulaca oleracea leaves added to the feed of layers play a very important role in reducing total lipids and total cholesterol in blood serum.

In our study, the omega-6/omega-3 ratio was significantly improved in the purslane-treated groups, a finding consistent with those of other researchers [8,9,46]. In agreement with the results of the present research, Dalle Zotte et al. [47] supplementing dried purslane meal to laying hens found a significant increase in omega-6 and omega-3 PUFA, as well as a significant improvement in the omega-6/omega-3 nutritional index. In addition, in the same study, a significant decrease in SFA content and an increase in PUFA content (LA, ALA, DHA) were observed. Similar results were found in the recent work of Dalle Zotte and Pranzo [14].

Lipid oxidation is a primary concern in the deterioration of foods like eggs. The addition of antioxidants to foods can inhibit lipid rancidity, delay the formation of toxic oxides, preserve nutritional value, and extend shelf life [48]. Purslane is known to contain various antioxidant compounds, including α-tocopherol, ascorbic acid, β-carotene, and 2,2-dyphenyl-1-picrylhydrazyl (DPPH). The total phenolic content (TPC) in different cultivars of Portulaca oleracea, expressed as total antioxidant capacity, ranges from 127 ± 13 to 478 ± 45 mg GAE/100 g of fresh plant weight [20]. However, in our study, the addition of purslane did not have a significant positive effect on the oxidative stability of egg yolk.

Essential oils (EO) from aromatic/medicinal plants contain active aromatic compounds that are widely used to prevent the peroxidation of egg yolk [49]. EO has antioxidant components that protect lipids from oxidation, thereby slowing lipid peroxidation [50]. The polyphenols carvacrol and thymol, which are major active compounds of oregano (Origanum vulgare) essential oil, can act as hydrogen donors to neutralize the excessive free radicals produced at the beginning of lipid oxidation [51]. The total phenolic content of the oregano extract amounts to 19.5 ± 0.2 mg gallic acid/g of dry sample [52]. When the diet of laying hens was supplemented with ground leaves and stems of dried oregano at a concentration of 5 g/kg feed, the oxidative stability of egg yolk was significantly increased [37]. According to the reports of Ghanima et al. [53], the addition of thymol formulation to layers’ diet significantly reduced the level of MDA in egg yolk.

Among the essential oils of aromatic plants is the essential oil of the plant species sage (Salvia officinalis). The content of the cultivated sage in ethereal extract amounts to 2.37%, with the main active antioxidant components being cis-thujone and trans-thujone, in percentages of 33.80 and 6.97%, respectively [26]. The total phenolic content of sage extract amounts to 15.6 ± 0.1 mg gallic acid/g of dry sample [52]. Galamatis et al. [26] found a significant increase in the oxidative stability of the egg yolk when laying hens were fed diets supplemented with ground dried sage leaves and stems at levels of 0.5 and 1.0%, compared to the control, at incubation times of 50, 100, and 150 min.

Another plant species of interest for its antioxidant properties is fennel (Foeniculum vulgare). The essential oil of fennel seeds contains 16.81% trans-anethole (phytosterol) and 47.20% estragol, active substances with antioxidant potential that contribute to the reduction of cholesterol and triglycerides [54]. The total phenolic content of the essential oil extracted from fennel seeds is extremely high and is measured to be 70.42 mg GAE/g [55]. Gharaghani et al. [56] showed that consumption of diets containing fennel significantly reduced cholesterol and triglyceride levels in eggs. Abou-Elkhair et al. [57] found that the addition of fennel to the diets of layers had no significant effect on the MDA levels in the egg yolk, despite the strong antioxidant potential of fennel.

In our research, the addition of oregano essential oil (ORE) or the blend of essential oils (BLEND) contributed to a significant increase in the oxidative stability of the egg yolk at all incubation times, which is in general agreement with the aforementioned studies. The greater antioxidant capacity of BLEND compared to ORE is likely due to the greater total phenolic content of BLEND or possibly to a synergistic effect of the active components of the three essential oils in the mixture.

In summary, the findings of the present research provide a positive response to the question posed in the introduction regarding the possibility of producing an egg with increased omega-3 PUFA content protected by natural antioxidants. Our results indicate that adding fresh purslane to the feed and essential oils to the drinking water of laying hens can help achieve the production of such a product.

5. Conclusions

Incorporating fresh purslane into the ration of layers significantly contributes to the enrichment of the egg yolk in omega-3 PUFA. The addition of oregano essential oil, or a mixture of oregano, sage, and fennel seed essential oils, to the drinking water of laying hens results in a significant increase in the oxidative stability of the egg yolk, with a prolonged antioxidant effect over time.

The combined administration of fresh purslane and essential oils of aromatic plants could be a promising option for poultry farmers, both economically and environmentally. This approach utilizes natural raw materials from native or cultivated plants that are abundant in nature and have little impact on the cost of nutrition. It also mimics the nutritional habits of laying hens, particularly those reared in organic or free-range farms, and adapts to the physiology of their digestive system. Furthermore, this method contributes to producing eggs with an improved PUFA proportion, which are protected with natural plant-based antioxidants and potentially have an extended shelf life. Additionally, this approach meets the modern nutritional requirements of consumers and may enable product certification, leading to increased added value, farm viability, and sustainable development.

While the proposed management model has several advantages, one potential drawback is the limited availability of fresh purslane year-round. However, this can be easily addressed by cultivating the plant in a greenhouse.