Effects of Marigold and Paprika Extracts as Natural Pigments on Laying Hen Productive Performances, Egg Quality and Oxidative Stability

Abstract

Enhancing the quality of eggs by using natural food sources has become a very important topic in the last decade. The objective of this study was to determine the influence of natural (marigold and paprika extracts) pigments on the shelf life of eggs from laying hens. This research was carried out for a 6-week period on 168 Lohmann Brown laying hens (45 weeks age) divided into four groups (C, E1, E2 and E3) to assess the performances, external and internal egg quality parameters, egg yolk color, and antioxidant profile. The control group (C) was fed a standard diet (16.39% PB, 2750 kcal EM/kg compound feed) and the experimental diets were supplemented with 0.07% marigold extract (E1), 0.07% paprika extract (E2), and a mixture containing 0.07% of both extracts (E3). In summary, the study demonstrated that adding natural pigments from marigold and paprika extract with highly antioxidant lipid capacity into the diets of laying hens improved egg quality when eggs were stored at 28 days, under both storage temperature conditions (4 °C and 20 °C).

1. Introduction

Eggs are a highly nutritious food that provides the consumer with necessary elements such as fatty acids, vitamins, minerals, and proteins [1]. Therefore, the egg has perishable qualities, much like any other food derived from animals, losing its interior quality from the time the hen lays it until it is consumed [2]. Conservation techniques become crucial to prevent premature perishability, with a focus on refrigeration, which tends to maintain the internal quality of eggs by delaying their degradation [1]. Temperature and relative humidity have the biggest effects on the internal quality of eggs. High temperatures during storage are associated with decreased albumen quality, which can be attributed to water and carbon dioxide loss that causes the albumen to become fluidified and the yolk membrane easier to crack when breaking eggs. The egg will lose water more quickly and lose weight as a result of the air chamber expanding if the relative humidity of the atmosphere around it is less than 99.6% [3].

The color of the egg yolk is another attribute associated with internal egg quality, and a factor contributing to customer decision-making, because it is typically linked to the nutritional value and quality of the egg. Corn is a notable source of xanthophylls, which are responsible for the yolk’s yellowish color. However, the amount of these pigments in corn varies throughout the year, primarily from harvest to harvest, which means that laying hens must use pigments in their diet to maintain the yolk’s color given that the birds cannot synthesize xanthophylls; they must only get them from their dietary ingredients [4].

Carotenoids are a group of pigments found in plants, algae, and photosynthetic bacteria, responsible for the vibrant red, orange, and yellow hues in various fruits and vegetables [5]. Due to their importance to nutrition, health, and well-being, carotenoids have recently attracted much attention in the food and feeding industries. Carotenoids also serve as essential precursors for synthesizing vitamin A in animals, contributing to various physiological functions such as vision and immune system support [6]. Additionally, their antioxidant properties make carotenoids valuable in promoting human health by neutralizing harmful free radicals, potentially reducing the risk of chronic diseases [7]. Including natural pigments in poultry feed enhances these antioxidant benefits in the eggs, maintaining their flavor, color, and nutritional profile. This is crucial since eggs are a significant source of essential nutrients like vitamins A and E [8].

As Darvin et al. [9] stated, antioxidant combinations can significantly enhance antioxidant protection through a synergistic complex effect when the composition and concentration of antioxidants are optimally balanced to neutralize free radicals and reduce their harmful effects. Being seen as a strategy in laying hen diets, the plant/herbal supplementation had a positive impact on poultry physiological, productive, reproductive, and immunological performances [10]. Therefore, natural pigment extract utilization in animal feed can provide a triple benefit, as observed in various studies: enhanced oxidative stability of food lipids, increased carotenoid concentration, and a more intense egg yolk color [11].

The marigold extract, derived from Tagetes erecta L. (Asteraceae family), is well-known as an ornamental plant (yellow or orange flowers grouped in solitary inflorescences), but also for the rich content of carotenoids, is easy to purchase and available to anyone. The total carotenoid content of marigold extract is 4200 mg/kg [12]. Additionally, it is utilized in the production of dietary supplements intended to stop age-related macular degeneration and other eye disorders from affecting visual acuity. The content of the plant in lutein means it has many uses, such as commercial purposes as a natural pigment for poultry feed [13].

Paprika extract (Capsicum annuum) is one of the most well-known and often-used natural food pigments [14]. The bright orange-red pigment known as capsanthin, which gives paprika its unique red color, belongs to the xanthophylls class of oxygen-containing carotenoids. The strong antioxidant activity of capsanthin results in its shown chemopreventive, antitumor, skin photoprotective, anti-inflammatory, and antidiabetic properties. Capsanthin provides a lot of health benefits, is naturally occurring, and could be developed into a pharmaceutical, nutraceutical, or cosmeceutical product [15]. Carotenoids are becoming feed additives preferred as human food natural pigments due to their positive and healthy effects on animal products when added to their diets [16]. The specific carotenoids, lutein and zeaxanthin, have demonstrated antioxidant, anti-inflammatory, light absorbing, and blue filtering effects. It has been suggested that they may help prevent immune-mediated macular degeneration and the development of age-related cataracts [17,18,19].

The trial aimed to assess the nutritional feeding qualities of the two natural pigments from plant extracts, marigold and red pepper, in the diets of laying hens, based on the premise that the aforementioned plants can be considered phyto-additives with beneficial effects on laying hens’ health, productivity and egg quality.

2. Materials and Methods

2.1. Ethical Statement

The feeding trial was carried out according to the protocol (No. 4096/23 September 2022) approved by the institute’s Commission of Ethics in the experimental halls of the National Research Development Institute of Animal Biology and Nutrition (IBNA-Balotesti, Romania) following the Romanian legislation (Law 206/2004, Ordinance 28/31 August 2011, Law 43/11 April 2014, Directive 2010/63/EU) for the feeding, handling, and slaughtering procedures of a study.

2.2. Carotenoids Purchasing and Their Proximal Chemical Analyses

The natural carotenoid powders (marigold and red pepper extracts) were provided by Animal Feed Consulting (Ilfov, Romania). The natural pigments were produced by Kaesler Nutrition GmbH (Cuxhaven, Germany) under EC Regulation no. 1831/2003, labelled accordingly and packed in aluminium plus polymer bags of 25 kg. Samples of marigold and red pepper powders, of 500 g each, were analyzed to determine the proximate qualities of dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF) and ash, the minerals copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn), and the factors contributing to antioxidant activity, including polyphenols, antioxidant capacity, tocopherols, and carotenoids. The natural carotenoids and feed samples were analyzed using standardized methods to determine the nutrient concentration. The DM concentration was measured using the gravimetric method (BMT model ECOCELL Blueline Comfort, Nuremberg, Germany), the CP concentration was assessed using the Kjeldahl method [Kjeltek auto 1030–Tecator (FOSS Tecator AB, Höganäs, Sweden)], the EE concentration was determined by extraction in organic solvents (FOSS Tecator AB, Höganäs, Sweden), the CF concentration was assessed using intermediary filtration, (Fibertec 2010 System-Foss Tecator, Sweden), and crude ash [Nabertherm Labotherm L15/11/P320 Comfort, (Bremen, Germany)] was determined according to the procedures described in Regulation (CE) No. 152/2009.

2.3. Birds Performances, Housing and Experimental Diets

The 6-week experiment used 168 Lohmann Brown layers (45 weeks old), assigned into four groups: control (C), experimental group 1 (E1), experimental group 2 (E2) and experimental group 3 (E3). They were housed in a hall under controlled microclimate conditions, where the temperature was recorded twice a day, at 8:00 a.m. and 3:00 p.m.

The registered daily average temperature was 22.44 °C ± 1.85, the humidity was 63.88% ± 6.02, and the ventilation was 27.4% ± 10.86. The hens were weighed individually (initial average weight of 1.760 ± 53.46 g) and housed in three-tier batteries, Big Dutchman digestibility cages, with 42 birds/group, 21 cages/group and 2 birds/cage, respectively, with a 16 h/24 h light regimen. Each cage (50 cm width × 40 cm height × 50 cm length) was considered an experimental unit and performance parameters were evaluated per pen. Feed and water were offered ad libitum throughout the experiment. Each of the four groups had a similar basal diet formulation (

Table 1. Diet formulation and proximal analysis results.

Specifications	C	E1	E2	E3
Red pepper extract, %	-	-	0.07	0.05
Marigold extract, %	-	0.07	-	0.02
Corn, %	55.07	54.93	54.93	54.93
Soybean meal, %	13.97	14.00	14.00	14.00
Sunflower meal, %	16.00	16.00	16.00	16.00
Lysine, %	0.26	0.26	0.26	0.26
Methionine, %	0.23	0.23	0.23	0.23
Calcium carbonate, %	8.77	8.77	8.77	8.77
Monocalcium phosphate, %	1.01	1.01	1.01	1.01
Salt, %	0.36	0.36	0.36	0.36
Soybean oil, %	3.26	3.31	3.31	3.31
Choline 60%	0.05	0.05	0.05	0.05
Phytase	0.01	0.01	0.01	0.01
Premix *, 1%	1.00	1.00	1.00	1.00
Total ingredients, %	100.00	100.00	100.00	100.00
Calculated Analysis
Metabolizable energy, kcal/kg	2.750	2.750	2.750	2.750
Crude protein, %	17.00	17.00	17.00	17.00
Calcium, %	3.90	3.90	3.90	3.90
Phosphorus, %	0.38	0.38	0.38	0.38
Lysine, %	0.94	0.94	0.94	0.94
Met + cist, %	0.85	0.85	0.85	0.85
Threonine, %	0.64	0.64	0.64	0.64
Chemical Analysis
Gamma tocopherol (mg/kg)	7.31	8.79	8.13	8.06
Alpha-tocopherol (mg/kg)	34.61	36.07	28.25	31.69
Lutein + zeaxanthin (ppm)	9.90	22.19	9.06	14.77
Antioxidant capacity (µM Trolox)	22.64	26.89	30.07	28.28
Total polyphenols, mg/g GAE	4.31	4.89	4.54	4.74

* 1 kg premix contains 1,100,000 IU/kg vitamin A; 200,000 IU/kg vitamin D3; 2700 IU/kg vitamin E; 300 mg/kg vitamin K; 200 mg/kg vitamin B1; 400 mg/kg vitamin. B2; 1485 mg/kg pantothenic acid; 2700 mg/kg nicotinic acid; 300 mg/kg vitamin B6; 4 mg/kg vitamin B7; 100 mg/kg vitamin B9; 1.8 mg/kg vitamin B12; 2000 mg/kg vitamin C; 8000 mg/kg manganese; 8000 mg/kg iron; 500 mg/kg copper; 6000 mg/kg zinc; 37 mg/kg cobalt; 152 mg/kg iodine; 18 mg/kg selenium.

). Compared to the C diet, the E1 group included 0.07% marigold extract, the E2 group included 0.07% red pepper extract, and the E3 group included a combination of marigold and red pepper extracts. The structure of the experimental diets was developed using dedicated software for the formulation of compound feed (Brill^® Formulation 2.5, AGRIFOOD, Lleida, Spain), in agreement with the feeding requirements of laying hens [20]. The diets were isoenergetic and isonitrogenous, containing 17% CP and 11.51 MJ metabolizable energy (ME) per kg diet (Table 1). Average body weight (BW, g/hen) was measured at the beginning and at the end of the experimental period. Daily production parameters were monitored: average daily feed intake (ADFI; g/hen/day); feed conversion rate (FCR; kg feed/kg egg); laying rate intensity (LRI; %); average egg weight (AEW; g) and viability (%). No medical treatment was applied to the hens throughout the six-week experimental period. The difference between the amount of feed administered and the amount of leftover feed that was not consumed each day was used to calculate the ADFI parameter. The production parameter for FCR was given as kg of feed eaten for every kilogram of eggs produced. By dividing the total number of eggs laid by the total number of laying hens, the LRI parameter was determined.

2.4. Egg Collection and Their Quality Measurement

After 3 and 6 experimental weeks, a total of 144 eggs were collected randomly (18 eggs/group/period) to assess the internal and external quality parameters of the eggs: egg weight and its components (albumen, yolk, shell) with a Kern EW6000-1M Electronic Balance, precision 0.001 (Kern & Sohn GmbH, D-72336 Balingen, Germany), egg freshness, Haugh unit, and eggshell breaking strength, using the Digital Egg Tester DET-6500 (NABEL Co., Ltd., Kyoto, Japan). The eggshell thickness was measured within a range of 0.10–0.60 mm, with an accuracy of ± 0.02 mm, with the concave part of the eggshell side down, using a digital micrometer in the range of 0–10 mm, with a measuring force of 1.5 N or less (Mitutoyo 547-360 ABSOLUTE Digimatic Thickness Gauge, NABEL Co., Ltd., Kyoto, Japan). The pH of the albumen and egg yolk was measured using a portable pH meter (Five Go F2-Food kit with LE 427IP67, Sensor Metler Tolledo, Greifensee, Switzerland). To assess accurately the yolk color, DET6500 uses natural light, as artificial light varies greatly between locations depending on its light source. The yolk color was estimated using DSM YolkFan^TM based on daylight, without artificial light. Furthermore, utilizing the CIE-Lab system (Commission Internationale de l’Eclaraige) with a customized aperture (8 mm/4 mm/1 × 3 mm), 2.6 s measuring time, high accuracy of 0.04 and an observer angle of 2°/10°, the egg yolk color (parameters L*, a*, and b*) was measured using the portable colorimeter 3nh YS3020 (Shenzhen Threenh Technology Co., Ltd., Beijing, China).

The oxidative stability of the fat yolk was measured at the end of the experiment and after 28 days when 144 eggs were collected randomly and kept at different storage temperatures: 18 eggs/group were kept at room temperature (20 °C ± 1) and another 18 eggs/group were kept in a refrigerator (4 °C) for 28 days and analyzed to assess the yolks’ oxidative stability.

After measuring the internal and external quality parameters of the eggs, 6 yolk samples (3 yolks/sample) per group were formed and assayed for the antioxidant profile (lutein content, alpha and gamma tocopherols, vitamins A and E, antioxidant capacity and total polyphenols) and oxidative status of the yolk, represented by thiobarbituric acid reactive substances (TBARS) assessment. Yolk samples were stored at −20 °C until analysis. Before analysis, the samples were allowed to reach room temperature. Chemical analysis, represented by vitamins, polyphenols and antioxidant capacity, was performed on the dry yolk samples, while the oxidative stability of eggs and color measurement were determined on the fresh yolk samples.

2.5. Minerals Assessment

Flame atomic absorption spectrometry [Thermo Electron—SOLAAR M6 Dual Zeeman Comfort (Cambridge, UK)] was utilized for the concentration assessment of zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) in marigold and red pepper powder extracts [21]. The results have been expressed as μg/g (ppm) of dried sample.

2.6. Determination of Total Polyphenols Content

The total polyphenol content of plants was spectrometrically determined using the Folin–Ciocalteu method, as described by [22]. The reading of absorbance was performed at 732 nm, and gallic acid was used for the calibration curve, the results being expressed as mg gallic acid equivalents per gram sample.

2.7. Total Antioxidant Capacity Assessment

The total antioxidant capacity of samples was evaluated by the DPPH method described by [22]. The antioxidant activity was expressed as Trolox equivalents. The total antioxidant capacity of the extracts was based on the reaction between the sample solution and DPPH reagent prepared in methanol and the absorbance recorded at 517 nm using a V-530 Jasco (Japan Servo Co., Ltd., Tokyo, Japan) spectrophotometer, as described elsewhere.

2.8. Determination of Vitamin E, Lutein and Zeaxanthin

Vitamin E determination for plants, compound feed and eggs were performed using high-performance liquid chromatography (HPLC Finningan Surveyor Plus, Thermo-Electron Corporation, Waltham, MA, USA) and a PDA-UV detector at a wavelength of 292 nm, as described by Vărzaru et al. [23]. The results have been expressed as mg/kg.

Lutein and zeaxanthin content were analyzed using a high-performance liquid chromatograph (Perkin Elmer 200 series, Shelton, CT, USA) with a UV detector (445 nm), and a Nucleodur C18 column (Macherey-Nagel, Dueren, Germany), as described by Vărzaru et al. [23]. The results have been expressed as mg/kg.

2.9. Determination of Thiobarbituric Acid Reactive Substances (TBARS)

The oxidative stability of yolk was indicated by the levels of thiobarbituric acid reactive substances (TBARS), according to the methods described by Untea et al. [22]. The TBARS values were calculated from a standard curve of malondialdehyde and expressed as milligrams of malondialdehyde (MDA) per kg of sample (mg MDA/kg). The absorbance of the prepared sample was read at 532 nm.

2.10. Statistical Analysis

All data were subjected to an analysis of variance using the GLM procedure of the Minitab software (version 17, Minitab^® Statistical Software). The level of significance was set at p < 0.05. The experimental results (production performances and antioxidant profile) were analyzed according to the following linear model:

Yij = µ + Aj + eij

where Yij means the value of the trait (the dependent variable); µ, overall mean; Aj, the treatment effect; and eij, random observation error.

The effect of feeding time on external and internal egg quality parameters and yolk color was analyzed according to a 2 × 2 factorial arrangement to determine whether the factors studied (treatment and feeding time) influenced the egg parameters for different periods. The data obtained were analyzed by two-way ANOVA using the Tukey test following the statistical model:

Yijk = µ + αi + βj + αiβj + eijk

where Yijk = variable measured for the kth observation of the ith treatment and jth feeding or storage time; μ is the sample mean; αi is the effect of the ith treatment; βj is the effect of the jth feeding or storage time; αiβj is the interaction of ith treatment and jth feeding or storage time, and eijk is the effect of error. The differences were highly significant when p < 0.001 and significant if p < 0.05.

The graphs for the oxidative stability parameters (TBARS) were statistically constructed using GraphPad Prism 9.1.2 software (GraphPad Software, La Jolla, CA, USA). Values were determined to be significant when * p < 0.05, ** p < 0.01 and *** p < 0.001, with different letters indicating significant differences between groups (p < 0.05).

3. Results

3.1. Nutritional Profile of Marigold and Red Pepper Powder Extracts

The nutritional compositions of natural pigments are presented in

Table 2. The nutritional profile of marigold and red pepper powder extracts.

Parameters	Red Pepper Extract Powder	Marigold Extract Powder
Proximate Composition
Dry matter (DM), %	92.79	94.84
Crude protein (CP), %	0.25	5.15
Ether extract (EE), %	4.21	0.23
Crude fiber (CF), %	0.21	1.17
Ash, %	55.54	50.74
Mineral Content
Cooper (Cu), %	n.d.	3.78
Iron (Fe), %	284.26	1296.77
Manganese (Mn), %	20.87	158.94
Zinc (Zn), %	n.d.	33.71
Antioxidant Activity
Total polyphenols, mg/g GAE	5.32	3.64
Antioxidant capacity (μM Trolox/kg)	22.80	52.82
Delta tocopherol (mg/kg)	n.d.	n.d.
Gamma tocopherol (mg/kg)	560.32	68.22
Alpha-tocopherol (mg/kg)	199.43	150.11
Lutein + zeaxanthin (ppm)	956.26	3786.66
Astaxanthin, (mg/kg)	660.05	n.d.
Canthaxanthin (mg/kg)	31.72	n.d.

where: n.d., not detectable.

. The marigold extract powder showed higher CP (5.15%) and CF (1.17%) contents, compared to red pepper extract powder (0.25% CP and 0.21 CF %). The red pepper extract registered a higher content of EE (4.21%) compared to marigold extract (0.23%). Also, the trace mineral content (Cu, Fe, Mn, Zn) was higher in marigold extract compared to red pepper extract powder. However, Cu and Zn could not be detected in red pepper extract powder samples.

Regarding the antioxidant profile, the marigold extract powder was the richest in lutein (3786.66 ppm) and antioxidant capacity (52.82 μM Trolox). The red pepper extract presented the highest values for total polyphenols content (5.32%) and the concentrations of astaxanthin (660.05 ppm) and canthaxanthin (31.72 ppm), not detectable in the red pepper extract’s case. The richest source of gamma-tocopherol (mg/kg) was red pepper extract powder, its content being 8.21 times higher compared to the concentration encountered in marigold extract powder.

3.2. Productive Performance Parameters

Table 3. Effect of marigold and red pepper extract powder in laying hens’ diets on the productive performances of layers.

Specifications	C	E1	E2	E3	SEM	p-Value
Initial body weight (g/layer)	1667.60	1624.60	1684.00	1657.00	18.500	0.71
Final body weight (g/layer)	1830.52	1825.00	1894.20	1830.20	17.080	0.43
Average daily feed intake (g/hen/day)	118.92 a	116.21 b	120.76 a	119.96 a	0.469	<0.0001
Feed conversion rate (kg feed/kg egg)	2.15 bc	2.21 b	2.22 b	2.11 c	0.017	<0.0001
Laying rate intensity (%)	89.02	89.21	89.02	90.33	0.531	<0.110
Average egg weight (g)	63.46 b	63.17 b	64.39 a	64.37 a	0.095	<0.0001
Total egg weight (g)	1393.65	1326	1406.95	1429.91	10.561	<0.081

C—control diet; E1—control diet supplemented with 0.07% marigold extract powder; E2—control diet supplemented with 0.07% red pepper extract powder; E3—control diet supplemented with a mix of 0.05% red pepper extract powder and 0.02% marigold extract powder; SEM, standard error of the mean; ^a–c mean values within a row not sharing the same superscripts are significantly different at p < 0.05.

presents the results related to the production performances. There were no significant differences (p > 0.05) between the groups C, E2 and E3 groups in terms of ADFI parameter, except for the E1 group, which recorded a significantly lower consumption (p < 0.0001). At the same time, the lowest FCR values were highly significant (p < 0.0001) for the C and E3 groups compared to the E1 and E2 groups. Regarding the LRI (%) parameter and total number of eggs laid, no significant differences (p < 0.110; p < 0.081) were observed between experimental groups. There were highly significant (p < 0.0001) differences recorded for the AEW parameter in the E2 and E3 groups compared to the C and E1 groups.

3.3. External and Internal Egg Quality Parameters

Table 4. Effect of marigold and red pepper extract powder in laying hens’ diets on external and internal egg quality parameters.

Specification		Egg Weight and Components						Albumen			Yolk
		Whole Egg Weight (g)	Albumen (g)	Yolk (g)	Eggshell (g)	Shell Thickness (mm)	Breaking Strengths (kgF)	pH Albumen	Albumen Height (mm)	Haugh Units	pH Yolk	Yolk Height (mm)	Yolk Diameter (mm)	Yolk Index
3 weeks	C	60.95	38.47	14.91	7.57	0.38	3.54 b	8.29 d	7.23 ab	84.42 ab	6.15	18.23 ab	40.83	0.45
	E1	62.25	40.44	14.38	7.44	0.38	4.08 ab	8.44 cd	8.23 a	90.07 a	6.37	17.78 ab	39.73	0.45
	E2	61.57	38.48	15.27	7.82	0.39	4.85 a	8.30 d	7.63 ab	86.68 ab	6.43	17.12 ab	41.78	0.41
	E3	61.57	38.67	15.32	7.58	0.37	4.57 ab	8.36 d	7.85 ab	88.23 ab	6.29	17.25 ab	40.98	0.42
6 weeks	C	61.29	38.95	15.07	7.27	0.39	4.51 ab	8.85 a	7.16 ab	83.60 ab	6.33	17.92 ab	39.91	0.45
	E1	62.14	39.40	15.40	7.34	0.38	4.23 ab	8.62 bc	7.76 ab	87.18 ab	6.25	18.32 a	40.67	0.45
	E2	61.32	38.97	15.22	7.13	0.39	4.42 ab	8.67 ab	6.68 b	80.67 b	6.22	17.51 ab	41.21	0.43
	E3	60.87	38.07	15.64	7.17	0.38	4.42 ab	8.69 ab	7.18 ab	83.90 ab	6.16	17.16 ab	41.63	0.41
Main effects
Group	C	61.12	38.71	14.99	7.42	0.38	4.02	8.57	7.19	84.01	6.24	17.83 a	40.37	0.45 a
	E1	62.19	39.92	14.89	7.38	0.37	4.15	8.53	8.00	88.62	6.31	17.81 a	40.20	0.45 a
	E2	61.44	38.73	15.24	7.47	0.38	4.64	8.49	7.16	83.68	6.33	17.32 ab	41.50	0.42 b
	E3	61.22	38.37	15.48	7.38	0.37	4.50	8.52	7.51	86.07	6.23	17.25 b	41.31	0.42 b
	SEM group	0.412	0.435	0.258	0.141	0.004	0.199	0.030	0.225	1.34	0.064	0.183	0.554	0.008
Period
	3 weeks	61.58	39.01	14.97	7.60 a	0.38	4.26	8.35 b	7.74 a	87.35 a	6.31	17.60	40.83	0.43
	6 weeks	61.41	38.85	15.33	7.23 b	0.39	4.40	8.70 a	7.19 b	83.84 b	6.24	17.72	40.85	0.44
	SEM period	0.292	0.307	0.182	0.099	0.003	0.140	0.021	0.159	0.951	0.045	0.130	0.392	0.005
Interaction (p-Value)
group		0.260	0.077	0.378	0.961	0.081	0.115	0.279	0.042	0.048	0.602	0.001	0.259	0.003
period		0.669	0.701	0.168	0.012	0.114	0.493	0.000	0.020	0.013	0.259	0.490	0.970	0.736
period×group		0.850	0.504	0.493	0.520	0.945	0.09	0.001	0.575	0.574	0.158	0.326	0.574	0.782

C—control diet; E1—control diet supplemented with 0.07% marigold extract powder; E2—control diet supplemented with 0.07% red pepper extract powder; E3—control diet supplemented with a mix of 0.05% red pepper extract powder and 0.02% marigold extract powder; SEM, standard error of the mean; ^a–d mean values within a row not sharing the same superscripts are significantly different at p < 0.05.

data presents the internal and external egg parameters over the two periods (3 weeks and 6 weeks) and groups (C, E1, E2, E3).

There were no significant differences registered (p > 0.05) concerning the whole egg weight, albumen and yolk weight, breaking strength, eggshell thickness, pH yolk, and yolk diameter across groups, periods, or their interaction. Significant differences were found regarding eggshell weight between periods (p < 0.012). Highly significant differences in values for pH albumen were found between periods (p < 0.0001) and between period*groups interactions (p = 0.001). For the albumen height parameter, significant differences (p < 0.05) were observed between groups (p = 0.042) and between periods (p = 0.02); significantly lower albumen heights were registered at 6 weeks compared to 3 weeks. Consequently, the same trend was noticed for the Haugh unit parameter, where significant differences were registered between groups (p < 0.048) and between periods (p = 0.013). Other significant differences between groups were observed for yolk height (p < 0.001) and yolk index (p < 0.003). For most of the analyzed parameters, there were statistically significant differences between groups, periods and their interaction, indicating that the observed changes were primarily due to periods rather than different treatment groups. Indeed, as time increased, lower values but without statistical significance were noticed for whole egg albumen, albumen and eggshell weight, and yolk pH.

3.4. Yolk Color

Table 5. Egg yolk color assessment at 3 weeks and 6 weeks (average values/group).

Specifications		Yolk Fan Color (DSM)	L*	a*	b*
3 weeks	C	4.83 d	42.84 a	0.49 d	20.54 bc
	E1	7.33 c	42.88 a	2.80 bc	27.51 a
	E2	9.00 ab	40.30 bc	4.44 a	20.62 bc
	E3	9.67 a	42.88 a	4.42 a	21.60 bc
6 weeks	C	4.50 d	42.06 ab	0.12 d	21.52 bc
	E1	6.89 c	39.79 c	2.21 c	28.18 a
	E2	8.78 ab	40.13 bc	3.52 ab	18.99 c
	E3	8.11 bc	39.74 c	3.00 bc	22.67 b
Main effects
Group	C	4.67 c	42.45 a	0.30 c	21.03 bc
	E1	7.11 b	41.34 ab	2.50 b	27.84 a
	E2	8.89 a	40.21 b	3.98 a	19.81 c
	E3	8.89 a	41.31 ab	3.71 a	22.14 b
	SEM group	0.222	0.331	0.169	0.458
Period
	3 weeks	7.71 a	42.23 a	3.04 a	22.57 a
	6 weeks	7.07 b	40.43 b	2.21 b	22.84 a
	SEM period	0.157	0.234	0.120	0.324
Interaction (p-Value)
group		0.000	0.000	0.000	0.000
period		0.006	0.000	0.000	0.549
period × group		0.139	0.001	0.149	0.123

C—control diet; E1—control diet supplemented with 0.07% marigold extract powder; E2—control diet supplemented with 0.07% red pepper extract powder; E3—control diet supplemented with powder a mix of 0.05% red pepper extract powder and 0.02% marigold extract powder; L* (lightness), a* (redness), b* (yellowness); SEM, standard error of the mean; ^a–d mean values within a row not sharing the same superscripts are significantly different at p < 0.05.

presents the effects of marigold and red pepper extracts’ inclusion in the diet on yolk color. The presence of these two carotenoids exhibited a significant effect on the L*, a*, and b* color parameters. Concerning the yolk fan color, highly significant differences (p < 0.0001) were noted between groups, with the darkest colors in E3 and E2 groups compared to C and E1 groups. Also, highly significant differences (p < 0.0001) regarding the darkest hue were measured at 3 weeks compared to 6 weeks. No significant differences (p = 0.139) were registered for the period and group interaction.

For the L* parameter, highly significant differences (p < 0.000) were noticed between the C and E2 groups, whereas a highly significant statistical difference with the lowest values of the L* parameter was registered on the 6 weeks. Concerning group and period interaction (p = 0.001), highly significant statistical differences were recorded. Highly significant differences (p < 0.000) were found in the a* parameter between the groups, with values of 3.98 for E2 and 3.77 for E3, compared to 2.50 for E1 and 0.30 for the control (C). These differences were also found to be significant in the period evaluation, with values of 3.04 at 3 weeks compared to 2.21 at 6 weeks. For the b* parameter, the only highly significant difference (p < 0.000) was observed in the group analysis, where the E1 group showed higher yellow values compared to the control (C), E2, and E3 groups.

3.5. Antioxidant Profile

Table 6. Effects of dietary marigold and red pepper extract powder on antioxidant profile (average values/group).

Specifications	Experimental Diets				SEM	p-Value
	C	E1	E2	E3
Lutein + zeaxanthin (ppm)	7.77 c	28.67 a	8.03 c	12.35 b	0.436	0.0001
Alfa tocopherol (ppm)	158.48	179.34	160.43	163.74	6.01	0.167
Gama tocopherol (ppm)	15.83	18.21	17.92	17.65	0.533	0.052
Vitamin E (ppm)	174.31	197.55	178.35	181.39	6.44	0.166
Vitamin A (ppm)	19.84	22.76	19.09	21.26	1.07	0.195
Antioxidant capacity (µM Trolox)	0.54 b	0.60 ab	0.69 a	0.60 ab	0.033	0.067
Total polyphenols, mg/g GAE	0.15 c	0.19 bc	0.29 a	0.24 ab	0.020	0.002

C—control diet; E1—control diet supplemented with 0.07% marigold extract powder; E2—control diet supplemented with 0.07% red pepper extract powder; E3—control diet supplemented with a mix of 0.05% red pepper extract powder and 0.02% marigold extract powder; SEM, standard error of the mean; ^a–c mean values within a row not sharing the same superscripts are significantly different at p < 0.05.

presents the effects of the two dietary extracts on the antioxidant profile of egg yolks. Concerning the lutein and zeaxanthin concentrations, diet E1 showed the highest concentration (p < 0.0001), whihc was significantly higher that that of the C, E2 and E3 groups. Group E3 had a higher concentration compared to the E2 group, but this was lower than in the E1 group. The antioxidant capacity of group E2 exhibited the highest antioxidant capacity (p < 0.0001), which was significantly higher compared to the C group. For the total polyphenols content, the E2 group registered significantly higher values (p < 0.002) compared to the C and E1 groups. For other parameters, such as alpha-tocopherol, gamma-tocopherol, vitamin E, and vitamin A, there were no significant differences among the experimental diets. Nevertheless, it is noteworthy that a trend towards significance was observed for gamma-tocopherol on experimenatl groups compared to the C group (p = 0.052).

3.6. Oxidative Stability

In Figure 1 are presented the differences registered between experimental groups regarding the TBARS values. Common practices in the scientific evaluation of egg stability and quality involve monitoring them at intervals of 7, 14, 21, or 28 days to understand the qualitative transformations that occur during the product’s shelf life under various storage conditions and temperatures (https://agriculture.ec.europa.eu/farming/animal-products/eggs_en (accessed on 17 June 2024). In our study, after 28 days, under both storage conditions (room temperature and refrigeration), the TBARS values were highly significant (p < 0.0001) when compared to the C group. The TBARS values decreased significantly (p < 0.0001), which indicates that the natural extracts of marigold and paprika demonstrated a high lipid oxidative capacity, delaying the degradation of yolk fat. There were no statistically significant differences (p > 0.05) between the experimental groups for the 4 °C versus the 20 °C conditions.

4. Discussion

4.1. Nutritional Composition of Powder Extracts of Marigold and Red Pepper

Both marigold and red pepper extracts provide a natural yellow/red hue to egg yolks, with a high efficiency and rate of transfer to eggs. Marigold extract contains a high content of polyphenols, lutein and zeaxanthin compared to red pepper extract. This makes it a potent source of antioxidants, valuable for enhancing immune function. On the other hand, red pepper extract registered a higher antioxidant capacity, higher concentrations of gamma-tocopherol, alpha-tocopherol, astaxanthin and canthaxanthin, and crucial trace minerals such as copper, iron, manganese, and zinc, compared to marigold extract. Other studies on marigolds [24] registered a concentration of 12.16% for Cu and 1262.54 mg for Fe, while the lutein + zeaxanthin was 11.221 ppm and the total polyphenols were 13.55 mg GAE/g. Varying results were found in the literature when assessing the antioxidant activities of plant extracts due to different factors such as extraction solvents, assay procedures, and sample processing methods used [25,26]. Also, within marigold fresh flowers, the lutein concentration can vary from 4 mg/g in greenish-yellow flowers to 800 mg/g in orange-brown flowers. Dark colors have roughly 200 times more lutein esters compared to light colors [27]. Panaite et al. [28] found in kappa pepper the highest concentrations of lutein at 5.631 mg/kg, zeaxanthin at 1.528 mg/kg, lycopene at 0.351 mg/kg, and β-carotene at 7.576 mg/kg, as well as a total carotenoid content of 16.719 mg/kg compared to sea buckthorn pomace and carrot as alternative carotenoid sources.

4.2. Dietary Effects of Powder Extracts of Marigold and Red Pepper on Productivity Parameters

The results of the study indicate that while the experimental groups were not significantly different concerning production indicators including total egg weight or laying rate index, they varied significantly in terms of feed conversion ratio, average egg weight, and average daily feed intake, respectively.

Similar favorable results were observed by Oliveira [29], who concluded that supplementing extracts of marigold (0.1%) and paprika (0.6%) showed no apparent effect on the production of laying hens (p > 0.05). Compared to the 0.6% inclusion and control groups, the authors reported that 0.7% red pepper extract enhanced average daily feed intake and average egg weight. Skrivan [30] examined the effects of 150, 250, and 350 mg/kg marigold extracts when included in laying hens’ diets, finding no differences concerning the productive performance compared to a corn-based treatment. In egg-laying quails, Oliveira [29] included 0.06% paprika extract and 0.01% marigold extract, and registered an increased nutritional digestibility of the small intestine, a lower average feed intake, and a higher feed/gain ratio. A slightly increased egg weight was noted as well by Jang et al. [31] in lutein-supplemented groups. However, Skřivan et al. [30] reported a decrease in egg weight in the group fed with marigold flower extract. Other researchers [32] concluded that lutein from marigold flower extract did not affect egg weight. Atai et al. [33] examined the effects of 100, 200, 400, and 800 ppm lutein on 70 Brown-Nick laying hens (39-week-old) over a 6-week trial period. They found no significant effects on final body weight, feed intake, or feed conversion ratio, but noted higher egg production in the 100, 200, and 400 ppm lutein groups compared to the control and 800 ppm groups. Wang et al. [34] observed that supplementation with 0.075%, 0.15%, 0.30%, and 0.60% of marigold extract did not affect feed intake, body weight gain, or feed conversion ratio. Other authors [35] also found improvements in egg mass, egg production and feed conversion rate in an experiment on 160 laying hens fed with a diet supplemented with 0.5, 1, and 1.5% red pepper extract.

4.3. Dietary Effects of Powder Extracts of Marigold and Red Pepper on External and Internal Egg Quality Parameters

Supplementation with the dietary marigold extract (E1), paprika (E2), and both extracts (E3) did not significantly affect whole egg weight, albumen and yolk weight, eggshell thickness, breaking strength, yolk pH, or yolk diameter. This suggests that the inclusion of these natural pigment extracts does not significantly impact the egg quality under the experimented conditions. Significant differences (p < 0.05) were observed in eggshell weight during the two different periods from 3 weeks to 6 weeks, indicating that time may influence the shell quality and the pH of the albumen, which might be related to changes in egg freshness or storage conditions. Significant differences in albumen height were observed between groups and periods, with a noted decrease at 6 weeks compared to 3 weeks and with a similar pattern reflected in the Haugh unit, suggesting a decline in freshness over time. Differences were also statistically significant for yolk height and index between groups, indicating that the diets might have some specific effects on yolk structure and quality. The changes observed during the two periods of time, with generally lower values in parameters such as albumen height and Haugh unit, emphasize that the observed variations were more influenced by the duration of the storage period rather than the type of dietary pigment. Grčević et al. [36], when using 1 g/kg of marigold extract (E1) and 2 g/kg of marigold extract supplementation (E2), noticed that the greatest values of albumen height and HU were measured in the E2 group, both in fresh and in stored eggs, but the authors could not explain the marigold extract’s influence on these egg quality parameters. Maia et al. [37] tested four levels of marigold flower extract (2.10; 2.40; 2.70; 3.00 ppm) and observed a linear reduction in albumen percentage. Chowdhury et al. [38] used 40 g of marigold flower in laying pullets, observing no statistical differences compared to the C group regarding the albumen index, weight and Haugh unit measured at 8 and 12 experimental weeks. Mixed results have been found concerning the effects of marigold extract on egg quality parameters, with some studies showing improvements and others not finding significant effects. The mixed results indicate that adding these plant extracts might have some potential benefits for productive performance, or no significant effect. In another similar study, Spasevski et al. [39] conducted a trial using marigold (1.5%, 1%, 0.5%) and paprika (1.5%, 1%, 0.5%), and a combination of the two plants (marigold 1% + paprika 0.5%), and found no significant differences (p > 0.05) in egg weight, shell, yolk, or albumen. Oliveira et al. [29] stated that none of the experimental groups (paprika vs. marigold or the mix of the two) produced changes in egg weight or Haugh unit. The inclusion of paprika extract improved egg quality, lowering egg pH values and increasing yolk height. A significant difference in laying rate and egg mass (p < 0.02) was registered in the experimental groups vs. the control group. Also, Moraleco et al. [40] included marigold flower (0.8%) and paprika (0.8%) extracts in the diets of 90 Black Avifran hens (60 weeks old) with no significant effects (p > 0.05) concerning egg/shell weight or shell percentage. Further, when 4% paprika extract was used in the diets of Lohmann Brown laying hens aged 30 weeks, the authors declared there were no significant differences either in the feed consumption of the hens or in the quality parameters of the eggs [41]. Hussain et al. [42] supplemented the diets of layer hens (27-week-old) with 4% marigold powder for 60 days, noticing that egg weight was significantly (p < 0.05) impacted by the dietary inclusion of marigold. Maia et al. [37] included four levels of marigold flower extract (2.10; 2.40; 2.70; 3.00 ppm) and noticed that the yolk and Haugh unit linearly increased concomitantly with levels of marigold, whereas the percentage of albumen decreased linearly. Also, Skřivan et al. [30] stated that the addition of marigold extract in the diets of laying hens increased hen egg production and egg weight.

4.4. Dietary Effects of Powder Extracts of Marigold and Red Pepper on Yolk Color

Although the yolk color does not significantly influence the egg’s nutritional value, consumers often associate a darker/intense yellow or golden-orange hue with a healthier egg, rich in natural carotenoids. Worldwide, preferences for yolk color can differ significantly across various countries and regions. The highest amounts of lutein and zeaxanthin, which provide a yellow hue, were registered in the E1 group for marigold extract, and the highest value for the a* redness parameter coincided with the E2 group, red paprika extract. Overall, dietary supplementation in the E1, E2, and E3 groups effectively enhanced yolk pigmentation compared to the C group. Authors Belyavin and Marangos [43] suggested that, for optimal yolk coloration, the hens’ diets should be supplemented with both yellow and red xanthophylls. The marigold flower contains 12 g/kg of total xanthophylls (80 to 90% lutein). Similarly, paprika meal contains 4 to 8 g/kg of total xanthophylls (50 to 70% capsanthin). Our results are similar to those of Skřivan M. et al. [30], who stated that the level of lutein and zeaxanthin increased yolk color parameters. Lokaewmanee et al. [32] noted that incorporating marigold extract into the diet of laying hens at a rate of 0.4% increased yolk redness. The other authors Englmaierová and Skrivan [44] and Skrivan et al. [30] stated that including marigold extract in hens’ diets led to an increased yolk coloration compared to diets based only on maize. Niu et al. [45] noticed a dose-dependent carotenoid concentration in the yolk ranging from 3.43 mg/g to 16.83 mg/g as paprika extract inclusion rates varied from 0.1% to 0.8%. Lokaewmanee et al. [46] found no combined effects of paprika and marigold on yolk coloration, although Moura et al. [47] reported an increase in coloration effects when the two plant extracts were combined.

Oliveira et al. [29] reported that a 0.6% red pepper inclusion rate significantly enhanced yolk color compared to groups uisng only marigold extract or a mixture of paprika and marigold. Furthermore, Lokaewmanee [46,48] considered that red pepper powder could be used as a potent natural colorant for poultry, enhancing yolk coloration. Santos-Bocanegra et al. [49] observed that hens fed with dietary red xanthophylls from capsicum (7.5 ppm) or yellow xanthophylls from Tagetes (4.0 ppm) exhibited intense yolk pigmentation, classified as 11.7, compared to synthetic carotenoids (citranaxanthin, canthaxanthin pigments) at various concentrations, resulting in yolk color ranging from 13 to 14 at the highest concentration. Panaite et al. [28] provided 2% kapia pepper, in a 4-week experiment, to Lohmann Brown layers (43 weeks of age), and registered the highest reddish yolk pigmentation. Furthermore, Grčević et al. [36] tested 0.2% and 0.4% dietary additions of marigold powder extract on laying hens at 31 weeks of age, observing a color intensification due to the positive correlation between the inclusion rate and the increased lutein concentration. Yolk color was evaluated using the RocheFan, rating the color from 1 (very light) to 15 (very dark orange), and the yolk color was found to be 12.66. Maia et al. [37] observed a quadratic effect concerning marigold inclusion (2.73 and 2.80 ppm/kg) when assessed using YolkFan DSM^®, especially regarding redness/yellowness.

4.5. Dietary Effects of Powder Extracts of Marigold and Red Pepper on Antioxidant Profile

In our study, we observed that while the yolks from the group receiving marigold extract exhibited the highest concentrations of lutein and zeaxanthin, the highest values for antioxidant capacity and total polyphenols were found in group E2 compared to group C.

The lutein concentration in red pepper was around 4 times higher compared to marigold, and similar results were found for the antioxidant capacity values, which were approximately 2.3 times higher compared to marigold. According to Biacs et al. [50], the level of antioxidant compound, and the content and composition of carotenoids, are linked to the quality and stability of paprika color. As Daood et al. [51] stated that the yellow color of paprika is attributed to β-carotene and zeaxanthin ester concentrations, while capsorubin, cryptocapsin esters, and capsanthin contribute to the red coloration of carotenoids in red peppers. Wang et al. [52] used 24 kg of dried marigold flower (170.2 g free lutein, 80% purity) and showed via PCL assay that lutein exhibited higher antioxidant activity (0.266 mM Trolox equivalent) compared to β-carotene (0.027 mM) and lycopene (0.018 mM). Additionally, using the b-CLAMS assay, only lutein displayed the inhibition of peroxidation.

4.6. Dietary Effects of Powder Extracts of Marigold and Red Pepper on Oxidative Stability

Our experiment highlighted the effects of marigold and red pepper extract supplementation on lipid oxidation in eggs, as measured by TBARS values, an indicator of lipid peroxidation. The significant TBARS values reduced after 28 days, for all experimental groups (E1, E2, and E3), compared to the C group, and this suggests that the two natural extracts have antioxidant properties, effectively delaying the degradation of yolk fat. No significant differences were found among the experimental groups (E1, E2, and E3), neither at 4 °C nor at 20 °C, after 28 days of storage, and this indicates that both marigold and red pepper extracts, whether used individually or in combination, presented similar effectiveness in reducing lipid oxidation. Other researchers found that natural sources of essential polyunsaturated fatty acid (flaxseed) can be successfully combined with dietary natural antioxidants such as kapia pepper, dried carrot, sea buckthorn pomace [28], thyme [53], pine wood [54] and grape pomace [55,56] to reduce lipid peroxidation in the yolk. The study conducted by Romero et al. [55] concluded that grape pomace demonstrated a higher antioxidant capacity compared to grape extract in yolk. Also, Panaite et al. [28] observed a significant decrease in TBARS values in the groups supplemented with 2% kapia pepper and 2% linseed meal (0.14 mg MDA/kg). Grčević et al. [36] noticed that dietary supplementation with 400 mg/kg of marigold extract led to a slightly reduced oxidation value compared to the control group, but no statistical differences were observed regarding the lipid oxidation levels among fresh egg yolks. Meanwhile, Englmaierová et al. [44] established a significant influence (p < 0.001) of lutein supplementation (250 mg/kg) on the enhancement of the oxidative stability of yolk lipids during a 28-day storage period at 18 °C. Rezaei et al. [57] observed a significant improvement in the oxidative stability of yolks in hens fed marigold pigments after 3 weeks (p < 0.05). Skřivan et al. [30] reported that the dietary inclusion of 950 mg marigold extract/kg reduced lipid peroxidation levels. Additionally, all supplementary levels of marigold flower extract (150, 350, 550 and 750 mg marigold extract/kg diet) notably enhanced the lipid oxidative stability in eggs stored for 28 days at 18 °C. According to Cadun [58], food that is intended for human consumption should have lipid oxidation levels below 3 mg MDA/kg of sample, with a maximum limit of 7–8 mg MDA/kg sample.

5. Conclusions

Marigold and red pepper represent natural sources of carotenoids with the capacity to improve yolk color pigmentation, which is an important criterion of consumer preference, without affecting the health and the productive performances of laying hens. In our study, we obtained decreased TBARS values after 28 days of storage at both 4 and 20 °C, and observed significantly increased yolk coloration in all experimental groups. Therefore, these two natural extracts could enhance egg quality during different storage periods and under different temperature conditions as an alternative to synthetic colorants, with potential applications in animal nutrition, and further benefits for food preservation and human health.