Silky Fowl (Gallus gallus domesticus) Dietary Supplementation with Dried Red Pepper (Capsicum annuum): Effects on Egg Quality, Blood Biochemical Parameters, and Egg Storage Stability

Abstract

Egg yolk coloration influences consumer perceptions of table eggs. Red pepper (Capsicum annuum) is a known dietary source of carotenoids, which enhances yolk pigmentation, but its effects in silky fowl (SF) remain unexplored. We examined how dried red pepper flakes influence blood biochemical parameters and egg quality in SF hens. Sixty hens were divided into three groups: basal ration (control), low supplementation (3.5 mg/100 g), and high supplementation (7.0 mg/100 g). During a 35-day feeding period, eggs collected in the final week were maintained at 4 or 25 °C for quality evaluations. Haugh units and the yolk index were decreased significantly in eggs maintained at 25 °C compared with those in the day 0 and 4 °C samples, whereas the yolk carotenoid content decreased with the storage duration, particularly in the high-supplementation group. CIELAB parameters (L*, a*, a/b), except for b*, were more strongly associated with the ZEN-NOH Yolk Color Chart Score than the DSM Yolk Color Fan Score, particularly in the high-supplementation group. In addition, serum high-density lipoprotein cholesterol was increased, and triglyceride levels were decreased in the high-supplementation group. In conclusion, dietary red pepper flakes enhance yolk pigmentation and lipid metabolism in SF hens. Moreover, ZEN-NOH YCCS provides a reliable indicator of the yolk color after dietary carotenoid supplementation.

1. Introduction

The yolk color is a key sensory and nutritional attribute that indicates egg quality, and it is primarily influenced by dietary carotenoids. In some European and Asian countries, the Roche Yolk Color Fan (RYCF) is applied to evaluate egg yolk pigmentation, with a preferred range of 10–14, and this correlates with both the carotenoid content and antioxidant capacity [1,2,3,4,5]. To achieve the desired yolk coloration, natural and synthetic pigments have been used; however, artificial pigments and saponified carotenoid extracts are prohibited in certain countries, including Sweden and the EU [6,7].

Red pepper (Capsicum annuum) and paprika extracts are well-established carotenoid sources that are particularly known for enhancing yolk redness (a* value) [8,9,10,11]. These additives have also been associated with improved blood lipid profiles in laying hens, including lower concentrations of total cholesterol and triglycerides, alongside elevated high-density lipoprotein cholesterol (HDL-C) levels [12,13,14]. Our previous study demonstrated breed-specific metabolic responses to paprika supplementation in silky fowl (SF; Gallus gallus domesticus), suggesting the potential for targeted nutritional strategies [14].

SF, native to China, are distinct in appearance and metabolism, including silky feathers, black bones, and a dark bluish skin, owing to melanin deposits and pigment granules [15,16]. Their meat and eggs are valued in traditional Chinese medicine for their various health benefits, and they are especially beneficial to women and individuals affected by lung-related diseases [17]. Breed-specific variations in yolk carotenoid contents have also been observed, with more lutein and zeaxanthin deposition within the yolks of SF hens than in those of Rhode Island Red hens [5].

Previous studies have examined the effects of different storage environments and durations on the yolk color of chicken eggs. Although some investigations have demonstrated that the storage duration influences egg yolk pigmentation [18,19,20], other studies found no such effect [21,22,23]. Moreover, studies on the effects of the storage temperature on yolk color are limited [14,18]. Meanwhile, extensive research has investigated the effect of dietary carotenoids on egg quality traits and serum biochemical profiles [3,23,24]. However, such effects in SF remain underexplored [14].

We hypothesized that carotenoid supplementation would enhance yolk carotenoid deposition and improve lipid metabolism, and that these effects would be reflected by the yolk color and egg quality traits. Accordingly, the primary objective of this study was to determine the effects of dietary carotenoid supplementation on the yolk carotenoid content and egg quality in SF hens. The secondary objectives were to: (1) evaluate the influence of the supplementation level on serum lipid profiles; (2) assess the egg quality after storage at 25 °C and examine the storage temperature effects within the high-supplementation group; (3) analyze correlations between yolk color scoring systems and CIELAB color parameters; and (4) improve accessibility for small-scale egg producers by evaluating dried red pepper flakes as a practical carotenoid source.

2. Materials and Methods

2.1. Study Design, Feeding Regimens, and Animal Management

All hens were obtained from the breeding colony of the Tokyo Metropolitan Agriculture and Forestry Research Center (Tokyo, Japan). They were not commercially purchased. Birds were maintained under standard housing conditions prior to the experiment. Experimental protocols were carried out in compliance with the animal experimentation standards of the Tokyo Metropolitan Agriculture and Forestry Research Center. The experiment involved 60 SF hens aged 60 weeks, housed individually in single-bird wire-floor cages (Model M906/4-395-625, Hermann GmbH, Fridingen, Germany) arranged in double-decker rows within a windowless poultry facility. Each cage compartment had internal dimensions of 227 mm (width) × 395 mm (depth) × 625 mm (height), providing a floor area of 896.7 cm² per hen. Each treatment consisted of four replicates, with five birds per replicate (equivalent to a statistical replicate). Prior to the feeding trial, SF hens were given a mash-based basal ration for a 2-week acclimation period (

Table 1. Formulation and nutrient composition of the basal ration (air-dried basis).

Ingredient	Content (%)
Corn	49.70
Brown rice	5.40
Wheat bran	1.00
Defatted rice bran	4.00
Distiller’s dried grains with solubles (corn DDGS)	1.00
Soybean meal	25.20
Fish meal	0.50
Animal fat	2.90
Calcium carbonate	9.10
Dicalcium phosphate	0.76
Salt	0.20
DL-Methionine	0.10
Paprika extract	0.02
Premix and others	0.12
Total	100.00
Nutrient Composition	Value
Metabolizable energy (kcal/kg)	2800
Crude protein (%)	17.20
Crude fat (%)	5.40
Crude fiber (%)	2.60
Crude ash (%)	13.00
Calcium (%)	4.00
Phosphorus (%)	0.57

; feed supplied by JA Zen-Noh Kumiai Feed Co., Ltd., Ota City, Gunma Prefecture, Japan). Following this adaptative period, the hens were subjected to the dietary treatments for 35 days (5 weeks): basal mash ration (Cont: control group), basal mash ration supplemented with 0.6% dried red pepper flakes (LS: low supplementation group, total carotenoid content of 3.5 mg/100 g diet), or basal mash ration supplemented with 1.2% dried red pepper flakes (HS: high supplementation group, total carotenoid content of 7.0 mg/100 g diet).

A commercially available product labeled “Tōgarashi,” consisting of food-grade red pepper flakes, was purchased from a supermarket in Tokyo, Japan. The product was manufactured in China and imported by Kobe Bussan Co., Ltd. (Kakogawa, Japan). Then, the flakes were sieved using No. 8 to 16 mesh screens (2.38–1.19 mm aperture sizes). The carotenoid contents of the two experimental feeds were determined according to a previous report [14]. Throughout the trial phase, hens had free access to feed and water. The cages were designed for individual housing under controlled experimental conditions. Additionally, birds were maintained under a photo period regimen of 16/8 h of light/dark throughout the study. The ambient temperature in the poultry facility was controlled at a constant 22 °C. Productive indicators, including the number of laying hens, egg production rate per day, egg weight, and number of broken and abnormal eggs, were monitored each day. Feed consumption was measured weekly.

2.2. Sample Preparation

Eggs laid during the last 7 days of the 35-day feeding period were used for analysis. They were either assessed within 12 h of laying (designated as day 0) or preserved at 25 °C and examined after 1, 2, 3, 7, 14, or 21 days of storage. The storage duration was defined as the interval between laying and measurements. In total, 210 eggs from 21 treatments (3 diets × 7 storage periods) were analyzed, with 10 eggs examined per treatment.

Reportedly, the egg quality changes during storage, as discussed in the review by Obianwuna et al. [25]. In Japan, consuming raw eggs is a common culinary practice. To prevent food poisoning caused by Salmonella Enteritidis bacteria, strict guidelines limit the consumption of raw eggs and to within 21 days of laying at storage temperatures of 25 °C or less. Therefore, 21 days were selected as the maximum storage duration.

To evaluate the effect of the storage temperature, referring to the report of Heng et al. [23], we compared storage at 25 and 4 °C (refrigerated condition). All collected eggs were preserved by placing them in plastic egg trays (Sanko Co., Ltd., Gifu, Japan), functionally equivalent to the plastic egg trays containers commonly used in poultry research, with the small end down. For each treatment, 10 eggs were analyzed to evaluate yolk pigmentation, the carotenoid concentration, and the pH of both the yolk and albumen.

2.3. Evaluation of Egg Quality Parameters

Egg quality was assessed based on Haugh units (HUs), the yolk index (YI), and yolk pigmentation. Immediately following egg breakage, HUs and the YI were determined using a Digital Egg Tester (DET-6500; NABEL Co., Ltd., Kyoto, Japan). Yolk pigmentation was evaluated using two distinct visual scoring methods: the DSM Yolk Color Fan Score (DSM-YCFS; 1–16) and the ZEN-NOH Yolk Color Chart Score (ZEN-NOH-YCCS; 1–18). For analytical consistency, DSM-YCFS values displayed as “>16” were treated as 17. The ZEN-NOH-YCCS, developed in Japan in 2019, is widely used domestically for quality-control and consumer-preference studies, particularly among small-scale egg producers because of its simplicity. In addition, yolk pigmentation was measured instrumentally with a spectrophotometer (CM-600d; Konica Minolta, Tokyo, Japan) and reported based on the CIELAB system (L*, a*, b*, a/b). These methods were employed not to compare the devices themselves but to provide both practical (subjective scoring) and objective (colorimetric) perspectives on yolk pigmentation. This approach was particularly relevant because dietary supplementation with dried red pepper flakes was expected to enhance yolk redness. This multi-method evaluation allowed for a more comprehensive assessment of this effect. All measurements were performed immediately after breaking the eggs to minimize post-breakage changes.

2.4. pH Measurement

The pH assessment was conducted after separating the yolk and albumen with a household separator, followed by direct measurements using a Horiba D-72 pH meter (Kyoto, Japan).

2.5. Blood Sampling and Biochemical Analysis

At the conclusion of the trial, blood was drawn from the wing veins of 20 hens in each treatment group (four replicates from five birds) without prior fasting. Samples were collected in serum separator tubes without an anticoagulant and centrifuged at 1630× g for 10 min at 4 °C to obtain the serum. The serum was stored at −80 °C until analysis. The total cholesterol (T-Cho), HDL-C, and triglycerides (TG) concentrations were determined using a FUJI DRI-CHEM NX700iV analyzer (Fujifilm, Tokyo, Japan) following the manufacturer’s protocol. Non-HDL cholesterol (T-Cho minus HDL-C) and the HDL/T ratio were subsequently calculated.

2.6. Determination of Yolk Carotenoids

Yolk samples collected after 5 weeks of feeding were analyzed for the total carotenoid content using a UV-visible spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). Specifically, a 1.5 g portion of yolk was placed into a centrifuge tube and vigorously shaken with 30 mL of an acetone–methanol mixture (1:1, v/v). Following homogenization, the extract was transferred to a brown flask. Subsequently, 30 mL of the same solvent mixture was added to the residue and allowed to stand after homogenization. This extraction was repeated thrice, and each fraction was transferred to a brown volumetric flask. After adjusting the volume to 100 mL with the acetone–methanol mixture, the combined extract was passed through a 0.45-μm Millipore filter disk (Merck KGaA, Darmstadt, Germany) and used as the test solution. The carotenoid concentration was determined from absorbance readings at 453 nm, and the total quantity of carotenoids was assessed based on the optical density at a λmax of 450–470 nm. Total carotenoids were quantified using the extinction coefficients (absorbance of 1% concentration: 2400), as previously described [26].

2.7. Statistical Analysis

All statistical procedures were conducted using R (version 4.4.2; accessed on 31 October 2024, http://www.R-project.org [27]). The effects of the storage duration and dietary treatment were assessed through two-way analysis of variance (ANOVA). Blood biochemical values were determined by performing a one-way ANOVA. The internal egg quality of freshly laid eggs (0 days of storage) and eggs stored for 21 days at either 4 or 25 °C was compared by performing a one-way ANOVA, treating the “storage condition” as a single fixed factor with three levels (fresh, 4 °C for 21 days and 25 °C for 21 days). Freshly laid eggs were not subjected to any storage treatment; therefore, the “storage temperature” and “storage duration” did not form a fully crossed factorial design and could not be analyzed by performing a two-way ANOVA. The overall effects of treatments were evaluated by performing a one- or two-way ANOVA, and when a significant main effect was detected, pairwise group differences were examined using Tukey’s honestly significant difference post hoc test. Pearson’s correlation coefficients were computed to evaluate associations among the variables. Additionally, correlations between the YCFS (DSM-YCFS and ZEN-NOH-YCCS) and CIELAB color parameters were analyzed across all dietary groups (Cont, LS, HS) using eggs kept at 25 °C for 0–21 days. Statistical significance was set at p < 0.05.

3. Results

3.1. Yolk Pigmentation and Carotenoid Levels in Raw Eggs Maintained at 25 °C

Table 2. Effects of storage at 25 °C on yolk color and total yolk carotenoid content in SF ¹ eggs.

		Storage Period (Day)							SEM	p-Value 2			r 4
Item	Diet 3	0	1	2	3	7	14	21		AN	L	Q
YCFS 5	Cont	10.2 ab,y	10.0 b,y	10.0 b,y	10.2 ab,y	10.2 ab,y	11.0 a,y	10.4 ab,y	0.09	0.02	0.01	0.07	0.41
	LS	14.6 x	14.5 x	13.9 x	14.0 x	14.6 x	14.2 x	13.7 x	0.14	0.43	0.18	0.34	−0.16
	HS	15.3 x	15.3 x	14.3 x	14.7 x	14.8 x	14.7 x	14.4 x	0.13	0.24	0.13	0.30	−0.18
		Dose effect, p < 0.00; Time effect, p = 0.07; Interaction, p = 0.77
T-Caro 6	Cont	3.1 z	2.9 z	3.0 y	3.0 y	3.1 z	3.1 z	2.8 z	0.07	0.98	0.80	0.64	−0.04
	LS	4.3 y	4.0 y	4.7 x	4.8 x	4.6 y	4.2 y	4.2 y	0.12	0.41	0.46	0.50	−0.09
	HS	6.6 a,x	6.3 ab,x	5.2 b,x	5.3 b,x	5.5 ab,x	5.3 b,x	5.3 ab,x	0.14	0.00	0.02	0.01	−0.27
		Dose effect, p < 0.00; Time effect, p = 0.19; Interaction, p = 0.02

Different superscript letters for the same items in rows (a–b) and columns (x–z) indicate significant differences (p < 0.05, n = 10). ¹ SF = silky fowl. ² AN = ANOVA; L = linear; Q = quadratic. SEM = standard error of the mean. ³ Cont: control group = basal ration; LS: low-supplementation group = 3.5 mg total carotenoid/100 g feed; HS: high-supplementation group = 7.0 mg total carotenoid/100 g feed. ⁴ Pearson’s r. ⁵ Yolk color was determined based on the DSM-YCF score (1–16) and set to 17 when the score exceeded 16. ⁶ Total carotenoid content in egg yolk (mg/100 g yolk).

summarizes the effects of storage at 25 °C on yolk pigmentation (DSM-YCFS) and the total yolk carotenoid content (T-Caro) in eggs produced by SF hens fed diets containing different levels of red pepper flakes. Two-way ANOVA revealed significant effects of dietary supplementation on both the YCFS (p < 0.001) and T-Caro (p < 0.001). Across all storage durations, the low- and high-supplementation groups maintained consistently higher YCFS values than the controls, indicating the clear dose-dependent enhancement of yolk color. The storage duration showed only a marginal effect on the YCFS (p = 0.07), with no significant diet × time interaction (p = 0.77).

In contrast, the T-Caro exhibited a significant diet × time interaction (p = 0.02). The high-supplementation group had the highest initial carotenoid levels (day 0), but the levels declined significantly during storage, particularly by days 2, 3, and 14, suggesting measurable degradation during short-term storage at 25 °C. By comparison, the carotenoid content in the control and low-supplementation groups remained stable throughout the 21-day period.

3.2. Influence of Storage Temperature on Yolk Carotenoid Levels and Egg Quality

Table 3. Influence of storage temperature on yolk carotenoid content and egg quality in SF ¹ eggs (high-supplementation group).

Storage		T-Caro 2	YCFS 3		L* 4	a* 4	b* 4	a/b 4	ApH 5	YpH 6	HUs	YI
Temp	Period		DSM	ZEN-NHO
--	0	6.57 a	15.3	15.5 ab	52.1	26.3	41.0	0.65	8.6 c	6.0 a	78.9 a	0.48 a
4 °C	21	5.92 ab	15.3	15.3 b	50.4	25.1	42.0	0.61	9.1 b	5.9 b	76.6 a	0.47 a
25 °C	21	5.30 b	14.4	16.3 a	50.6	28.2	43.6	0.67	9.8 a	6.1 a	58.2 b	0.23 b
SEM		0.19	0.24	0.17	0.57	0.59	0.59	1.16	0.10	0.02	1.83	0.02
p-Value		0.02	0.21	0.03	0.42	0.10	0.10	0.66	<0.001	<0.001	<0.001	<0.001

^a–c Values in the same column marked with different letters are statistically distinct (p < 0.05, n = 10). ¹ SF = silky fowl. SEM = standard error of the mean; HUs = Haugh units; YI = yolk index. ² Total carotenoid content in egg yolk (mg/100 g yolk). ³ DSM = the yolk color was determined based on the DSM-YCF score (1–16) and set to 17 when the score exceeded 16; ZEN-NHO = the yolk color was determined based on the ZEN-NHO-YCC score (1–18). ⁴ L* (lightness), a* (redness), b* (yellowness), a/b (redness-to-yellowness ratio). ⁵ ApH: albumen pH. ⁶ YpH: yolk pH.

shows the influence of the storage temperature on yolk carotenoid levels and the internal quality of eggs from SF hens in the high-supplementation group. A significant difference (p < 0.05) in total carotenoid content of egg yolk was observed between eggs on the day of laying (day 0) and those stored at 25 °C, whereas eggs stored at 4 °C did not differ significantly (p > 0.05) from either day-0 eggs or those stored at 25 °C. Yolk color measurements based on the ZEN-NOH YCCS revealed significant differences among the storage groups (p = 0.03). Eggs kept at 25 °C had higher ZEN-NOH YCCS values than those kept at 4 °C, suggesting a perceptible shift in pigmentation despite a decrease in the carotenoid content.

Among the yolk color parameters, the a* and b* values tended to change during storage (p = 0.10), suggesting possible shifts in the intensity of red and yellow coloration. Although the L* value did not change significantly, the a/b ratio tended to increase in samples kept at 25 °C. Egg quality traits, particularly the albumen pH and HUs, were markedly influenced by the storage conditions. The albumen pH was significantly higher in eggs kept at 25 °C (p < 0.001), which was accompanied by a substantial decline in HU scores, reflecting deterioration of the albumen quality. Additionally, the YI was significantly decreased (p < 0.001) in the 25 °C samples compared with that in the day 0 and 4 °C storage groups, suggesting a weakened yolk structure during higher-temperature storage.

3.3. Correlations Between Yolk Color Scores and CIELAB Parameters

As the egg yolk color is a key quality trait that is directly influenced by dietary carotenoids, we further examined the relationship between subjective fan scores (YCFS) and objective CIELAB parameters. This analysis determined whether supplementation-induced changes in yolk pigmentation could be consistently captured using different evaluation methods. Correlations between YCFS (DSM-YCFS and ZEN-NOH-YCCS) and CIELAB parameters were analyzed across all dietary groups (Cont, LS, HS) using eggs kept at 25 °C for 0–21 days (

Table 4. Correlations between YCFS and CIELAB parameters for different red pepper supplementation levels: comparison of DSM and ZEN-NOH methods.

Group 1	Method 2	L* 3 (r, Cl, p) 4	a* 3 (r, CI, p)	b* 3 (r, CI, p)	a/b 3 (r, CI, p)
Cont	DSM	−0.35 [−0.61, −0.02], 0.039	0.55 [0.26, 0.74], <0.001	−0.01 [−0.34, 0.33], 0.961	0.69 [0.46, 0.83], <0.001
	ZEN-NHO	−0.58 [−0.77, −0.31], <0.001	0.56 [0.27, 0.75], <0.001	−0.08 [−0.41, 0.26], 0.632	0.80 [0.64, 0.90], <0.001
LS	DSM	−0.49 [−0.65, −0.29], <0.001	0.44 [0.23, 0.61], <0.001	−0.51 [−0.66, −0.31], <0.001	0.72 [0.59, 0.82], <0.001
	ZEN-NHO	−0.51 [−0.67, −0.31], <0.001	0.78 [0.67, 0.86], <0.001	−0.27 [−0.47, −0.03], 0.025	0.88 [0.81, 0.92], <0.001
HS	DSM	−0.46 [−0.62, −0.25], <0.001	0.47 [0.26, 0.63], <0.001	−0.55 [−0.70, −0.37], <0.001	0.60 [0.42, 0.73], <0.001
	ZEN-NHO	−0.77 [−0.85, −0.66], <0.001	0.86 [0.78, 0.91], <0.001	−0.46 [−0.62, −0.24], <0.001	0.85 [0.77, 0.90], <0.001
Pooled	DSM	−0.72 [−0.78, −0.63], <0.001	0.85 [0.80, 0.89], <0.001	−0.55 [−0.65, −0.44], <0.001	0.88 [0.85, 0.91], <0.001
	ZEN-NHO	−0.73 [−0.80, −0.66], <0.001	0.91 [0.88, 0.93], <0.001	−0.48 [−0.59, −0.36], <0.001	0.90 [0.87, 0.93], <0.001

Storage temperature and sampling days: 25 °C; days 0, 1, 2, 3, 7, 14, and 21 (n = 70). ¹ Cont: control group = basal ration; LS: low-supplementation group = 3.5 mg total carotenoid/100 g feed; HS: high-supplementation group = 7.0 mg total carotenoid/100 g feed. ² DSM = the yolk color was determined based on the DSM-YCF score (1–16) and was set to 17 when the score exceeded 16; ZEN-NHO = the yolk color was determined based on the ZEN-NHO-YCC score (1–18). ³ L* (lightness), a* (redness), b* (yellowness), a/b (redness-to-yellowness ratio). ⁴ r = Pearson’s correlation coefficient; CI = 95% confidence interval for the correlation coefficient (r); p = p-value.

).

In the high-supplementation group, the DSM-YCFS showed moderate negative correlations with L* (r = −0.46, p < 0.001) and b* (r = −0.55, p < 0.001) and moderate positive associations with a* (r = 0.47, p < 0.001) and a/b (r = 0.60, p < 0.001). The ZEN-NOH YCCS was associated with stronger correlations: a marked inverse correlation with L* (r = −0.77, p < 0.001), a very strong positive correlation with a* (r = 0.86, p < 0.001), a moderate negative correlation with b* (r = −0.46, p < 0.001), and a marked positive correlation with a/b (r = 0.85, p < 0.001). Similar patterns were observed in the control and low-supplementation groups, with ZEN-NOH consistently showing stronger correlations with L*, a*, and a/b values, but not for b*, than DSM.

3.4. Blood Biochemical Parameters

Dietary carotenoid levels significantly influenced selected serum lipid parameters in SF hens (

Table 5. Effect of feeding red pepper flakes on biochemical values in SF ¹ hens.

	T-Cho	HDL-C	TG	non-HDL	HDL/T
Diet 2	mg/dL	mg/dL	mg/dL	mg/dL	Ratio
Cont	108.9	47.2 b	1374.9 a	61.7	0.47
LS	113.2	52.8 b	1169.7 a	60.4	0.50
HS	136.9	76.5 a	192.9 b	60.4	0.58
SEM	6.4	3.6	133.3	5.5	0.02
ANOVA 3	0.16	<0.001	<0.001	1.00	0.16
Linear	0.06	<0.001	<0.001	0.94	0.06
Quadratic	0.16	<0.001	<0.001	1.00	0.16
r 4	0.27	0.49	−0.54	−0.01	0.27

^a,b Values in the same column marked with different letters are statistically distinct (p < 0.05, n = 20). T-Cho, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride; non-HDL, T-Cho minus HDL-C; HDL/T, ratio of HDL-C to T-Cho. ¹ SF = silky fowl. ² Cont: control group = basal ration; LS: low-supplementation group = 3.5 mg total carotenoid/100 g feed; HS: high-supplementation group = 7.0 mg total carotenoid/100 g feed. ³ One-way analysis of variance. ⁴ Pearson’s r.

). High carotenoid supplementation caused marked alterations in HDL-C and TG concentrations. HDL-C concentrations were markedly increased in the high-supplementation group (76.5 mg/dL), compared with those in the control (47.2 mg/dL) and low-supplementation (52.8 mg/dL) groups (p < 0.05). Additionally, the HDL-C-to-total cholesterol ratio (HDL/T) tended to increase with increasing dietary carotenoid levels (0.47, control; 0.50, low; 0.58, high), showing a linear trend (p = 0.06), although differences among groups were not significant (p = 0.16). Similarly, T-Cho and non-HDL cholesterol levels were not significantly different (p > 0.05). Pearson’s correlation values indicated a moderate positive correlation between carotenoid levels and HDL-C (r = 0.49) and a moderate negative correlation between carotenoid levels and TGs (r = −0.54), confirming that increased carotenoid intake improved blood lipid profiles in SF hens.

4. Discussion

In this study, we investigated the effect of dietary carotenoid addition, using dried red pepper flakes, on egg quality traits and blood lipid profiles in SF hens. This supplementation enhanced yolk pigmentation, consistent with previous reports using paprika extract or other carotenoid-rich additives [8,14,23]. Similar to findings by Heng et al. [23] and Shevchenko et al. [28], the higher yolk color scores were maintained throughout storage in the supplemented groups, reinforcing the well-established relationship between dietary carotenoid intake and yolk pigmentation stability. Although the total carotenoid content declined during storage at 25 °C, yolk color scores remained elevated, particularly in the high-supplementation group. This pattern aligns with earlier observations that the visual yolk color does not always decline proportionally with carotenoid degradation during storage [21,29], suggesting that pigment stability and visual perception may be associated with different kinetics. Differences from previous studies may also be attributable to the carotenoid source, as saponified paprika extract contains free carotenoids with higher bioavailability, whereas red pepper flakes provide bound carotenoids with greater stability during storage [14,30,31].

Egg quality traits were also affected by the storage conditions. HUs and the YI remained stable over the 21-day period at 4 °C, whereas the albumen pH increased significantly at 25 °C, accompanied by declines in HUs and the YI. These results are consistent with classical reports showing that elevated storage temperatures accelerate CO₂ loss and albumen thinning [32,33], ultimately reducing HUs and the YI. Our findings therefore support previous conclusions that refrigerated storage is essential for maintaining egg freshness and functional properties [34,35], while also demonstrating that yolk carotenoid retention is similarly temperature-dependent. Of note, shell quality traits such as the shell strength and thickness were not evaluated in this study, and we acknowledge this as a limitation that should be addressed in future research.

A correlation analysis across all dietary groups (Table 4) revealed that the ZEN-NOH YCCS showed stronger associations with CIELAB parameters than the DSM-YCFS, particularly for L*, a*, and a/b ratios, supporting its utility as a region-specific tool for evaluating yolk pigmentation. This is relevant for markets where high redness (a*) is preferred, such as East Asia and Southern Europe.

Dietary carotenoid supplementation also influenced blood lipid metabolism. High supplementation (7.0 mg/100 g feed) significantly increased HDL-C and decreased TG levels, consistent with previous findings in poultry research [36]. Similar lipid-modulating effects of carotenoids, such as astaxanthin and capsanthin, have been reported in laying hens [3,23], supporting the notion that carotenoid-rich feed ingredients exert antioxidative and hypolipidemic effects. Moreover, the moderate correlations between dietary carotenoid levels and HDL-C (r = 0.49) and TG (r = −0.54) levels suggest a dose-dependent physiological response. This trend aligns with earlier reports demonstrating dose-responsive changes in lipid metabolism following carotenoid supplementation [3]. No significant changes were observed in T-Cho or non-HDL cholesterol levels, which may reflect the breed-specific metabolic characteristics of SF hens [14,16]. A similar pattern—where carotenoid supplementation primarily affects HDL-C and TG levels but not total cholesterol—has been noted in previous studies [36], suggesting that carotenoid-induced lipid modulation may selectively target specific lipoprotein pathways. The reproducibility of the low TG value in the HS group was confirmed based on a re-analysis of stored samples, strengthening confidence in these results. Previous studies have demonstrated that red pepper supplementation can reduce yolk TG and cholesterol levels in laying hens [37]. Although yolk cholesterol was not measured in the present study, these findings suggest that Capsicum additives may influence both the serum and yolk lipid fractions. Red pepper contains bioactive compounds with antioxidant and metabolic regulatory properties, which may contribute to the observed modulatory effect on lipid metabolism, including reductions in serum cholesterol and improvements in HDL levels [37].

Although the gut morphology and inflammatory markers were not directly assessed in this study, previous work has shown that moderate levels of Capsicum-derived ingredients do not induce intestinal inflammation in poultry and may even exert anti-inflammatory and mucosal-supportive effects [38]. Therefore, the dried red pepper levels used in our experiment are unlikely to have caused adverse gastrointestinal responses. Furthermore, because the basal diet contained a trace amount of paprika extract (0.02%), the control feed did not represent a completely carotenoid-free diet but rather contributed to a condition with background-level carotenoids. This should also be recognized as a limitation of the study, and the present findings should therefore be interpreted as the effects of additional carotenoid supplementation relative to this low-dose background level, rather than as absolute differences between carotenoid-free and carotenoid-supplemented diets. Overall, the use of commercially available dried red pepper flakes provided carotenoid concentrations that were sufficient to improve yolk pigmentation and blood lipid profiles, offering a practical alternative to bulk paprika extracts for small-scale producers. However, this study was limited to a 35-day feeding period and a single breed. Long-term and multi-breed trials are warranted to confirm the broader applicability of red pepper supplementation. In addition, the basal diet contained a small amount of paprika extract (0.02%), and the potential contributions of other bioactive compounds in red pepper flakes, such as vitamins, minerals, or capsaicin, were not assessed. Further studies are required to clarify these additional effects.

5. Conclusions

Dietary supplementation with dried red pepper flakes enhanced the yolk color and improved selected blood lipid parameters in SF hens. Moreover, supplementation at 7.0 mg of carotenoids/100 g of feed significantly increased HDL-C levels and decreased TG concentrations, indicating beneficial effects on lipid metabolism. Further, the yolk color was consistently elevated in supplemented groups, and the strong correlations between CIELAB parameters and the ZEN-NOH Yolk Color Chart Score suggest that this region-specific chart provides a reliable tool for evaluating yolk pigmentation, particularly in eggs with high values induced by dietary carotenoids. Together, these findings highlight the value of incorporating carotenoid-rich natural additives to promote egg quality and support poultry health.