Resveratrol supplementation mitigates age-related declines in egg production, quality, and ovarian health of laying hens

Abstract

This investigation focused on how resveratrol (RES) influences egg production, quality, reproductive hormone, and antioxidant capacity in laying hens of different ages. A completely randomized experiment was conducted with a 2 × 2 factorial arrangement of treatments, including 47- and 67-week-old Lohmann Gray laying hens (initial body weight 1.58 ± 0.12 kg) and two dietary treatments (0 and 400 mg/kg RES) for 12 weeks. The study involved 216 hens, randomly allocated with nine replicates per treatment and six hens per replicate. Results indicated that older (67-week old) laying hens exhibited a higher feed-to-egg ratio and defective egg rate during weeks 1 to 12 than 47-week-old without RES supplementation (interaction, P = 0.012 and P = 0.047), while addiction with RES significantly reduced feed-to-egg ratio in 67-week-old hens (P < 0.05). Significant interactions between age and RES were observed for egg quality parameters including albumen height, eggshell strength, and eggshell thickness (P < 0.05). The RES supplementation improved feed efficiency and egg quality (P < 0.05), particularly in older hens. Haugh unit was restored by RES in aged birds during weeks 9 to 12 (interaction, P = 0.023). Plasma levels of melatonin (MT), estradiol (E2), and anti-Müllerian hormone (AMH) also showed significant interactions (P < 0.05), with RES increasing these hormones only in older hens. Age and RES supplementation had a significant interaction for ovarian malondialdehyde (MDA) levels (P < 0.001), where RES significantly reduced MDA concentrations in older hens (P < 0.05). At the molecular level, RES significantly upregulated the mRNA expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and NAD(P)H quinone oxidoreductase 1 (NQO1; P < 0.01), while aging upregulated tumor necrosis factor-α (TNF-α) expression (P = 0.018). RES also suppressed IL-6, IL-8, TNF-α, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) expression, and enhanced anti-apoptotic genes B-cell lymphoma-extra-large and B-cell lymphoma 2 expression (P < 0.05). In conclusion, RES supplementation (400 mg/kg) effectively improved laying performance, egg quality, and oxidative and inflammatory responses in laying hens, suggesting its potential as a nutritional strategy to support laying performance during the late production stages.

1. Introduction

Age-related deterioration in egg-laying performance and egg quality presents a major challenge in the laying hen industry, directly impacting economic efficiency and sustainability (Liu et al., 2025; Sinclair-Black et al., 2023). This decline is driven by age-related physiological deterioration, primarily characterized by accelerated ovarian follicular atresia, impaired hepatic antioxidant capacity, and chronic low-grade inflammation (Bendikov-Bar et al., 2021; Gu et al., 2021). In commercial flocks, hens typically achieve peak egg production (85% to 90% laying rate) at 50 weeks of age, with a progressive downturn occurring thereafter, with egg production dropping to 60% to 70% during 60 to 70 weeks (Bell and Adams, 1992). During this critical transition, oxidative stress accumulation and dysregulated immune responses further exacerbate follicular degradation and metabolic dysfunction, ultimately reducing eggshell and albumen quality (Xu et al., 2023). Collectively, these age-associated changes not only compromise reproductive efficiency but also escalate production costs, underscoring the urgent need for targeted interventions to mitigate physiological decline and extend laying cycles.

Current research in laying hens primarily focuses on strategies to sustain egg quality and productivity (Bain et al., 2016). Among these approaches, nutritional interventions have gained increasing attention, as dietary composition significantly influences systemic health and reproductive efficiency observed within the laying period of hens (Cai et al., 2013; Wang et al., 2025). It has been well documented that phytogenic feed additives can enhance laying performance, egg characteristics and intestinal health in aging hens, making them a valuable nutritional strategy for improving laying performance (Sharma et al., 2020). Additionally, the use of phytogenic additives contributes to better antioxidant activity and lipid metabolism in laying hens, which are critical factors in sustaining egg quality as hens age (Li et al., 2022).

Resveratrol (RES)–classified as a non-flavonoid polyphenol and is widely distributed among phytonutrient-rich foods such as fruits and edible greens–has emerged as a bioactive molecule exhibiting antioxidant capacity and inflammation-modulating properties, and is thought to be effective in preventing senescence and its associated pathologies (Kuršvietienė et al., 2016; Li et al., 2018; Vikal et al., 2024). The aging process is significantly influenced by oxidative stress. Previous studies have reported that RES supplementation mitigates oxidative stress-induced declines in ovarian physiology, digestive function, and overall productivity by enhancing antioxidant enzyme activity, reducing inflammation, and upregulating barrier-related and reproductive hormone receptor genes in laying hens (Ding et al., 2022; Wang et al., 2022). Ortega et al. (2012) observed that RES can promote rat ovarian granulosa cell viability and proliferation.

Although RES has garnered growing attention as a dietary supplement, its impact on productivity traits, egg characteristics, and ovarian physiology across different laying stages has not been fully elucidated. Elucidating how RES influences these physiological functions at varying ages is essential to refine its nutritional application in poultry management. Therefore, the present study was designed to evaluate the effects of dietary RES to enhance performance, maintain egg quality, and support ovarian health in laying hens of different ages, primarily via its antioxidative and anti-inflammatory actions. Based on previous studies demonstrating that 400 mg/kg RES effectively improved hepatic antioxidant status and alleviated inflammation in laying hens (Wang et al., 2020; Xing et al., 2020), this dosage was adopted to investigate its age-specific effects on ovarian function and reproductive performance. The results are expected to inform targeted nutritional strategies to extend laying lifespan and improve reproductive outcomes in commercial poultry operations.

2. Materials and methods

2.1. Animal ethics statement

Experimental protocols involving animals complied with the Chinese guidelines for animal welfare and received ethical approval from the Animal Care and Use Committee of Sichuan Agricultural University (approval No. 20221116004).

2.2. Birds, experiment design, and management

A 2 × 2 factorial arrangement of treatments was applied in a completely randomized design to evaluate the effects of RES supplementation on Lohmann Gray laying hens at ages 47 and 67 weeks. The initial average body weight was 1.58 ± 0.12 kg. A total of 216 healthy hens, sourced from the same parental generation, were housed under uniform environmental conditions and fed identical basal diets. The experimental setup comprised two dietary treatments: a control diet (CON; 0 mg/kg RES) and a RES-supplemented diet (RES; 400 mg/kg RES), administered over a 12-week period. Each age group (47- and 67-week-old hens) contained 108 birds, which were randomly assigned to two distinct feeding regimens, each comprising nine replication units with six hens per units. The RES used in this study, procured from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) with a chemical purity reaching 98%.

A multi-tier stacked cage system (45 cm × 45 cm × 43 cm) was used, with three birds per cage and each group consisting of two adjacent cages. All experimental hens were placed in the middle tier to eliminate positional differences. The environmental temperature was maintained at 20 to 22 °C, 16:8 light–dark cycle, stable ventilation, and weekly hygiene management.

2.3. Nutrient composition of the diet

A powdered diet with a mean granule diameter of approximately 4.5 mm was employed in this research. The basal diet, composed primarily of corn and soybean meal, was designed conformed to NRC (1994) and the Chinese Chicken Feeding Standard NY/T 33-2004 (Ministry of Agriculture of the People's Republic of China, 2004). The nutrient composition of the diet is detailed in Table 1. The metabolizable energy (ME) was calculated based on standard feed composition values from the 32nd edition of the Feed Composition and Nutritional Value Table (China Feed Database, 2021), using the formula:

$$\text{ME}=\text{Corn}\times {\text{ME}}_1+\text{Soybean}\text{meal}\times {\text{ME}}_2+\dots +\text{Vitamin}\text{and}\text{mineral}\text{premix}\times {\text{ME}}_{\mathrm{n}}\text{.}$$

Table 1.

Composition and nutrient levels of basal diet (%, as-fed basis)¹.

Items	Content
Ingredients
Corn	56.40
Soybean meal (43%)	25.50
Wheat bran	4.80
Soybean oil	2.34
Calcium carbonate	8.30
Calcium hydrophosphate	1.50
L-Lysine hydrochloride	0.04
DL-Methionine	0.14
Threonine	0.04
Tryptophan	0.01
NaCl	0.30
Choline chloride (50%)	0.10
Vitamin and mineral premix1	0.53
Total	100.00
Nutrients
ME2, MJ/kg	11.31
CP	16.58
Ca	3.55
Available P	0.38
Lysine	0.79
Methionine	0.36
Threonine	0.58
Tryptophan	0.18
Methionine + cysteine	0.59
Digestible AA3
Lysine	0.78
Methionine	0.58
Threonine	0.74
Tryptophan	0.46

ME = metabolizable energy; CP = crude protein; AA = amino acid.

¹Provided per kg of diet: vitamin A, 10,000 IU; vitamin D₃, 3000 IU; vitamin E, 30 IU; vitamin K₃, 4.8 mg; thiamin, 3.0 mg; riboflavin, 9.6 mg; pyridoxine, 6 mg; vitamin B₁₂, 0.3 mg; folic acid, 1.5 mg; niacin, 60 mg; pantothenic acid, 18 mg; biotin, 0.6 mg; iron, 60 mg; copper, 8 mg; manganese, 60 mg; zinc, 80 mg; selenium, 0.30 mg; iodine, 0.35 mg.

²Calculated according to the Chinese Feed Composition and Nutritional Value Table (China Feed Database, 2021).

³Digestible amino acid concentrations were estimated based on standardized ileal digestibility coefficients reported in the Chinese Feed Composition and Nutritional Value Table (China Feed Database, 2021).

Crude protein (CP) was determined using the Kjeldahl method (method 990.03; AOAC, 2005) with a nitrogen analyzer (LECO CNS-2000, LECO Corp., St. Joseph, MI, USA). Ca concentration was quantified by flame atomic absorption spectrometry (ZEEnit 700P, Analytik Jena AG, Jena, Germany), following GB/T 13885-2017 (China National Standard, 2017), while total P was measured spectrophotometrically (UV-2700, Shimadzu Corp., Kyoto, Japan) according to GB/T 6437-2018 (China National Standard, 2018). Amino acids including lysine, methionine, threonine, and tryptophan were included via crystalline forms (L-lysine hydrochloride, DL-methionine, threonine, and tryptophan) and analyzed based on calculated inclusion levels and confirmed by acid hydrolysis and subsequent analysis using high-performance liquid chromatography (HPLC; Agilent 1260 Infinity II, Agilent Technologies Inc., Santa Clara, CA, USA) where applicable (ISO, 2005). The sum of methionine and cysteine was reported as total sulfur amino acids (TSAA). Digestible amino acid contents were calculated by multiplying the total contents of lysine, methionine, threonine, and tryptophan by their respective standardized ileal digestibility (SID) coefficients (Adedokun et al., 2014). These coefficients were adopted from the Chinese Feed Composition and Nutritional Value Table (China Feed Database, 2021).

2.4. Sample collection

Daily monitoring of egg quality included measurements of egg weight and classification of shell defects. Weekly records per replicate were compiled, and productivity metrics such as laying performance, feed conversion capacity, and egg quality indicators were calculated in accordance with the Poultry Performance Measurement Standards (NY/T 823–2020, Ministry of Agriculture of the People's Republic of China, 2020). At the conclusion of the 12-week feeding trial, blood (10 mL) was obtained by venipuncture of either the jugular or brachial vein from one randomly selected hen per replicate (n = 9 per treatment). After clotting at ambient temperature for 1 h, samples were centrifuged (3000 × g, 15 min) to separate serum and plasma; both fractions kept at −20 °C until subjected to biochemical evaluation. Ovarian and liver samples were obtained post-exsanguination, frozen at once using liquid nitrogen, and kept at −80 °C until further analysis.

2.5. Determination of egg quality

Egg quality was evaluated at weeks 4, 8, and 12, based on albumen height, egg weight, Haugh unit, and shell strength and thickness. For each time-point, 27 eggs (three per replicate) were obtained from each treatment group over three consecutive days. All eggs were analyzed individually, with each egg serving as a biological replicate for quality assessment. Freshly laid eggs (within 2 h of oviposition) were cleaned, allowed to stabilize at ambient temperature (25 ± 1 °C) for 30 min, and analyzed using standardized procedures. Egg weight was determined in triplicate using a precision digital scale (Model FA1604N, Shanghai Precision Scientific Instruments, Shanghai, China). Albumen height and Haugh units determined with an Egg Multi-tester (EMT-5200, Robotmation Co., Ltd., Tokyo, Japan). Eggshell strength tested using an eggshell force gauge (Model II, Robotmation Co., Ltd., Japan). Eggshell thickness was assessed using a Peacock dial gauge (Model P-1, Meg Co., Ltd., Tokyo, Japan) after membrane separation. Thickness values were averaged from three positions: blunt pole, apex, and equatorial zone. For eggshell analysis, shells were isolated from albumen and yolk, cleaned of residual material, after drying at 65 °C for 4 h in an oven, the samples were subjected to weighing. All procedures followed the NY/T 823-2020 Poultry Performance Measurement Standards (Ministry of Agriculture of the People's Republic of China, 2020). Instruments were calibrated prior to measurements to ensure accuracy.

2.6. Determination of plasma reproductive hormone

Reproductive hormone in plasma, including progesterone (Prog; H089-1-2), melatonin (MT; H256-1-2), luteinizing hormone (LH; H206-1-2), follicle-stimulating hormone (FSH; H101-1-2), estradiol (E2; H102-1-2), and anti-Müllerian hormone (AMH; H324-1-2), were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bio Engineering Institute, Nanjing, Jiangsu, China). Absorbance was measured using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA).

2.7. Determination of antioxidant capacity

The antioxidant capacity of ovarian and hepatic tissues was assessed using commercial ELISA kits (Nanjing Jiancheng Bio Engineering Institute, Nanjing, Jiangsu, China). For ovarian tissues, the following parameters were quantified: glutathione (GSH; A061-1-2), glutathione peroxidase (GSH-Px; A005-1-2), glutathione S-transferase (GST; A004-1-1), and malondialdehyde (MDA; A003-1-2). For hepatic tissues, the antioxidant profile included GSH (A061-1-2), GSH-Px (A005-1-2), superoxide dismutase (SOD; A001-3-2), and MDA (A003-1-2). Absorbance was measured using a microplate reader (Synergy H1).

2.8. Quantitative real-time PCR analysis of ovarian function–related gene expression

Total RNA was isolated from ovarian tissues (one hen per replicate, nine replicates per treatment) using TRIzol reagent (Takara Bio Inc., Dalian, Liaoning, China) and quantified spectrophotometrically (NanoDrop 2000; Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA integrity was verified by ensuring A260/A280 ratios between 1.8 and 2.0. Complementary DNA (cDNA) synthesis was performed with the PrimeScript RT Reagent Kit (RR047A, TaKaRa Bio Inc., Dalian, Liaoning, China) with genomic DNA removal performed. Quantitative real-time PCR amplification was conducted on an ABI Prism 7000 system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq II (Takara Bio Inc., Dalian, Liaoning, China). Target genes included HO-1, Nrf2, NQO1, IL-6, IL-8, TNF-α, Bcl-XL, Bcl-2, and NF-κB, with primer sequences provided in Table S1. The full names of the abbreviations of all genes can be found in Table S2. To ensure reliable normalization in gene expression analysis, two reference genes, β-actin and GAPDH, were initially tested across all experimental samples. Expression stability was evaluated, and β-actin was selected as the reference gene due to its consistent Ct values and minimal variation. All target gene expressions were normalized using β-actin and calculated via the 2^−ΔΔCt method. The PCR amplification was performed with an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. A melt curve analysis was performed to confirm amplification specificity.

2.9. Statistical analysis

Statistical analyses were performed through two-way ANOVA employing the general linear model (GLM) in SAS software (version 9.2, SAS Institute Inc., Cary, NC, USA) complemented by GraphPad Prism (v10.0, GraphPad Software LLC., San Diego, CA, USA). The statistical model used was:

$$Y_{ijk}=\mu +A_i+T_j+{\left(A\times T\right)}_{ij}+{\epsilon }_{ijk}\text{,}$$

where Y_ijk represents the observed value of the dependent variable for the k-th replicate under the i-th level of hen age and j-th level of RES supplementation; μ is the overall mean; A_i is the fixed effect of hen age; T_j is the fixed effect of RES supplementation level; (A × T)_ij represents the interaction effect between hen age and RES supplementation; ε_ijk is the random error term assumed to be normally distributed with mean zero and constant variance.

Data normality and homogeneity of variance were assessed using the Shapiro–Wilk and Levene's tests, respectively. A two-way ANOVA based on least squares means was performed to evaluate the effects of age, RES supplementation, and their interaction. When a significant interaction was detected (P < 0.05), multiple comparisons among the four treatment groups were conducted using Tukey–Kramer's HSD test. Differences were considered significant at P < 0.05 and a trend at 0.05 ≤ P < 0.10.

3. Results

3.1. Egg production performance

During weeks 1 to 12, feed-to-egg ratio was elevated in older hens, particularly in the absence of RES supplementation, resulting in a significant age × RES interaction (P = 0.012; Table 2, Table 3). Supplementation with RES improved feed efficiency in 67-week-old hens (interaction, P = 0.012), narrowing the gap between age groups. Although a significant interaction was also detected for feed intake (P = 0.043), no clear differences were found among the four treatment groups during this period (P > 0.05). A significant interaction between age and RES supplementation was observed for the unqualified egg rate during weeks 1 to 4 (P = 0.047), where older hens exhibited a markedly higher rate of defective eggs compared to younger birds (P < 0.05). RES supplementation mitigated this increase, particularly in the aged group. Independent of interaction, at 67 weeks of age, hens showed a significant decline in egg-laying rate at all timepoints (P < 0.001), as well as reduced feed intake during weeks 1 to 4 (P = 0.038) and weeks 9 to 12 (P = 0.027), and lower egg mass throughout the trial (P < 0.001) compared to those at 47 weeks of age. In contrast, older hens had significantly increased egg weight and a higher defective egg rate during the entire experimental period (P < 0.001). Dietary supplementation with RES significantly improved egg-laying rate during weeks 5 to 8 (P = 0.028) and weeks 9 to 12 (P = 0.006). Moreover, RES improved feed intake during weeks 5 to 8 (P = 0.037) and increased egg mass during both weeks 5 to 8 (P = 0.036) and weeks 9 to 12 (P = 0.014).

Table 2.

Effects of resveratrol (RES) on laying rate, egg weight, and feed intake in laying hens of different ages.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
Laying rate, %
1–4 week	92.53	94.78	68.12	72.31	1.931	93.66	70.22	80.33	83.55	<0.001	0.106	0.619
5–8 week	86.98	89.85	68.06	76.51	2.454	88.42	72.29	77.52	83.18	<0.001	0.028	0.264
9–12 week	84.39	90.31	64.09	70.26	2.064	87.40	67.18	74.24	80.29	<0.001	0.006	0.952
1–12 week	87.97	91.65	66.75	74.47	1.330	89.81	70.61	77.36	83.06	<0.001	<0.001	0.139
Egg weight, g
1–4 week	61.30	61.04	63.89	63.31	0.544	61.17	63.6	62.60	62.18	<0.001	0.445	0.768
5–8 week	61.55	61.46	64.05	63.33	0.577	61.51	63.69	62.80	62.40	<0.001	0.486	0.593
9–12 week	61.38	61.03	63.77	64.07	0.619	61.21	63.92	62.58	62.55	<0.001	0.973	0.603
1–12 week	61.41	61.18	63.90	63.57	0.523	61.30	63.74	62.66	62.38	<0.001	0.592	0.926
ADFI, g
1–4 week	109.20	110.81	109.04	105.86	1.183	110.01	107.45	109.12	108.34	0.038	0.514	0.052
5–8 week	102.73	103.29	102.09	103.00	0.337	103.01	102.55	102.41	103.15	0.179	0.037	0.605
9–12 week	101.46	103.02	103.29	103.41	0.479	102.24	103.35	102.38	103.22	0.027	0.089	0.141
1–12 week	104.46ab	105.71a	104.81ab	104.09b	0.464	105.09	104.45	104.64	104.90	0.181	0.573	0.043

ADFI = average daily feed intake; RES = resveratrol; SEM = standard error of the mean.

Means with different superscripts within a column differ significantly (interaction P < 0.05).

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

Table 3.

Effects of resveratrol (RES) on feed conversion, egg mass, and unqualified egg rate in laying hens of different ages.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
Feed-to-egg ratio
1–4 week	1.93	1.92	2.53	2.33	0.060	1.93	2.43	2.23	2.13	<0.001	0.080	0.122
5–8 week	1.93	1.88	2.36	2.16	0.069	1.91	2.26	2.15	2.02	<0.001	0.064	0.268
9–12 week	1.98	1.87	2.55	2.31	0.067	1.93	2.43	2.27	2.09	<0.001	0.014	0.304
1–12 week	1.94c	1.89c	2.46a	2.20b	0.039	1.92	2.33	2.20	2.05	<0.001	<0.001	0.012
Egg mass, g/hen per d
1–4 week	56.70	57.85	43.55	45.70	1.172	57.28	44.63	50.13	51.78	<0.001	0.168	0.673
5–8 week	53.56	55.17	43.54	48.39	1.471	54.37	45.97	48.55	51.78	<0.001	0.036	0.279
9–12 week	51.84	55.13	40.86	45.01	1.429	53.49	42.94	46.35	50.07	<0.001	0.014	0.766
1–12 week	54.03	56.06	42.66	47.32	0.899	55.05	44.99	48.35	51.69	<0.001	<0.001	0.153
Unqualified egg rate, %
1–4 week	1.07b	1.92b	12.65a	7.84a	1.368	1.50	10.25	6.86	4.88	<0.001	0.159	0.047
5–8 week	0.86	0.63	11.72	8.27	1.459	0.75	10.00	6.29	4.45	<0.001	0.217	0.279
9–12 week	1.29	1.23	12.42	8.79	1.511	1.26	10.61	6.86	5.01	<0.001	0.231	0.246
1–12 week	1.07	1.06	12.71	9.01	1.370	1.07	10.86	6.89	5.04	<0.001	0.210	0.167

SEM = standard error of the mean.

Means with different superscripts within a column differ significantly (interaction P < 0.05).

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

3.2. Egg quality

As shown in Table 4, during weeks 1 to 4 and 9 to 12, 67-week-old hens exhibited lower albumen height compared with younger birds, but RES supplementation effectively improved this parameter in aged hens, resulting in a significant age × RES interaction (P = 0.021 and P = 0.025, respectively). A similar pattern was observed for eggshell strength during weeks 5 to 8 and 9 to 12 (interaction, P = 0.019 and P = 0.016, respectively), as older hens without RES supplementation exhibited reduced shell strength, which was mitigated by RES treatment. Eggshell thickness also responded to the interaction between age and RES across all observed periods (P < 0.05). Aged hens had thinner shells in the control group, while RES improved shell thickness particularly during weeks 5 to 8 and 9 to 12 (P < 0.05). For Haugh units, a notable interaction emerged during weeks 9 to 12 (P = 0.023), where a decrease in older control hens was offset by RES supplementation, restoring Haugh unit values to levels similar to younger birds.

Table 4.

Effects of resveratrol (RES) on egg quality of different age laying hens.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
Albumen height, mm
1–4 week	7.88a	7.67ab	6.86b	7.84a	0.245	7.78	7.35	7.37	7.76	0.092	0.128	0.021
5–8 week	7.23	7.62	6.89	7.98	0.240	7.43	7.44	7.06	7.80	0.958	0.004	0.157
9–12 week	7.78a	7.74a	6.70b	7.86a	0.257	7.76	7.28	7.24	7.80	0.072	0.037	0.025
Eggshell strength, kg/cm2
1–4 week	3.96	4.39	3.32	4.34	0.171	4.18	3.83	3.64	4.37	0.051	<0.001	0.092
5–8 week	4.05a	4.28a	3.47b	4.30a	0.122	4.17	3.88	3.76	4.29	0.026	<0.001	0.019
9–12 week	4.22a	4.46a	3.46b	4.31a	0.119	4.34	3.88	3.84	4.39	<0.001	<0.001	0.016
Eggshell thickness, mm
1–4 week	0.38a	0.38a	0.31b	0.39a	0.008	0.38	0.35	0.34	0.39	<0.001	<0.001	<0.001
5–8 week	0.36a	0.37a	0.32b	0.38a	0.007	0.36	0.35	0.34	0.37	0.041	<0.001	0.002
9–12 week	0.37a	0.37a	0.33b	0.36a	0.006	0.37	0.35	0.35	0.36	<0.001	0.014	0.030
Yolk color
1–4 week	6.48	5.89	6.77	5.89	0.257	6.19	6.33	6.63	5.89	0.578	0.008	0.578
5–8 week	6.11	6.41	6.34	6.34	0.230	6.26	6.34	6.22	6.37	0.720	0.519	0.519
9–12 week	5.66	5.63	5.58	5.69	0.224	5.64	5.64	5.62	5.66	0.973	0.860	0.768
Haugh units
1–4 week	88.18	87.44	83.65	83.45	1.869	87.81	83.55	85.92	85.45	0.029	0.802	0.885
5–8 week	83.84	86.27	80.89	87.98	1.452	85.06	84.44	82.37	87.12	0.673	0.003	0.118
9–12 week	86.77a	86.77a	79.61b	87.02a	1.546	86.77	83.32	83.19	86.89	0.032	0.023	0.023

SEM = standard error of the mean.

Means with different superscripts within a column differ significantly (interaction P < 0.05).

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

3.3. Plasma reproductive hormone

Age and RES supplementation both influenced plasma reproductive hormone levels in laying hens (Table 5). Significant interactions between age and RES were observed for MT (P < 0.001), E2 (P = 0.049), and AMH (P = 0.026). In the control group (RES-0), the concentrations of these hormones were significantly lower in aged hens compared to 47-week-old hens (P < 0.05). RES supplementation significantly increased plasma MT and E2 levels in 67-week-old laying hens (P < 0.05), while no such effect was observed in the younger group. Regardless of interaction effects, older hens exhibited markedly lower levels of Prog (P < 0.001), LH (P = 0.002), and FSH (P = 0.003) compared with younger hens. Similarly, RES supplementation significantly elevated plasma levels of FSH (P = 0.011) concentrations, with a tendency to increase Prog (P = 0.050) and LH (P = 0.086).

Table 5.

Effects of resveratrol (RES) on plasma reproduction related hormone levels of different age hens.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
Prog, ng/mL	54.01	63.23	32.75	33.66	2.458	58.62	33.21	43.38	48.45	<0.001	0.050	0.106
MT, ng/L	4514.71a	4055.08a	3579.21c	4107.43b	24.133	4284.90	3843.32	4046.96	4081.26	<0.001	0.171	<0.001
LH, mIU/mL	49.74	61.75	43.43	44.16	3.526	55.75	43.80	46.59	52.96	0.002	0.086	0.125
FSH, mIU/mL	62.09	75.39	50.97	59.92	3.937	68.74	55.45	56.53	67.66	0.003	0.011	0.586
E2, ng/L	245.39a	242.17a	205.78b	234.37a	8.248	243.78	220.07	225.58	238.27	0.009	0.038	0.049
AMH, ng/L	2218.25a	2172.49ab	2155.45b	2205.87ab	19.939	2195.37	2180.66	2186.85	2189.18	0.469	0.908	0.026

Prog = progesterone; MT = melatonin; LH = luteinizing hormone; FSH = follicle-stimulating hormone; E2 = estradiol; AMH = anti-Müllerian hormone; SEM = standard error of the mean.

Means with different superscripts within a column differ significantly (interaction P < 0.05).

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

3.4. Ovarian antioxidant capacities

Ovarian antioxidant status was affected by both RES supplementation and age (Table 6). A significant interaction between age and RES was observed for MDA (P < 0.001). In the control group, MDA levels were markedly elevated in older hens (67 weeks) compared to younger ones (P < 0.05). However, RES supplementation significantly reduced MDA concentrations in aged hens (P < 0.05). A tendency for age × RES interaction was observed for GSH (P = 0.051), GSH-Px (P = 0.087), and GST (P = 0.066). Based on the main effects, older hens exhibited lower GSH (P = 0.022) and GST (P = 0.025) levels compared to younger hens. RES supplementation significantly increased GSH (P < 0.001), GSH-Px (P < 0.001), and GST (P = 0.037) levels.

Table 6.

Effects of resveratrol (RES) on ovarian antioxidant capacity in laying hens of different ages.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
GSH, μmol/g prot	19.06	21.68	14.39	21.28	1.055	20.37	17.84	16.73	21.48	0.022	<0.001	0.051
GSH-Px, U/mg prot	34.36	57.91	25.06	63.72	4.300	46.13	44.39	29.71	60.81	0.688	<0.001	0.087
GST, U/mg prot	2.93	4.07	2.81	2.88	0.282	3.50	2.84	2.87	3.48	0.025	0.037	0.066
MDA, nmol/g prot	4.74b	4.31bc	5.80a	3.95c	0.160	4.53	4.87	5.27	4.13	0.037	<0.001	<0.001

GSH = glutathione; GSH-Px = glutathione peroxidase; GST = glutathione S-transferase; MDA = malondialdehyde; prot = protein; SEM = standard error of the mean.

Means with different superscripts within a column differ significantly (interaction P < 0.05).

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

3.5. Liver antioxidant capacity

The antioxidant activity of GSH (P = 0.002) was significantly lower at 67 weeks than at 47 weeks (Table 7). Dietary RES contributed to a pronounced elevation in GSH-Px activity (P < 0.001), accompanied by a decrease in MDA levels (P < 0.001). However, the interaction effect between age and RES supplementation was not statistically significant in the expression of hepatic antioxidant-related parameters (P > 0.05).

Table 7.

Effects of resveratrol (RES) on liver antioxidant capacity of different age hens.

Items1	47 week		67 week		SEM	Age, week		RES, mg/kg		P-value
	RES-0	RES-400	RES-0	RES-400		47	67	0	400	Age	RES	Age × RES
GSH-Px, U/mg prot	67.82	98.10	66.70	95.91	5.609	82.96	81.3	67.26	97.01	0.770	<0.001	0.925
GSH, μmol/g prot	58.97	58.73	50.35	49.24	2.633	58.85	49.79	54.66	53.99	0.002	0.801	0.870
SOD, U/mg prot	168.77	183.82	174.69	162.24	7.968	176.3	168.47	171.73	173.03	0.334	0.871	0.096
MDA, nmol/g prot	0.78	0.48	0.75	0.54	0.052	0.63	0.64	0.77	0.51	0.838	<0.001	0.410

GSH-Px = glutathione peroxidase; GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; prot = protein; SEM, standard error of the mean.

¹Each mean represents one layer/replicate, nine replicates/treatment. RES-0 and RES-400, diets with 0 and 400 mg/kg RES, respectively.

3.6. Ovarian antioxidant related gene expression

As evidenced by the data in Fig. 1, the expression of antioxidant-related gene exhibits significant age and treatment effects. Compared to younger hens (47 weeks old), the expression of HO-1 in the ovarian of older individuals (67 weeks old) was significantly decreased (P = 0.018), whereas Nrf2 mRNA levels were markedly upregulated in the 67-week-old group (P = 0.039). Notably, RES supplementation led to a significant upregulation of HO-1 (P < 0.001), Nrf2 (P = 0.002), and NQO1 (P < 0.001) mRNA levels.

Fig. 1.

Effects of dietary resveratrol (RES) supplementation on ovarian antioxidant capacity related gene expression of different age hens. (A) HO-1, (B) Nrf2, and (C) NQO1 relative mRNA expression. CON and RES, diets with 0 and 400 mg/kg RES, respectively. CON = control. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; n = 9.

3.7. Expression of ovarian inflammation–associated genes

Compared to 47-week-old hens, TNF-α expression was markedly higher in the 67-week group (Fig. 2, P = 0.018). Additionally, RES supplementation suppressed the mRNA expression of NF-κB (P = 0.015) and pro-inflammatory cytokines including IL-6 (P = 0.011), IL-8 (P < 0.001), and TNF-α (P < 0.001), while enhancing the transcription levels of the anti-apoptotic markers Bcl-2 (P < 0.001) and Bcl-XL (P = 0.001).

Fig. 2.

Effects of dietary resveratrol (RES) supplementation on ovarian inflammation and cell apoptosis related gene expression of different age hens. (A) IL-6, (B) IL-8, (C) TNF-α, (D) Bcl-XL, (E) Bcl-2, and (F) NF-κBrelative mRNA expression. CON and RES, diets with 0 and 400 mg/kg RES, respectively. CON = control. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; n = 9.

4. Discussion

Aging significantly impacts the reproductive efficiency and overall health of laying hens, leading to declines in egg production, egg quality, and ovarian function. This decline is consistent with the well-documented age-associated ovarian senescence and hormonal dysregulation that contribute to reproductive inefficiency in late-phase laying hens (Lebedeva et al., 2025; Liu et al., 2018; Xu et al., 2023). These findings extend this understanding by demonstrating that feed efficiency and defective egg rate were not only affected by age, but also significantly modulated by RES supplementation in an age-dependent manner. This observation align with previous studies that report age-associated reproductive declines due to ovarian senescence and hormonal imbalances in laying hens (Wu et al., 2023). Specifically, 400 mg/kg RES supplementation significantly improved feed efficiency and reduced the rate of defective eggs in 67-week-old hens, particularly during the early phases (weeks 1-4). These findings are consistent with previous research indicating that RES, a polyphenolic compound, can enhance mitochondrial function and antioxidant capacity in aging animals, which may contribute to improved nutrient metabolism and reproductive performance (Wu et al., 2022). The age-specific improvements may reflect increased susceptibility of aged hens to oxidative damage, which RES helps mitigate through its antioxidant action. Notably, RES also enhanced eggshell strength, thickness, and albumen height in older hens, with multiple indicators showing significant interaction effects, indicating that these benefits are not merely additive but age-contingent. This age-related decline was primarily due to a reduced capacity for calcium absorption and decreased expression of intestinal calcium-binding proteins, resulting in inadequate calcium intake and compromised eggshell quality in the late laying period (Sun et al., 2025; Yosefi et al., 2003). While previous studies confirmed that RES enhances Ca metabolism (Abd El-Hack et al., 2023), this study adds evidence that such effects are functionally translated into measurable improvements in shell strength and thickness, especially under an aged condition. Similarly, the restoration of Haugh units in RES-treated older hens may be attributed to estrogen-mimicking properties of RES, which has been linked to enhanced albumen protein synthesis in the magnum region via estrogen-responsive genes (Jeong et al., 2018). Thus, the functional recovery in egg quality is likely mediated through both metabolic and endocrine modulation, particularly effective in older hens.

Following the post-peak laying period in hens, follicle numbers decrease, and the increased atresia rate leads to reduced ovarian function (Lillpers and Wilhelmson, 1993). Age-related ovarian decline is associated with changes in FSH, LH, inhibin B, and AMH levels, though the specific patterns may vary between species (He et al., 2023; Sinha et al., 2022). The reduction in LH and FSH suggests impaired follicular recruitment, while lower E2 levels indicate diminished estrogenic stimulation, which is necessary for yolk precursor synthesis and oviduct function (Ebeid et al., 2008; Meng et al., 2013; Rangel and Gutierrez, 2014). Consistent with this, these results revealed that aging led to significant declines in plasma levels of MT, E2, and AMH, while RES supplementation selectively reversed these reductions only in the older hens, indicating a strong age × RES interaction. This supports the hypothesis that RES acts as a modulator of age-related endocrine decline, potentially through restoration of ovarian steroidogenic capacity. Previous studies have shown that RES enhances ovarian steroidogenesis via sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) activation (Nishigaki et al., 2020), and these findings suggest that this pathway may be particularly relevant in aged birds, where mitochondrial dysfunction and oxidative stress impair hormone biosynthesis. Similar age-dependent effects of RES were reported in 12-month-old female mice, where it improved fertility outcomes more significantly than in middle-aged mice (Ziętek et al., 2021). Interestingly, although Prog, LH, and FSH showed no significant interaction, a main effect analysis indicated that RES supplementation increased FSH and Prog levels and tended to elevate LH. This suggests that RES also exerts basal stimulatory effects on gonadotropin production, independent of age, but its restorative impact on downstream targets like E2 and AMH appears to be conditioned by the extent of age-related damage. These findings align with previous studies demonstrating that RES can enhance steroidogenesis by upregulating the expression of ovarian steroidogenic enzymes and increasing hormone synthesis (Ding et al., 2022). Similar findings have been reported in studies on other polyphenols, such as tea polyphenols and flavonoids, which have been shown to enhance reproductive performance by modulating ovarian function and hormone secretion (Amevor et al., 2021; Xu et al., 2023).

The aging process contributes to oxidative stress accumulation, thereby disturbing the balance between pro-oxidant and antioxidant systems, leading to cellular damage and functional decline in multiple organs (Luderer, 2014). The present study demonstrated organ-specific responses to oxidative stress and RES supplementation in laying hens of different ages. A significant age × RES interaction was observed for ovarian MDA levels, indicating that lipid peroxidation in the ovary was more pronounced in aged hens and effectively mitigated by RES supplementation. In contrast, the liver showed no such interaction, although aging led to a decrease in GSH levels and RES supplementation significantly increased GSH-Px activity. These findings indicate that both organs experience oxidative damage with age, yet their responses and susceptibilities to oxidative stress differ, likely due to organ-specific metabolic functions, antioxidant enzyme expression, and mitochondrial activity (Wang et al., 2020). The liver, as a central metabolic organ, possesses a highly efficient antioxidant system, including GSH-Px, SOD, and CAT, which counteract reactive oxygen species (ROS) to maintain homeostasis (Olsvik et al., 2005). However, age-related oxidative stress depletes GSH reserves, impairing the liver's detoxification capacity and leading to lipid peroxidation. Interestingly, RES supplementation enhanced GSH-Px activity while simultaneously decreasing MDA levels, suggesting that RES enhances hepatic antioxidant defence, possibly through activation of the Nrf2 pathway, a master regulator of antioxidant gene expression (Ding et al., 2023; Farkhondeh et al., 2020; Meng et al., 2018). In contrast, the ovary is more vulnerable to oxidative stress as a result of its high lipid content and fluctuating hormonal environment, which make ovarian follicles particularly vulnerable to ROS-induced damage (Yan et al., 2022). These findings align with previous studies suggesting that ovarian antioxidant systems such as GST and mitochondrial enzymes decline more sharply with age compared to hepatic systems (Chen et al., 2024). This is supported by the observed decline in ovarian GST and elevated MDA in aged hens in this study. Unlike the liver, which has extensive detoxification pathways, the ovary relies on mitochondrial SOD2 and GST activity to neutralize oxidative stress, and these pathways appear to decline with age (Lim and Luderer, 2010; Tatone et al., 2024). The significant increase in GSH-Px and GST levels following RES supplementation suggests that RES effectively enhances ovarian antioxidant capacity, likely by modulating the SIRT1/Nrf2 pathway, improving mitochondrial function, and reducing ROS accumulation (Gao et al., 2023; Grzeczka and Kordowitzki, 2022). The upregulation of HO-1, Nrf2, and NQO1 gene expression in RES-treated hens further supports the role of RES in activating endogenous antioxidant defences. Nrf2 functions as a central regulator in orchestrating cellular antioxidant mechanisms by inducing the transcription activation of key detoxification enzymes including GST, GSH-Px, and HO-1 (Ma et al., 2018; Wang et al., 2018; Yuan et al., 2024; Zhang et al., 2018). Interestingly, this study found that Nrf2 expression was already elevated in older hens compared to younger ones, suggesting a compensatory response to age-related oxidative stress. However, the further increase in Nrf2 and HO-1 expression after RES supplementation indicates that RES may act as a direct activator of the Nrf2/HO-1 pathway, enhancing the transcription of antioxidant genes and boosting cellular defence beyond baseline levels.

Beyond oxidative stress, chronic inflammation is another major contributor to ovarian aging. In current study, 67-week-old hens exhibited significantly higher TNF-α levels, suggesting an inflammatory shift that may accelerate follicular dysfunction. This finding is consistent with evidence that aging is associated with increased pro-inflammatory cytokine production in ovarian tissue (Zeng et al., 2024). Interestingly, dietary supplementation with RES exerted an anti-inflammatory response by significantly inhibiting the mRNA expression of pro-inflammatory markers such as IL-6, IL-8, and TNF-α while upregulating anti-apoptotic genes (Bcl-XL and Bcl-2). Additionally, the mRNA expression of NF-κB was notably reduced following RES supplementation, suggesting that RES exerts its protective effects via NF-κB pathway-mediated signaling modulation, thereby suppressing inflammatory responses and promoting cell survival (Ibrahim et al., 2021; Malaguarnera, 2019). The dual regulation of inflammation and apoptosis observed here suggests that RES not only suppresses inflammatory signaling, but also promotes cell survival within ovarian follicles, potentially preserving follicular viability in aging hens.

5. Conclusion

Aging impairs reproductive performance, egg quality, and antioxidant status in laying hens. Dietary RES supplementation alleviated oxidative damage, improved reproductive hormone levels, and supported ovarian and hepatic antioxidant defence. These effects were particularly evident in older hens for several key parameters. This may be due to RES promoting mitochondrial biogenesis, activating the Nrf2/HO-1 pathway, and regulating NF-κB signaling to reduce oxidative stress and inflammation. Future studies should explore its long-term effects on ovarian reserve and potential synergy with other antioxidants.

Credit Author Statement

Li Zhang: Writing – original draft, Visualization, Validation, Formal analysis, Conceptualization. Wenwen Xu: Writing – review & editing, Visualization, Validation, Investigation, Formal analysis. Keying Zhang: Supervision, Methodology. Xuemei Ding: Validation, Supervision. Qiufeng Zeng: Validation, Software. Shiping Bai: Project administration, Conceptualization. Jingbo Liu: Writing – review & editing, Data curation. Jianping Wang: Writing – review & editing, Software, Funding acquisition, Conceptualization.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.