Ethical approval was not needed for this study because it involved secondary analysis of published data. All included studies adhered to institutional animal care and use guidelines.

The meta-analysis was carried out from October 2024 to March at the National Research and Innovation Agency (BRIN), Indonesia, in collaboration with the Faculty of Animal and Agricultural Sciences, Diponegoro University, Indonesia.

This meta-analysis compiled evidence from previously published studies assessing the effects of dietary quercetin supplementation in laying hens. The research question was developed using the PICO framework [36], where the population (P) consisted of laying hens, the intervention (I) was quercetin supplementation, the comparator (C) was a control diet without quercetin, and the outcomes (O) included egg production and egg quality traits. The study protocol adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, with reporting aligned to the PRISMA 2020 statement (https://doi.org/10.5281/ zenodo.17718238) [37]. Formal protocol registration was not performed because the study began before registration was required.

A comprehensive literature search strategy is summarized in Table 1. Searches were conducted in the Scopus and Web of Science databases on October 7, 2024. The Scopus search string was: TITLE-ABS-KEY (hen*) AND TITLE-ABS-KEY (quercetin) AND TITLE-ABS-KEY (egg*). For Web of Science, the query was: TS = (hen*) AND TS = (quercetin) AND TS = (egg*). No restrictions were applied regarding publication year. Only original research articles were included, while reviews, preprints, and conference proceedings were excluded. Articles not published in English were also excluded. The initial search yielded 116 records, comprising 58 articles from each database.

Study eligibility was determined based on the PICO framework. Studies were included if they met these criteria: (1) full-text available; (2) investigated quercetin supplementation in laying hens; (3) assessed production performance, egg quality, antioxidant status, and/or blood metabolite parameters; (4) included a control group; and (5) reported measures of variability, such as confidence intervals (CI), standard errors (SE), or standard deviations (SD). Studies using mixed antioxidant formulations (for example, quercetin combined with vitamin E or other antioxidants) were excluded. When studies reported multiple quercetin doses, each dose was considered an independent comparison to evaluate dose-dependent effects.

The study selection process followed the PRISMA flow diagram (Figure-1). Zotero [38] and Microsoft Excel version 16.91 [39] were used to identify and remove duplicate records and to support title and abstract screening. Six reviewers (SH, HK, HPW, FRPH, AP, and BH) independently screened titles and abstracts for eligibility. Full-text articles were independently assessed by two reviewers (SH and HK), with discrepancies resolved by a third reviewer (HPW). After duplicate removal, 84 records remained. Following title and abstract screening, 28 studies were retained for full-text evaluation, of which 24 met the inclusion criteria. Manual screening of reference lists between October 20 and October 24, 2024, identified three additional eligible studies. In total, 27 studies were included in the final meta-analysis.

The characteristics of the included studies are shown in Table 2 [7–33]. Data collected included performance indicators ([FI] and FER), egg production parameters (LR and EW), egg quality traits (ST, YC, EW, and HU), blood metabolites (SGPT, glucose, total cholesterol, HDL, and LDL), oxidative stress markers (malondialdehyde [MDA]), and antioxidant enzyme activities (catalase [CAT] and SOD). Eight reviewers independently extracted data in duplicate using a standardized extraction form. Any disagreements were resolved through discussion, with a third reviewer (SH) consulted if consensus was not reached.

Mean values and SDs were obtained or calculated for meta-analysis. When SDs were not directly reported, they were computed using the following formulas: (1) SD = SE × √N, where N is the number of replicates; and (2) SD = √N × (upper CI − lower CI)/3.92, which corresponds to a 95% CI. For small sample sizes (<60), the constant 3.92 was replaced with the appropriate t-distribution value based on degrees of freedom [40].

Graphical data were digitized using WebPlotDigitizer software [41]. Reference axes were calibrated before extracting mean values and corresponding CI limits. Extracted data were transferred to Microsoft Excel, where SDs or SEs were calculated and cross-checked against original sources. All measurement units were standardized before analysis.

Two reviewers (HPW and HK) independently assessed the risk of bias using the SYRCLE risk of bias tool for animal studies [42]. The evaluation covered domains such as selection bias, performance bias, detection bias, attrition bias, reporting bias, and other possible biases. Each domain was rated as low, unclear, or high risk based on set criteria. Disagreements were resolved through discussion or by consulting a third reviewer (SH).

All statistical analyses were performed using R software version 4.2.2 [43], with the “metafor” package version 4.8-0 [44]. Subgroup analysis results were visualized with the “ggplot2” package version 3.5.2 [45] and Microsoft Excel [39]. Effect sizes were calculated as MDs along with 95% CI using a restricted maximum likelihood (REML) random-effects model [46]. REML was chosen for its robustness and reliability in situations of high heterogeneity [47]. Statistical significance was defined as p < 0.05.

Heterogeneity was evaluated using the I² statistic, with values over 50%, between 25% and 50%, and below 25% indicating high, moderate, and low heterogeneity, respectively [48]. Additionally, a Q-test p-value less than 0.10 was deemed indicative of significant heterogeneity [49].

Subgroup and meta-regression analyses were performed when three criteria were satisfied: (1) a significant overall effect size (p < 0.05); (2) considerable heterogeneity (I² > 50%, p < 0.001); and (3) adequate data availability (≥ 10 comparisons) [50]. Quercetin dose (mg/kg) was considered a continuous covariate, while initial hen age (≤ 50 vs. > 50 weeks), treatment duration (≤ 4, 5–8, and ≥ 8 weeks), and quercetin form (plant powder or extract) were treated as categorical covariates. A mixed-effects model was used for subgroup analysis [51].

Publication bias was evaluated using funnel plot visualization and Egger’s regression test [52], with p < 0.05 indicating a high risk of bias. Sensitivity analysis was conducted with a leave-one-out approach to assess the robustness of the pooled estimates. The trim-and-fill method was used to estimate the potential effect of missing studies [53].

All extracted datasets, statistical scripts, and supplementary Figures 1A–1P are publicly accessible at https://doi.org/10.5281/zenodo.17626656.

A total of 27 eligible studies were included in this meta-analysis. The dietary quercetin doses evaluated across studies ranged from 0 to 2,378 mg/kg. Most studies involved hens with an initial age of ≤ 50 weeks (70.38%), while the remaining 29.62% included hens older than 50 weeks. Treatment duration varied significantly, with 11.11% of studies administering quercetin for less than 4 weeks, 40.74% for 5–8 weeks, and 48.15% for more than 8 weeks. Regarding formulation, quercetin was primarily provided as extract powder (81.48%), while plant powder made up 18.52% of the treatments. The nutrient composition of the basal diets used in the included studies is summarized in Table-3. Risk of bias assessment outcomes are illustrated in Figure-2, showing that all included studies had a low risk of bias regarding baseline characteristics and selective reporting domains.

Dietary quercetin supplementation significantly affected (p < 0.001) various performance, production, and egg quality parameters in laying hens (Table 4). Significant effects were seen for LR, FER, EW, HU, ST, and YC. In contrast, FI was not influenced by quercetin supplementation (p > 0.05). Overall, quercetin increased LR (MD = 2.819%), EW (MD = 1.209 g/unit), HU (MD = 1.838%), ST (MD = 0.014 mm), and YC (MD = 0.526), while significantly decreasing FER (MD = −0.146), indicating improved feed efficiency.

The influence of dietary quercetin on blood metabolites is shown in Table 5. Quercetin supplementation significantly impacted serum glucose, SGPT, HDL, and total cholesterol levels (p < 0.001). In contrast, LDL levels did not differ between control and quercetin-treated hens (p > 0.05). Specifically, quercetin supplementation decreased SGPT (MD = −7.009 U/L), glucose (MD = −17.589 mg/dL), and total cholesterol (MD = −20.834 mg/dL), while significantly increasing HDL levels (MD = 32.590 mg/dL).

Dietary quercetin supplementation significantly influenced malondialdehyde (MDA) and SOD levels in hens (p < 0.001) (Table 6). In contrast, catalase (CAT) activity was not significantly affected (p > 0.05). Quercetin treatment led to a notable decrease in MDA concentration (MD = −7.373 nmol/mL), indicating reduced lipid peroxidation, and a significant increase in SOD activity (MD = 8.114 U/mL), reflecting enhanced antioxidant defense.

All evaluated parameters showed significant heterogeneity (p < 0.001), with high inconsistency values (I² > 50%) across studies (Tables 4–6). Subgroup and meta-regression analyses identified quercetin dose as a significant moderator affecting LR (R² = 11.44%), FER (R² = 9.62%), SGPT (R² = 33.07%), glucose (R² = 29.42%), total cholesterol (R² = 28.01%), and SOD (R² = 30.71%) (Table 7). Treatment duration significantly influenced YC (R² = 9.33%), SGPT (R² = 75.29%), glucose (R² = 60.63%), and total cholesterol (R² = 44.79%). Initial hen age significantly impacted LR (R² = 5.18%), ST (R² = 24.58%), and SOD (R² = 24.89%). Additionally, the form of quercetin significantly moderated LR (R² = 4.99%), FER (R² = 9.75%), YC (R² = 16.98%), total cholesterol (R² = 26.17%), and SOD (R² = 22.11%).

Meta-regression analysis showed that the quercetin dose had a linear effect on SGPT, glucose, and total cholesterol levels (Table 8; Figures 3A–C). In contrast, LR, FER, and SOD showed quadratic responses to increasing quercetin doses. The highest LR was observed at 600 mg/kg (Figure 3D), while the optimal FER occurred between 400–600 mg/kg (Figure 3E). The highest SOD activity was recorded at approximately 500 mg/kg of dietary quercetin (Figure 3F).

Treatment durations of 5–8 weeks and over 8 weeks significantly improved YC (p < 0.05) (Figure 4A), while supplementation for less than 4 weeks had no significant impact. Short-term (<4 weeks) and medium-term (5–8 weeks) supplementation significantly decreased SGPT, glucose, and total cholesterol levels (p < 0.001) (Figures 4B–D). However, these blood metabolite improvements were not observed when supplementation was extended beyond 8 weeks.

Subgroup analysis based on initial hen age showed significant effects on LR, ST, and SOD levels (Figures 5A–C). Hens older than 50 weeks had higher LR and ST compared to both the control and the ≤ 50-week groups (p < 0.05). Additionally, quercetin supplementation in hens aged ≤ 50 weeks significantly increased LR, ST, and SOD activity (p < 0.05).

The form of quercetin significantly affected LR, FER, YC, total cholesterol, and SOD levels (Figures 6A–E). Quercetin provided as an extract powder resulted in the highest LR and the lowest FER (p < 0.05), indicating better productivity and feed efficiency. In contrast, plant powder supplementation produced the highest YC scores and the lowest total cholesterol levels. Extract powder had no notable effect on SOD activity, while plant powder significantly increased SOD levels.

Assessment of publication bias was conducted using funnel plots and Egger’s regression test (Suppl. Figures 1A–P; https://doi.org/10.5281/zenodo.17626656). A low risk of publication bias (p > 0.05) was found for LR, FER, EW, HU, ST, YC, SGPT, HDL, LDL, and CAT. Conversely, significant publication bias (p < 0.05) was observed for glucose, total cholesterol, MDA, and SOD outcomes. Leave-one-out sensitivity analysis verified the robustness of the results, as removing individual studies did not significantly change the pooled effect estimates.

Dietary quercetin supplementation significantly enhanced productivity and egg quality in laying hens, as shown by improvements in FER, LR, and egg quality traits such as EW, HU, ST, and YC. These results align with those reported by Liu et al. [7], who found that quercetin supplementation positively affected LR, EW, FER, HU, and ST in hens. Additionally, the better YC scores in quercetin-fed hens were accompanied by improvements in LR, FER, HU, and ST. Similar gains were reported by Ahmad et al. [10], who observed that quercetin-rich mulberry leaf supplementation significantly increased LR, HU, and ST. The improvements in productivity and egg quality may be due to quercetin’s effects on blood metabolites, antioxidant capacity, and reproductive physiology in laying hens.

Quercetin supplementation had significant effects on blood metabolite profiles, especially lipid metabolism. Serum total cholesterol and HDL concentrations are important markers of lipid metabolic health in poultry [54]. In this study, quercetin notably lowered total cholesterol levels and increased HDL concentrations. These findings align with those of El-Saadany et al. [12] and Liu et al. [24], who observed decreased serum total cholesterol and higher HDL levels in hens supplemented with quercetin. Since hepatic lipoprotein synthesis is crucial for follicular development in laying hens [55], improved serum lipid profiles indicate better liver function and reproductive performance. Lower total cholesterol and elevated HDL also suggest a decreased risk of fatty liver syndrome [56].

Mechanistically, quercetin has been shown to promote cholesterol homeostasis by enhancing the selective uptake of HDL-derived lipids [57]. Maintaining cholesterol balance is essential for liver health, as dysregulation can cause intrahepatic lipid buildup and subsequent liver injury [58]. The significant decrease in SGPT observed in this analysis further supports the hepatoprotective role of quercetin. Previous research has similarly indicated reduced SGPT activity following flavonoid supplementation, which suggests improved liver health [59]. Notably, Amevor et al. [22] reported that quercetin alleviated hepatic steatosis in aging hens. Overall, these findings suggest that quercetin improves liver function and lipid metabolism, thereby enhancing egg production and quality.

Dietary quercetin significantly improved antioxidant capacity in laying hens, as shown by increased SOD activity and decreased malondialdehyde (MDA) levels. These findings agree with those reported by Liu et al. [14], who saw lower MDA levels and higher SOD activity in hens supplemented with quercetin. Similar antioxidant effects have also been reported in aging hens [22] and hens exposed to heat stress [21]. Lin et al. [17] further showed that mulberry leaf supplementation with quercetin increased SOD activity while also decreasing MDA levels in hen serum.

SOD is a crucial first-line antioxidant enzyme that safeguards cells against oxidative damage [60] and has an important role in ovarian development and reproductive function [61]. In contrast, MDA is a widely used biomarker of lipid peroxidation and oxidative stress in serum and tissues [62]. Although limited data prevented a meta-analysis of glutathione peroxidase (GSH-Px) and catalase (CAT) activities, the observed changes in SOD and MDA strongly indicate that quercetin supplementation effectively reduces oxidative stress by enhancing endogenous antioxidant defenses. Reduced oxidative damage likely contributed to the noted improvements in performance, productivity, and egg quality.

The current meta-analysis showed that the effects of quercetin on productivity and antioxidant status depend on the dose. Supplementation between 400–600 mg/kg most effectively improved LR, FER, and SOD activity. These results match those of El-Saadany et al. [12], who found maximum egg production, feed efficiency, and antioxidant activity in hens given 600 mg/kg of quercetin compared to lower or higher doses. Similarly, Liu et al. [14] noted that 500 mg/kg dietary quercetin increased laying performance and antioxidant defenses while decreasing lipid peroxidation.

Yang et al. [11] further reported that hens receiving 400 mg/kg quercetin had LR similar to those receiving 600 mg/kg, with better feed-to-egg efficiency at the lower dose. These findings suggest that moderate supplementation offers the best benefits while reducing potential inefficiencies or safety issues associated with higher doses. Based on the overall evidence, the safe and effective dietary range for quercetin supplementation in laying hens is 400–600 mg/kg.

Several limitations of this study should be acknowledged. First, significant heterogeneity was observed across all analyzed parameters, with I² values exceeding 75%, which could affect the accuracy of pooled effect estimates. Second, breed-specific subgroup analyses were not possible due to a lack of data for individual hen breeds. Third, most of the included studies (77.77%) were conducted in Asian countries, which may limit the applicability of the findings to other production systems and regions. Finally, this meta-analysis focused solely on quercetin as a single dietary intervention and did not assess its combined effects with other antioxidants, enzymes, vitamins, or minerals.

Future research should fill these gaps by conducting well-designed, multi-regional studies that investigate breed-specific responses and evaluate the synergistic effects of quercetin when combined with other functional feed additives.