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. 2026 Aug 26;105(12):107689. doi: 10.1016/j.psj.2026.107689

Age-related succession of the gut microbiome drives ovarian function decline in laying hens

Lianchi Wu a,b, Tzuhan Lin a, Ziyu Wang a, Shenao Zhan a, Xuanyang Sun a, Dongyou Yu a,b,c,⁎, Bing Liu a,b,c,⁎
PMCID: PMC13634777  PMID: 42777374

Abstract

The precipitous decline in egg production and the deterioration of egg quality induced by aging in laying hens pose significant challenges to the poultry industry; however, the underlying systemic mechanisms remain incompletely elucidated. This study aimed to comprehensively delineate the trajectory of ovarian function decline during the aging of laying hens and to explore the underlying mechanisms involving age-related intestinal injury and the succession of the intestinal microbiome. Laying hens at the peak, mid, and late laying periods were utilized as the experimental models. The results showed that with the increase of age, the production performance of laying hens decreased significantly, the follicle reserve was exhausted, and the secretion of reproductive hormones was significantly reduced, and the expression of key steroid synthases and hormone receptors was significantly down-regulated. Furthermore, severe oxidative stress and inflammatory responses were observed in the ovaries during the late laying period, which subsequently triggered ovarian cell apoptosis. Concurrently, the normal intestinal architecture and physical barrier function were compromised in aging hens, evidenced by a significant downregulation of tight junction proteins and accompanied by marked intestinal dysbiosis. Moreover, aging induced profound alterations in the intestinal microbial community. The late laying period was characterized by a reduced abundance of beneficial bacteria, including Parabacteroides and Lactobacillus, alongside a substantial enrichment of pathogenic taxa such as Helicobacter. In order to verify whether intestinal microbes are involved in regulating ovarian function, a fecal microbiota transplantation (FMT) experiment was conducted in this study. The FMT results indicated that receiving fecal microbiota from peak-laying donors effectively ameliorated the ovarian condition and alleviated ovarian functional decline in aged recipient hens, thereby confirming the regulatory role of the gut microbiota. In conclusion, by delineating ovarian functional degradation, systematically evaluating age-related intestinal tissue damage and microbiota succession, and revealing the potential link between age-associated gut microbes and core indicators of reproductive decline, this study provides comprehensive insights into hen aging. Importantly, the FMT trial substantiates the regulatory effect of the gut microbiota on ovarian aging, offering a crucial theoretical basis for targeting gut microecology to delay reproductive senescence and prolong the laying cycle in poultry.

Keywords: Laying hen, Reproductive senescence, Ovarian function, Intestinal microbiome

Introduction

Commercial laying hens serve as a crucial pillar for the agricultural economy and the supply of animal protein, but their economic value and production cycle are limited by their reproductive physiology (Zhang et al., 2026b). After commercial laying hens reach the peak of egg production and maintain a high-yield state for a period of time, with the increase of age, their production performance and egg quality inevitably gradually decrease, especially after entering the late laying period, the egg production performance will decline sharply (Wang et al., 2026a). The biological essence of this compromised performance is ovarian aging, which is primarily characterized by marked ovarian atrophy and a decrease in the number of developing follicles at all levels (Zhang et al., 2026a).

Ovarian aging is an irreversible and complex systemic biological process. With the increase of age, the rates of cellular proliferation and regeneration progressively decelerate, accompanied by a significant reduction in metabolic efficiency (Zhang et al., 2023b). Concurrently, the accumulation of damage at the molecular level further leads to the functional decline of the whole organ. While previous studies primarily attributed the decline in laying performance to localized ovarian changes, researchers are increasingly recognizing that the paradigm of single-organ aging is insufficient to comprehensively explain the systemic physiological deterioration observed in hens during the late laying period (Dai et al., 2022; Nafady et al., 2025).

With the development of systems biology and microecology, the gut-ovary axis provides a new perspective for understanding reproductive senescence. The intestine is not only the center of nutrient absorption, but also the largest immune and microecological barrier of the organism. Under youthful and healthy physiological conditions, the intestinal microecology and the host maintain a harmonious symbiotic relationship, and beneficial microbial populations not only provide some energy support for the intestinal epithelium, but also play a pivotal role in preserving mucosal barrier integrity (Ma et al., 2022). However, during the natural aging process, this microecological balance is broken, the diversity and structure of the intestinal microbial community significantly evolve and become disordered, accompanied by intestinal structural damage, leading to the destruction of barrier integrity (Wilmanski et al., 2022; Okumura and Takeda, 2024). Such age-dependent microbial dysbiosis and barrier dysfunction not only precipitate intestinal inflammation and oxidative stress but also trigger systemic inflammation via the translocation of inflammatory mediators into the bloodstream. This systemic cascade ultimately disrupts distal ovarian homeostasis, exacerbating ovarian inflammation and accelerating functional decline (Qi et al., 2021; Zhang et al., 2023a).

Therefore, elucidating the synergistic relationship between age-dependent intestinal microbiome succession and ovarian functional decline is of profound significance for developing novel nutritional and microecological interventions aimed at extending the laying period of hens. Utilizing laying hens at the peak, mid, and late laying stages, this study investigated the pathological and cytological features of ovarian functional decline during aging. We systematically evaluated the trajectory of intestinal tissue damage, barrier dysfunction, and microbiome succession across the aging process, aiming to uncover the potential correlations between age-specific intestinal microbiota and key indices of reproductive senescence in the ovary. This study not only enriches the theoretical framework of poultry reproductive physiology but also provides a scientific basis for future strategies targeting gut microbiome modulation to delay ovarian senescence.

Materials and methods

Animal ethics statement

All experimental procedures were approved by the Animal Care and Use Committee of Zhejiang University (Hangzhou, China, approval number ZJU20260189).

Animals and sample collection

A total of 270 Hy-Line Brown laying hens were utilized in this study and housed under standardized conditions in accordance with the Hy-Line Brown Commercial Management Guide (2018 edition). All diets were formulated to meet or exceed the recommendations provided in Hy-line brown Management Guide (Hy-Line, 2018).Based on their chronological age and physiological laying stages, the experimental birds were allocated into three groups: the Peak-laying period (32 weeks of age, PLP), mid-laying period (58 weeks of age, MLP), and late-laying period (84 weeks of age, LLP). Each group comprised 90 hens. At the time of sampling, 15 hens from each group were randomly selected for blood collection via the wing vein, and serum was subsequently separated by centrifugation at 4°C. After euthanasia, the ovary was removed and its macroscopic morphological photos were taken. The intact ceca were harvested and preserved at −80°C to facilitate downstream microbiome analyses (n = 15). Concurrently, ovarian and intestinal tissues from a subset of 6 hens were selectively fixed in paraformaldehyde and glutaraldehyde for histological and ultrastructural evaluations. The remaining tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C for subsequent testing (n = 6).

Fecal microbiota transplantation

Twenty recipient hens from the LLP group were randomly allocated into FMT-CON and FMT-PLP groups. To deplete their endogenous microbiota, all birds received a daily oral gavage of an antibiotic cocktail (10 mg/kg ampicillin, 100 mg/kg metronidazole, 50 mg/kg vancomycin, 100 mg/kg neomycin, and 1 mg/kg amphotericin B) for one week. Following the antibiotic treatment, a 48-hour washout period was implemented, during which the birds were provided with standard, antibiotic-free drinking water to ensure the clearance of residual antibiotics from the gastrointestinal tract. Subsequently, fresh feces of hens in the PLP group were collected every day. After removing the white uric acid part, the samples were transferred to sterile tubes. As previously described (Yang et al., 2023; Gao et al., 2025; Liu et al., 2025a), the feces were rapidly suspended in a sterile PBS solution at a concentration of 0.2 g/mL (10 g of feces dissolved in 50 mL of PBS), followed by vortexing and shaking to ensure full homogenization. The homogenate was centrifuged to pellet large particulate matter, and the resulting supernatant was filtered to eliminate any remaining debris, yielding the final fecal microbiota suspension. Birds in the FMT-PLP group were orally gavaged daily with the freshly prepared fecal suspension at a dose of 1 mL/kg body weight for three consecutive weeks. Conversely, the control birds (FMT-CON) received a daily oral gavage of an equivalent volume of sterile PBS.

Laying performance

Egg weight and egg number were recorded daily, and feed consumption was recorded at each stage with 15 birds as a replicate (n = 6). Data collected over the 2 consecutive weeks prior to sampling were utilized to determine the laying rate (LR), average daily egg mass (ADEM), average daily feed intake (ADFI), and feed conversion ratio (FCR).

Egg quality assessment

An automated egg quality analyzer (DET-6000, Nabel Co., Ltd., Kyoto, Japan) was utilized to precisely determine egg weight (including total, albumen, and yolk weights), eggshell strength, yolk color, Haugh unit, and albumen height. Furthermore, the longitudinal and transverse diameters of the eggs were measured using a vernier caliper.

Histological observation

As previously reported (Zhou et al., 2021), the paraformaldehyde-fixed ovarian and intestinal tissues were embedded in paraffin, sectioned, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Intestinal villus height (VH) and crypt depth (CD) were quantified utilizing ImageJ software, and the corresponding VDR (VH/CD) was calculated as previously reported (Cao et al., 2023a). For transmission electron microscopy (TEM) examinations, the glutaraldehyde-fixed ovarian tissues were trimmed, post-fixed with osmium tetroxide, dehydrated through a graded series of ethanol and acetone, embedded, and sectioned, as previously reported (Wang et al., 2026c). The resultant ultrathin sections were double-stained with uranyl acetate and lead citrate, and ultimately observed and photographed using a Hitachi H-7650 transmission electron microscope (Hitachi, Yokohama, Japan).

Evaluation of redox status

Ovarian and intestinal tissues were homogenized, and the resulting supernatants were utilized to determine the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), alongside the malondialdehyde (MDA) content, using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), with all measurements normalized to the total protein concentration, as previously reported (Wang et al., 2026b).

ELISA analysis

Serum hormone levels were quantified using commercial ELISA kits (Aifang Biological, China). Specifically, estradiol (E2), progesterone (P4), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined via a two-step double-antibody sandwich ELISA, whereas anti-Müllerian hormone (AMH) and androstenedione (ASD) were evaluated using a competitive ELISA method. The final hormone concentrations were interpolated from their respective standard curves. The concentrations of IL-1β, IL-6, IL-8, and TNF-α in both tissues and serum were assayed strictly in accordance with the manufacturer's protocols, as previously described (Lv et al., 2024). The tissue measurement results were standardized by the total protein content to ensure the accuracy of comparison between samples.

Immunofluorescence analysis

Paraffin sections were deparaffinized, hydrated, and placed in citrate buffer for high-pressure antigen retrieval for 3 minutes. Following blocking with BSA for 30 min, sections were incubated overnight at 4°C with a primary antibody cocktail against Occludin and Claudin-1 (Servicebio, China). Subsequently, sections were incubated with Alexa Fluor 488- and CY3-conjugated secondary antibodies for 50 min at room temperature in the dark. Nuclei were counterstained with DAPI, and imaged utilizing a fluorescence microscope (Eclipse C1, Nikon, Japan).

16S rRNA gene sequencing analysis

Total genomic DNA was extracted from the cecal contents utilizing the E.Z.N.A. Soil DNA Kit. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using barcoded primers (338F/806R) and subsequently sequenced on the Illumina MiSeq platform. The quality-filtered and merged sequences were denoised utilizing the DADA2 plugin within the QIIME 2 pipeline. Subsequently, the abundance data from the 45 samples were rarefied to a uniform sequencing depth of 28,002 sequences per sample, ultimately yielding a total of 7,953 amplicon sequence variants (ASVs). Non-metric multidimensional scaling (NMDS) and principal coordinate analysis (PCoA) were employed to evaluate beta diversity. Finally, linear discriminant analysis effect size (LEfSe) was utilized to identify differentially abundant microbial taxa, with a threshold of LDA > 3 and P < 0.05.

Quantitative real-time PCR

Total RNA was extracted from ovarian and intestinal tissues using Trizol reagent. cDNA was synthesized using the HiScript II Q RT SuperMix kit (Vazyme, Nanjing, China), and RT-qPCR was performed on a StepOne Plus Real-Time PCR System. Target gene mRNA expression was normalized Nto β-actin and calculated using the 2−ΔΔCt method. Primer sequences are listed in Table S1.

Statistical analysis

Data were analyzed using SPSS 26.0 software. Results are presented as mean ± standard error of the mean (SEM). Data normality was assessed using the Shapiro-Wilk test. Differences among multiple groups were analyzed using one-way ANOVA followed by Duncan's multiple range test, while comparisons between two groups were performed using Student's t-test. Statistical significance was set at P < 0.05, indicated by asterisks (*). Figures were generated using GraphPad Prism 8.0 software.

Results

Aging in laying hens is accompanied by compromised production performance and deteriorated egg quality

The production performance of hens during the peak (PLP), mid (MLP), and late (LLP) laying periods is presented in Fig. 1A. The laying rate of the hens decreased significantly with advancing age (P < 0.01). Furthermore, the average daily egg mass in the LLP group was significantly lower than that in the PLP and MLP groups (P < 0.01). Moreover, although the average daily feed intake of the hens showed no significant differences among the three periods, the feed conversion ratio in the LLP group was significantly higher than those in the PLP and MLP groups (P < 0.01).

Fig. 1.

Fig. 1

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Production performance and egg quality of hens at different ages. (A) Laying rate, average daily egg mass, average daily feed intake and feed conversion ratio of hens. (B) Egg quality indices. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Regarding egg quality (Fig. 1B), no significant differences were observed among the groups in terms of egg shape index, relative eggshell weight, relative yolk weight, and yolk color across the different laying periods. However, compared to the PLP group, the LLP group exhibited significantly decreased eggshell thickness, eggshell strength, and albumen height (P < 0.05). Concurrently, the Haugh unit in the LLP group was significantly lower than those in the PLP and MLP groups (P < 0.01).

Hen aging leads to significant ovarian atrophy and a decrease in the number of developing follicles at all levels

The overall morphology of the ovary, the developmental hierarchy of the follicles, and their quantification are illustrated in Fig. 2. With advancing age, the overall ovarian volume gradually atrophied, accompanied by a noticeable reduction in the number of attached follicles (Fig. 2A). Quantitative analysis of the developing follicles (Fig. 2B) revealed that the numbers of large yellow follicles and small yellow follicles declined significantly as the laying period progressed, being significantly lower in both the MLP and LLP groups compared to the PLP group (P < 0.05 or P < 0.01). Concurrently, the number of preovulatory follicles in the LLP group was significantly lower than that in the PLP and MLP groups (P < 0.01). Furthermore, while the number of large white follicles showed no statistical difference among the groups, the number of small white follicles in the LLP group was significantly lower than those in the MLP and PLP groups (P < 0.05 or P < 0.01). Overall, the total number of ovarian follicles exhibited a significant decline with advancing hen age (P < 0.01). Similarly, the absolute ovarian weight and ovarian percentage also decreased as the hens aged. Significant differences in ovarian weight were observed among all three laying periods (P < 0.01). The ovarian percentage differed significantly between the PLP and MLP groups (P < 0.05) and was significantly lower in the LLP group compared to the preceding two stages (P < 0.01).

Fig. 2.

Fig. 2

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Ovarian morphological changes and follicle quantification. (A) Representative images of ovaries and the attached follicles. (B) Quantitative analysis of follicles and the ovarian weight and ovarian percentage. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Hen aging exacerbates systemic inflammation and suppresses reproductive hormone secretion

As shown in Fig. 3A, the levels of FSH, LH and AMH significantly decreased with increasing age (P < 0.05 or P < 0.01). The concentrations of E2, P4, and ASD showed no statistical differences between the PLP and MLP groups, but declined significantly in the LLP group (P < 0.05 or P < 0.01). Analysis of serum inflammatory cytokines (Fig. 3B) revealed that pro-inflammatory cytokine levels exhibited an increasing trend with advancing age. The concentrations of IL-6 and IL-1β differed significantly among all three groups (P < 0.05 or P < 0.01). TNF-α levels showed no significant differences between the PLP and MLP groups but were significantly elevated in the LLP group (P < 0.01). Furthermore, IL-8 concentrations in both the MLP and LLP groups were significantly higher than those in the PLP group (P < 0.01).

Fig. 3.

Fig. 3

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Serum reproductive hormone and inflammatory cytokine concentrations. (A) Serum concentrations of FSH, LH, E2, P4, ASD and AMH. (B) Serum concentrations of pro-inflammatory cytokines. FSH: follicle-stimulating hormone; LH: luteinizing hormone; E2: estradiol; P4: progesterone; AMH: anti-Müllerian hormone; ASD: androstenedione; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; IL-1β: interleukin-1β; IL-8: interleukin-8. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Aging significantly downregulates the expression of genes associated with ovarian hormone receptors and steroidogenic pathways in hens

As illustrated in Fig. 4A, the relative mRNA expression levels of LHR and FSHR were significantly lower in the LLP group compared to the PLP and MLP groups (P < 0.01). The expression of ESR2 in the LLP group was significantly lower than that in the MLP and PLP groups (P < 0.05 or P < 0.01), whereas the relative expression of ESR1 showed no significant differences among the three groups. The expression of genes involved in ovarian steroidogenesis (Fig. 4B) revealed that the expression levels of STAR and CYP19A1 in the MLP group were significantly lower than those in the PLP group (P < 0.05), and these levels declined further in the LLP group (P < 0.05 or P < 0.01). The expression levels of CYP11A1 and HSD17B1 in the LLP group were significantly downregulated compared to those in the MLP and PLP groups (P < 0.05 or P < 0.01). Furthermore, compared to the PLP group, the expression levels of CYP17A1 and HSD3B1 were also significantly downregulated in the LLP group (P < 0.05 or P < 0.01).

Fig. 4.

Fig. 4

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Gene expression of ovarian hormone receptors and steroidogenic enzymes. (A) Relative mRNA expression of LHR, FSHR, ESR1, and ESR2. (B) Relative mRNA expression of genes involved in ovarian steroidogenesis. LHR: luteinizing hormone receptor; FSHR: follicle-stimulating hormone receptor; ESR1: estrogen receptor 1; ESR2: estrogen receptor 2; STAR: steroidogenic acute regulatory protein; CYP19A1: cytochrome P450 family 19 subfamily A member 1; CYP11A1: cytochrome P450 family 11 subfamily A member 1; HSD17B1: 17β-hydroxysteroid dehydrogenase 1; CYP17A1: cytochrome P450 family 17 subfamily A member 1; HSD3B1: 3β-hydroxysteroid dehydrogenase 1. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Hen aging induces ovarian morphological degeneration and activates inflammation

Representative H&E staining (Fig. 5A) revealed progressive histological degeneration of the ovaries and follicles with advancing age. In the PLP group, a large number of structurally intact and well-developed follicles at all levels can be seen in the ovarian tissue. Conversely, LLP ovaries showed fewer normal follicles, loosened stroma, and evident inflammatory infiltration (blue arrows). Microscopic observation of follicles further showed that from the PLP stage to the LLP stage, the granulosa cell layer and theca cell layer became thinner, and the cell arrangement gradually became loose. Ovarian IL-1β and IL-6 concentrations increased significantly with age (Fig. 5B; P < 0.05 or P < 0.01). The LLP group exhibited significantly higher IL-8 than the PLP group, and elevated TNF-α compared to both PLP and MLP groups (P < 0.01). Furthermore, the mRNA expression of inflammation-related genes (Fig. 5C) mirrored the trends in cytokine concentrations. NF-κB expression was significantly upregulated with age (P < 0.01). Both IL-1β and IL-6 were significantly upregulated in the LLP group versus the PLP and MLP groups (P < 0.05 or P < 0.01). Compared to the PLP group, TNF-α mRNA levels increased significantly in both MLP and LLP groups (P < 0.05 or P < 0.01). IL-8 was significantly elevated in the LLP group (P < 0.05). No significant differences were observed in INF-γ expression across groups.

Fig. 5.

Fig. 5

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Histological assessment and inflammatory gene expression in ovarian tissues. (A) Representative HE staining of ovarian and follicle tissues (B) Ovarian concentrations of inflammatory cytokines. (C) Relative mRNA expression of inflammation-related genes. Blue arrows indicate inflammatory infiltration. GL: granulosa layers; TL: theca layers; NF-κB: nuclear factor kappa B; INF-γ: interferon-gamma. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Aging exacerbates ovarian ultrastructural deterioration and induces oxidative stress in hens

TEM revealed alterations in the ovarian ultrastructure (Fig. 6A). The PLP group exhibited normal cellular morphology with intact nuclei, alongside abundant endoplasmic reticulum, lipid droplets, and mitochondria with clear cristae. Conversely, the LLP group showed swollen or ruptured mitochondria, accompanied by a reduction in ER structures. Ovarian oxidative stress biomarkers (Fig. 6B) indicated a profound state of oxidative stress in LLP hens. MDA content, a lipid peroxidation marker, increased significantly with age (P < 0.05 or P < 0.01). In contrast, the activities of T-SOD, GSH-PX, and CAT in the LLP group were significantly lower than those in the PLP and MLP groups (P < 0.01). Gene expression analysis (Fig. 6C) revealed that CAT, GPX1, and SOD2 were significantly downregulated in the LLP group compared to the PLP and MLP groups (P < 0.01). NQO1 expression was significantly lower in the MLP group than in the PLP group (P < 0.05) and further declined in the LLP group (P < 0.01). Additionally, SOD1 expression in the LLP group was significantly lower than that in the PLP group (P < 0.05). No significant differences were observed in HO-1 expression across the three stages.

Fig. 6.

Fig. 6

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Ovarian ultrastructure and oxidative stress indices. (A) Representative transmission electron microscopy images of ovarian cells. (B) Biochemical indices of oxidative stress. (C) Relative mRNA expression of antioxidant-related genes. MDA: malondialdehyde; T-SOD: total superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase; GPX1: glutathione peroxidase 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; NQO1: NAD(P)H quinone dehydrogenase 1; HO-1: heme oxygenase-1. N: cell nucleus; M: mitochondrion; ER: endoplasmic reticulum; LD: lipid droplet. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Hen aging accelerates ovarian decline by suppressing cell proliferation and promoting apoptosis

Analysis of cell proliferation-related genes in ovarian tissue (Fig. 7B) revealed that Ki67 expression was significantly downregulated in the MLP and LLP groups (P < 0.05). Compared to the PLP group, PCNA expression was significantly decreased in both MLP and LLP groups (P < 0.05 or P < 0.01). CCND1 expression in the LLP group was significantly lower than that in the MLP and PLP groups (P < 0.05 or P < 0.01). Additionally, the mRNA levels of CDK2 and CCND2 were significantly downregulated in the LLP group compared to the PLP group (P < 0.05). Apoptosis-related gene expression profiles are presented in Fig. 7C. The expression of P53, a key pro-apoptotic factor, was significantly higher in both MLP and LLP groups than in the PLP group (P < 0.05 or P < 0.01). The expressions of Caspase-10 and BAK were significantly higher in the LLP group than in the MLP and PLP groups (P < 0.05 or P < 0.01), and Caspase-3 was also significantly elevated compared to the PLP group (P < 0.01). Conversely, the anti-apoptotic gene BCL2 was significantly downregulated with age (P < 0.05 or P < 0.01). No statistical differences were observed in BCL9 expression across groups. Ovarian apoptosis was further evaluated using TUNEL immunofluorescence (Fig. 7A). The results indicated a progressive exacerbation of tissue apoptosis with advancing age. While the PLP group exhibited few apoptotic-positive cells, TUNEL-positive fluorescent signals increased markedly in the ovaries of the MLP and LLP groups.

Fig. 7.

Fig. 7

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Ovarian cell proliferation and apoptosis. (A) Representative images of TUNEL immunofluorescence assay. (B) Relative mRNA expression of cell proliferation-related genes. (C) Relative mRNA expression of apoptosis-related genes. PCNA: proliferating cell nuclear antigen; CCND1: cyclin D1; CDK2: cyclin dependent kinase 2; CCND2: cyclin D2; P53: tumor protein P53; Caspase-10: caspase 10; BAX: BCL2 associated X, apoptosis regulator; Caspase-3: caspase 3; BCL2: B-cell lymphoma 2; BCL9: B-cell lymphoma 9. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Aging induces jejunal structural damage and exacerbates inflammation and oxidative stress in hens

H&E staining of jejunal tissues (Fig. 8A) revealed relatively normal and intact morphology in both PLP and MLP groups. However, the LLP group exhibited severe intestinal structural deterioration, characterized by significantly decreased VH and CD. Jejunal inflammatory cytokine analysis (Fig. 8C) showed that IL-8 concentrations increased significantly with age (P < 0.05 or P < 0.01). While IL-1β, IL-6, and TNF-α concentrations showed no statistical differences between the PLP and MLP groups, they were significantly elevated in the LLP group (P < 0.05 or P < 0.01). The expression profiles of jejunal inflammation-related genes are presented in Fig. 8D. NF-κB expression was significantly upregulated with age (P < 0.05 or P < 0.01). IL-1β expression was significantly higher in the LLP group than in the PLP and MLP groups (P < 0.01). Additionally, the expression levels of IL-8 and INF-γ were significantly upregulated in the MLP and LLP groups compared to the PLP group (P < 0.05 or P < 0.01). TNF-α expression in the LLP group was also significantly higher than that in the PLP group (P < 0.05). In contrast, IL-6 expression showed no significant differences among the groups. Regarding oxidative stress-related genes (Fig. 8E), the expression levels of CAT, GPX1, and SOD1 were significantly downregulated in the LLP group compared to the PLP and MLP groups (P < 0.05 or P < 0.01). No significant changes were observed in the expression of HO-1, NQO1, and SOD2 across the three groups.

Fig. 8.

Fig. 8

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Jejunal histological damage, inflammation, and oxidative stress. (A) Representative HE staining of jejunal tissues. (B) Villus height and crypt depth measurement. (C) Jejunal concentrations of inflammatory cytokines. (D) Relative mRNA expression of inflammation-related genes. (E) Relative mRNA expression of oxidative stress-related genes. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Hen aging disrupts intestinal barrier integrity and downregulates tight junction gene expression

As shown in Fig. 9D, the jejunal mRNA expression levels of ZO-1, OCCLUDIN-1, and CLAUDIN-1 were significantly lower in the LLP group compared to the PLP and MLP groups (P < 0.05 or P < 0.01). Similarly, the expression of MUC-2, a marker of intestinal mucus barrier function, was significantly downregulated in the LLP group versus the PLP group (P < 0.05). No significant differences were observed in the expression of these genes between the PLP and MLP groups. Jejunal expression of Occludin and Claudin-1 was further evaluated using immunofluorescence (Fig. 9A). Both PLP and MLP groups exhibited strong positive fluorescent signals for Occludin and Claudin-1, displaying a clear and continuous distribution. Conversely, the LLP group showed markedly weakened, fragmented, and discontinuous fluorescent signals, indicating compromised jejunal tight junction structures in late-laying hens.

Fig. 9.

Fig. 9

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Intestinal barrier integrity and tight junction gene expression. (A) Representative immunofluorescence staining of Occludin and Claudin-1 proteins in the jejunum. (B) Relative mRNA expression of tight junction genes and the MUC-2 gene. ZO-1: zonula occludens-1; MUC-2: mucin 2. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Diversity and structural evolution of the gut microbiota during hen aging

Cecal microbiota were analyzed via 16S rRNA sequencing (Fig. 10). The Sobs index-based rarefaction curves plateaued across all samples (Fig. 10A), indicating adequate sequencing depth. ASV-level alpha diversity analysis (Fig. 10B) revealed no significant differences among the groups in Sobs, Simpson, Chao, and Ace indices, which represent community richness and evenness (P > 0.05). However, beta diversity analysis revealed a reorganization of the community structure. Both principal coordinate analysis (PCoA, Fig. 10C) and non-metric multidimensional scaling (NMDS, Fig. 10D) demonstrated distinct structural separation of microbial communities across the groups. Linear regression analysis further validated the relationship between hen age and gut microbiota structural shifts (Figs. 10E and F). The scores for PCoA principal coordinate 1 (PC1) and NMDS dimension 1 (NMDS1) both exhibited a highly significant negative correlation with hen age (Days) (P < 0.001). These findings confirm that the gut microecological structure alters significantly as hens age.

Fig. 10.

Fig. 10

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Gut microbiota diversity and structural analysis. (A) Rarefaction curves based on the Sobs index. (B) Alpha diversity analysis. (C) Principal coordinate analysis. (D) Non-metric multidimensional scaling analysis. (E, F) Linear regression analysis. ASV: amplicon sequence variant; PCoA: principal coordinate analysis; NMDS: non-metric multidimensional scaling.

Hen aging induces alterations in the gut microbiota composition

Community composition and LEfSe analysis (Fig. 11) further elucidated the temporal changes in cecal microbiota and identified differentially abundant taxa. Circos plots visually present the distribution of dominant taxa at the phylum (Fig. 11A) and genus (Fig. 11B) levels. At the phylum level, Bacteroidota and Bacillota predominated across all stages. At the genus level, Bacteroides, Rikenellaceae_RC9_gut_group, unclassified_o__Bacteroidales, and Streptococcus constituted the core shared genera among hens of different ages. LEfSe analysis (Figs. 11C and D) further identified differentially abundant taxa for each stage. Specifically, the PLP group was significantly enriched with p_Bacillota, g_Parabacteroides, and f_Tannerellaceae. The characteristic taxa in the MLP group primarily included f_Prevotellaceae, g_Campylobacter, and c_Verrucomicrobiia. Conversely, the LLP group exhibited significant enrichment of p_Bacteroidota and its subordinate taxa (e.g., Rikenellaceae_RC9_gut_group), along with g_Helicobacter.

Fig. 11.

Fig. 11

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Composition and differential taxa of microbiota. (A) Circos plots displaying the distribution of dominant taxa at the phylum levels. (B) Circos plots displaying the distribution of dominant taxa at the genus levels. (C) The cladogram of LEfSe analysis. (D) The histogram of LEfSe analysis.

Potential correlations between aging-related gut microbiota and indicators of ovarian functional decline

Spearman correlation analysis was employed to evaluate the potential associations between differentially abundant microbial genera and the expression of genes related to ovarian inflammation (Fig. 12A), antioxidant enzymes (Fig. 12B), apoptosis (Fig. 12C), and cell proliferation (Fig. 12D). Regarding inflammation (Fig. 12A), Helicobacter abundance was positively correlated with TNF-α, NF-κB, and IL-1β expressions (P < 0.05). Conversely, Parabacteroides and Lactobacillus exhibited significant negative correlations with multiple pro-inflammatory genes (TNF-α, IL-8, IL-1β, and NF-κB). In terms of antioxidant genes (Fig. 12B), Helicobacter abundance correlated negatively with SOD1, SOD2, and NQO1 (P < 0.05). In contrast, Alistipes and Parabacteroides abundances showed significant positive correlations with these antioxidant genes, including SOD2, SOD1, and NQO1 (P < 0.05). Regarding apoptosis (Fig. 12C), Helicobacter correlated positively with P53 and Caspase-10, but negatively with BCL2 (P < 0.05). Conversely, Lactobacillus and Parabacteroides were negatively correlated with pro-apoptotic genes such as Caspase-3 and Caspase-10. For cell proliferation (Fig. 12D), Parabacteroides, Lactobacillus, and Mediterraneibacter exhibited significant positive correlations with multiple proliferation marker genes, including PCNA and Ki67. In contrast, Helicobacter showed significant negative correlations with these proliferation genes.

Fig. 12.

Fig. 12

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Correlation analysis between aging-related gut microbiota and ovarian function indicators. Spearman correlation analysis between differentially abundant gut microbial genera and gene expression related to: (A) ovarian inflammation, (B) antioxidant enzymes, (C) apoptosis, and (D) cell proliferation.

FMT from peak-laying donors improves ovarian status in late-laying recipient hens

To elucidate whether the intestinal microbiota is directly implicated in the modulation of ovarian function in aging hens, a FMT trial was conducted (Fig. 13A). Prior to the FMT trial, the recipient gut microbiota was confirmed to be adequately depleted by the two-week antibiotic cocktail treatment, achieving a bacterial clearance rate of >99% (Figure S1). Histological evaluation revealed that while the overall structural of the ovarian tissue did not differ remarkably among the groups, the microstructural integrity of the follicles was notably ameliorated in the FMT-PLP group (Fig. 13B). Compared with the FMT-CON group, the granulosa and theca layers of the follicles in hens receiving the PLP microbiota exhibited a more intact and compact morphological organization. Furthermore, FMT profoundly modulated the inflammatory and oxidative stress status within the ovaries. The mRNA expressions of pro-inflammatory cytokines (NF-κB, IL-1β, and TNF-α) were significantly downregulated in FMT-PLP ovaries relative to CON (P < 0.05, Fig. 13C). Concurrently, the expressions of antioxidant genes (NQO1 and SOD2) were significantly upregulated in the FMT-PLP group (P < 0.05, Fig. 13D). In addition, FMT also slowed down the decline process of ovarian cells. The results showed that FMT-PLP significantly inhibited the expression of pro-apoptotic genes Caspase-10 and BAK (P < 0.05, Fig. 13E). Conversely, proliferation marker genes (PCNA and CCND1) were significantly upregulated in the FMT-PLP group (P < 0.05, Fig. 13F). In conclusion, the gut microbiota from peak-laying hens effectively mitigates aging-induced ovarian inflammation and apoptosis, thereby restoring cellular proliferation and antioxidant capacities.

Fig. 13.

Fig. 13

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FMT from peak-laying donors improves ovarian status in late-laying recipient hens. (A) Schematic diagram of the FMT trial. (B) Representative HE staining of ovarian and follicle tissues. (C) Relative mRNA expression of inflammation-related genes. (D) Relative mRNA expression of antioxidant-related genes. (E) Relative mRNA expression of apoptosis-related genes. (F) Relative mRNA expression of cell proliferation-related genes. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01.

Discussion

Aging is a continuous and intricate biological process characterized primarily by a precipitous decline in egg production and the deterioration of egg quality in laying hens, representing a persistent challenge within the poultry industry (Hu et al., 2025). The present study multidimensionally delineates the trajectory of ovarian functional decline during hen aging and investigates the critical roles of intestinal barrier impairment and gut microbial community succession in this process. These findings not only confirm the presence of inflammaging in the avian ovary but also underscore the pivotal role of the gut-ovary axis in mediating ovarian functional decline, thereby providing a robust theoretical foundation for extending the laying cycle via targeted interventions in the gut microecology.

Phenotypic data regarding the production performance of laying hens across the peak, mid, and late laying phases serve as the most intuitive indicators of reproductive aging. Our longitudinal analysis of egg production and quality across these three periods demonstrates that both the laying rate and average daily egg mass decrease significantly with advancing age, accompanied by the deterioration of egg quality metrics such as eggshell strength. Gross anatomical observation of the hen ovary shows that the direct cause of the decline in production performance is the significant reduction in the number of follicles in the ovary. Follicular recruitment, selection, and ovulation constitute an orderly cascade essential for sustaining high-intensity and prolonged egg production in laying hens (Nie et al., 2024). In this study, the numbers of hierarchical follicles exhibited varying degrees of age-related depletion, primarily characterized by inadequate recruitment of small white follicles, impeded selection of small yellow follicles, and compromised maturation of preovulatory follicles; these findings provide a histological explanation for the drastic drop in egg production during the late laying phase.

The normal development and continuous ovulation of follicles are influenced by the regulation of the endocrine system, however, aging can disrupt the balance of reproductive hormones in hens (Zhang et al., 2025). In our study, serum concentrations of anterior pituitary-derived FSH and LH declined significantly with advancing age. The decrease in FSH levels attenuates the recruitment of early follicles, while the decrease in LH leads to the loss of ovulation signals (Bosch et al., 2021; Longo et al., 2025). In addition, AMH, as a hormone specifically secreted by granulosa cells, supports the regulation of follicle selection, and its significant decrease in concentration indicates an insufficient follicle reserve in the ovary (Li et al., 2026). Similarly, steroid hormones that affect follicle development, such as E2, P4, and ASD, which are mainly secreted by the ovary, also significantly decreased in the LLP group, accompanied by the downregulation of key steroidogenic enzymes. Ovarian steroidogenesis relies on an enzymatic cascade wherein the STAR protein serves as the rate-limiting carrier transporting cholesterol from the outer to the inner mitochondrial membrane, representing a crucial step in the entire biosynthetic pathway (Koganti et al., 2025). The significant decrease in ovarian STAR content in the LLP group limits subsequent hormone synthesis. The expressions of enzymes such as CYP11A1, which is responsible for cleaving cholesterol into pregnenolone (Liu et al., 2024), CYP19A1, which aromatizes androgens into estrogens in granulosa cells (Baddela et al., 2023), and HSD17B1, which catalyzes the conversion of precursor estrogens into highly active estradiol (Heinosalo et al., 2019), were significantly downregulated in the ovaries of the LLP group, further revealing the decline in the hormone secretion function of the aging ovary. In addition, the regulation of hormones relies on the cooperation of corresponding receptors on target cells (Wang et al., 2022). The significant downregulation of receptors such as FSHR, LHR, and ESR during the late laying phase obstructs downstream signaling pathways, thereby exacerbating follicular developmental disorders in aging hens.

As previously noted, the diminished production performance in late-phase laying hens is directly correlated with impaired follicular development and ovarian functional decline. It is imperative to analyze the driving factors underlying ovarian senescence and dysfunction. Increasing research focus is shifting to inflammaging, a phenomenon where advancing age subjects the organism to a persistent state of chronic inflammation, widely recognized as a hallmark of aging (Li et al., 2023). The increase of inflammatory factors in the serum of the LLP group confirmed that this systemic inflammation is related to age. Correspondingly, ovarian tissues from the late laying period exhibited a marked increase in both pro-inflammatory cytokine concentrations and their respective mRNA expression levels. The expression of NF-κB was significantly upregulated with hen aging, followed by its nuclear translocation (Miao et al., 2024; Guo et al., 2024), which subsequently activated the transcription of downstream target genes including IL-1β, IL-6, and TNF-α.

Another critical factor driving ovarian senescence is oxidative stress (Ammar et al., 2024). Ovulation in laying hens is a highly active metabolic process, which easily induces the generation of excessive reactive oxygen species (ROS) (Lin et al., 2025). TEM observations of ovarian tissues revealed mitochondrial swelling and endoplasmic reticulum degeneration in hens during the late laying phase, indicative of organelle damage induced by oxidative stress. Intact mitochondria and endoplasmic reticula provide the structural basis for STAR-mediated steroidogenesis (Witt et al., 2023), elucidating the mechanism underlying restricted steroid synthesis in aging ovaries at the subcellular level. The significant elevation of MDA, an end product of lipid peroxidation, in the ovaries of the LLP group further substantiated the oxidative damage inflicted by ROS (Wu et al., 2024). More importantly, as hens age, the activities of antioxidant enzymes such as CAT, GSH-PX, and T-SOD in the ovary decrease, breaking the oxidative stress balance in the ovary, and these changes were further confirmed at the gene level by qPCR.

Persistent chronic inflammation coupled with intense oxidative stress precipitates extensive apoptosis in ovarian cells (Chen et al., 2024). Our findings demonstrated a significant downregulation of genes associated with cell proliferation and the cell cycle in the ovaries of late-phase laying hens, including the proliferation markers PCNA and Ki67, as well as the cyclins CDK2, CCND1, and CCND2, which drive the G1-to-S phase transition. This indicates that the cell division and renewal ability of the aging laying hen ovary is inhibited, making it difficult to support the proliferation and maturation of early follicles. Furthermore, a profound upregulation in the expression of P53, a crucial pro-apoptotic transcription factor, was observed in the LLP group. P53 orchestrates apoptosis by modulating the ratio of Bcl-2 family proteins (Hafner et al., 2019). Correspondingly, the upregulation of the pro-apoptotic factor BAK in aging ovaries was accompanied by elevated expression of the apoptotic marker caspase-3 and caspase-10. TUNEL fluorescence also confirmed the widespread cell apoptosis in the LLP group. Large-scale cell apoptosis in follicles leads to the stagnation of follicle development and moves towards atresia, ultimately causing a decline in the egg production rate (Ru et al., 2024).

While substantial evidence indicates that the aging of laying hens in the late laying period is mainly manifested in ovarian tissue, many studies have focused on the intestine in recent years, especially the age-related changes of gut microbiota and intestinal mucosal barrier (Su et al., 2026). To elucidate the factors exacerbating ovarian senescence, this study evaluated the characteristic changes in the intestinal tract of laying hens across different production phases. Analogous to the observations in ovarian tissues, an upregulation of markers associated with inflammation and oxidative stress was observed in the intestinal tissues of the LLP group. The results showed that in the peak and middle laying periods, the intestinal mucosal barrier still maintained a high degree of integrity. However, the LLP group exhibited compromised intestinal structure and barrier function, characterized primarily by villus atrophy, crypt degradation, and a significant downregulation of tight junction protein expression. The damaged physical barrier of the intestinal mucosa leads to increased permeability, making it easier for harmful substances in the intestinal lumen to translocate into the blood circulation, and then affect the ovary through the systemic circulation, inducing inflammatory reactions, and finally leading to a decline in production performance (Yue et al., 2026).

Intestinal barrier impairment is frequently associated with gut microbiota dysbiosis (Yan et al., 2022). Our 16S rRNA sequencing results indicated no significant differences in microbial species richness and evenness in the cecal contents of hens across the different phases. This suggests that the overall microbial species diversity remains relatively stable throughout the entire laying cycle. However, PCoA and NMDS analyses of the microbial structure revealed that the cecal microbiota underwent significant reorganization as the hens aged, and regression analysis further confirmed that this segregation was correlated with the chronological age of the hens. Further compositional analysis revealed that Bacteroidota and Bacillota (at the phylum level), as well as Bacteroides, Rikenellaceae_RC9_gut_group, and Bacteroidales (at the genus level), were the predominant taxa throughout the laying cycle, consistent with previous studies on various hen breeds (Su et al., 2026). LEfSe analysis was employed to identify the characteristics of microbial succession occurring with the progression of hen age. In PLP group, characterized by optimal production performance, representative taxa of a healthy microbiome were dominant. This phase was significantly enriched with the phylum Bacillota, the genus Parabacteroides, and the genus Monoglobus. Monoglobus is a taxon of plant pectin- and polysaccharide-degrading bacteria (Kim et al., 2019; Puhlmann et al., 2024), and given the high content of plant cell wall components in the feed, Monoglobus can efficiently utilize and decompose these substrates. When in the MLP group in the middle of egg laying, the microbiota showed a transitional change. The characteristic taxa shifted to Prevotellaceae, Campylobacter, and Verrucomicrobiia. Prevotellaceae are typically capable of fermenting complex polysaccharides (Tett et al., 2021; Wang et al., 2025), and their increased abundance may reflect an adaptive metabolic shift in the hen's gut to the continuous, monotonous diet. The concurrent emergence of Campylobacter suggests that potential opportunistic pathogens have initiated colonization within the gut (Rasschaert et al., 2020). Notably, upon entering the late laying phase, there was an aberrant increase in the abundance of Helicobacteriaceae and Helicobacter. Helicobacter is a recognized pathogen capable of colonizing mucosal surfaces, secreting cytotoxins, and inducing severe mucosal injury and systemic inflammation (Ménard and Smet, 2019; Ochoa and Collado, 2021).

By performing correlation analysis between the gut microbiota and ovarian functional indicators, we sought to identify the marker species that drive pathological features of aging. Results demonstrated that the abundances of Parabacteroides and Lactobacillus were significantly positively correlated with the maintenance of ovarian function, while being significantly negatively correlated with ovarian inflammation and apoptosis. Lactobacillus a widely acknowledged probiotic, has been established as being enriched in the gut of highly productive laying hens (Cao et al., 2023b; Wang et al., 2024; Zhan et al., 2025). Parabacteroides is recognized as a next-generation probiotic with potential benefits for host health. It possesses numerous polysaccharide utilization loci, enabling it to coordinate the degradation of complex polysaccharides, maintain intestinal homeostasis, and suppress inflammation (Chen et al., 2025; Liu et al., 2025b). Conversely, the proliferation of Helicobacter during the LLP phase was significantly positively correlated with ovarian inflammatory outbreaks, the downregulation of antioxidant genes, and extensive apoptosis. When the intestinal barrier ruptures in the late laying period, these harmful bacterial groups and their secreted toxic substances are very easy to translocate and spread through the blood to other tissues including the ovary, which explains why this bacterial group is highly positively correlated with ovarian inflammation indicators. In summary, our findings suggest that aging-driven enrichment of intestinal pathobionts and the concomitant loss of beneficial commensals may exacerbate ovarian oxidative stress and chronic inflammation, leading to impaired ovarian function and defective follicular development, which ultimately manifests phenotypically as a precipitous decline in egg production.

Correlation analysis suggested a link between age-related gut microbiota succession and ovarian reproductive decline; to further substantiate the causal role of the gut microbiota in this process, a FMT trial was conducted. The results demonstrated that transplanting fecal microbiota from peak-laying hens into late-laying recipients restored follicular morphology, alleviated ovarian inflammation and oxidative stress, suppressed cell apoptosis, and enhanced cellular proliferation capacity in the recipient hens. These results demonstrate that age-related succession of the gut microbiota plays a pivotal role in the progression of ovarian aging. This suggests that restoring a youthful and healthy gut microbial community can modulate ovarian health, thereby delaying or even partially reversing ovarian functional decline.

Based on these findings, specific microbial taxa, notably Parabacteroides and Lactobacillus, are closely associated with the alleviation of ovarian senescence. Consequently, they may serve as promising candidates for the development of targeted microbial products. Given their potential beneficial effects on host health, utilizing these specific microbes as functional feed additives could offer a practical strategy to maintain ovarian function and extend the productive lifespan of laying hens.

Conclusion

This study elucidated the age-related progressive characteristics of ovarian function decline in laying hens through multidimensional analysis of health-related ovarian function indicators. Furthermore, it revealed the potential driving roles of intestinal physical barrier impairment and specific microbial community succession in the progression of ovarian senescence (Fig. 14). The decline in production performance during the late laying phase is attributed not only to a depletion of the follicular reserve and neuroendocrine dysfunction but also to localized oxidative stress and inflammaging, which collectively drive ovarian apoptosis and follicular atresia. Moreover, gut microbiota dysbiosis and intestinal mucosal barrier damage are important factors promoting ovarian senescence. Advancing age is characterized by a reduction in beneficial gut commensals and an expansion of pathogenic populations, which collectively exacerbate oxidative damage and inflammatory responses within the ovary. More importantly, the FMT trial demonstrated that a young and healthy gut microbiota can mitigate the progression of ovarian decline in aged hens, firmly substantiating the critical role of the gut microbiome in this process. In summary, reproductive senescence in hens is a systemic process modulated by the gut microbiome and intestinal barrier function. This study provides a theoretical basis for alleviating ovarian aging and extending the egg-laying cycle of laying hens through targeted intervention of gut microbiota.

Fig. 14.

Fig. 14

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Succession of the intestinal microbiome during hen aging and its impact on ovarian function decline.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines as well as the following guidelines for animal welfare and/or reporting: Regulations for the Administration of Affairs Concerning Experimental Animals (State Science and Technology Commission of the People's Republic of China); Guiding Opinions on the Treatment of Laboratory Animals (Ministry of Science and Technology of the People's Republic of China); Zhejiang Provincial Measures for the Administration of Experimental Animals (Zhejiang Provincial People's Government). This study was approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang University. (Approval No. ZJU20260186).

Disclosures

The authors confirm that they do not have any discernible conflicting financial interests or personal affiliations that could it have been regarded as having no impact on the research presented within this paper.

Acknowledgments

The research was financially supported by the National Natural Science Foundation of China (grant no. 32402779).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.107689.

Contributor Information

Dongyou Yu, Email: dyyu@zju.edu.cn.

Bing Liu, Email: bing.liu@zju.edu.cn.

Appendix. Supplementary materials

mmc1.docx (559.2KB, docx)

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