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. 2026 Mar 13;105(6):106775. doi: 10.1016/j.psj.2026.106775

Fasting-induced physiological remodeling alleviated fatty liver in laying hens through regulating hepatic lipid metabolism

Yujie Gong a,b,†, Qingduo Zeng a,†, Xiaoqing Geng a, Jun Zhang a, Xiangtao Kang a,b, Yadong Tian a,b, Ruirui Jiang a,b,⁎
PMCID: PMC13052085  PMID: 41887173

Abstract

Aging laying hens in the late production phase are prone to fatty liver syndrome due to dysregulated lipid metabolism. Although fasting has been shown to reduce hepatic lipid accumulation in mammals, its mechanistic effects in poultry remain unclear. This study investigated whether fasting-induced physiological remodeling alleviates fatty liver in late-phase laying hens and explored the underlying molecular mechanisms. Fasting significantly reduced liver size, liver index, and hepatic triglyceride content (p < 0.05), accompanied by decreased Oil Red O staining, indicating attenuated lipid deposition. Serum estradiol (E2) concentrations decreased during fasting, whereas triiodothyronine (T3) and thyroxine (T4) concentrations increased and returned to baseline following refeeding (p < 0.05). Fasting induced transient oxidative stress, evidenced by elevated MDA levels, whereas hepatic GSH-Px activity and the mRNA expression of SOD, CAT, and GSH-Px increased during the recovery phase, indicating enhanced antioxidant capacity (p < 0.05). Liver function markers (ALT and AST) increased in serum but decreased in liver tissue during fasting, returning to normal levels after refeeding, thereby reflecting a reversible adaptive response (p < 0.05). Transcriptome and qPCR analyses revealed that autophagy-related genes (PIK3CB, ERN1) were upregulated during fasting, whereas pro-apoptotic genes (CASP-3, CASP-7) were downregulated, and the anti-apoptotic gene BCL2 was elevated (p < 0.05), indicating that fasting activated autophagy while suppressing apoptotic signaling, thereby potentially promoting hepatocellular survival during metabolic stress. The expression of the lipid synthesis gene ACAT2 decreased, while the decomposition gene ACSL1 increased (p < 0.05). Western Blot further confirmed enhanced expression of key autophagy proteins (PI3K, Beclin1, LC3) and suppression of mTOR, demonstrating that lipids were partially degraded via the autophagy pathway to supply energy. In conclusion, fasting-induced physiological remodeling effectively alleviated fatty liver in late-phase laying hens. This study provides mechanistic insight into nutritional regulation of hepatic lipid homeostasis and offers a theoretical basis for extending the laying cycle in commercial production.

Keywords: Fasting-induced physiological remodeling, Lipid metabolism, Liver, Autophagy, Apoptosis

Introduction

Under natural conditions, the periodic physiological remodeling of feathers in poultry is an inherent biological process that regulates body temperature and protect the organism. It also represents an adaptive mechanism that evolved through long-term evolutionary processes, enabling chickens to better respond to environmental fluctuations (Baker et al., 1983; Berry, 2003). Natural molting typically requires 14-16 weeks to complete natural physiological remodeling (Soe et al., 2007). In commercial production, induced molting is widely applied to synchronize flock remodeling, shorten molting duration, and restore laying performance (Alodan and Mashaly, 1999;). This approach significantly shortens the molting duration, typically to about 50-60 days (McDANIEL, 1985). Beyond improving post-molt egg quality and production efficiency (Andreatti Filho et al., 2019; Roberts et al., 2013), induced molting also promotes systemic metabolic adjustments that may influence organ function. Additionally, induced molting facilitates standardized flock management, reduces feed intake, decreases the frequency of flock replacement, and consequently improves overall production efficiency (Garlich et al., 1984). Recent studies have further demonstrated that induced molting improves intestinal health in aged hens, enhances calcium metabolism, and reduces the incidence of cartilage-related disorders (Han et al., 2019). Therefore, artificially induced physiological remodeling has been widely adopted in modern poultry production as an effective and practical technique to extend the laying cycle and enhance breeding efficiency.

Caloric restriction has been demonstrated to attenuate the decline of multiple age-associated physiological functions and extend the maximum lifespan across various animal species (de Cabo et al., 2014; Mitchell et al., 2019). Fasting, a specific form of caloric restriction, has long been practiced both as a medical intervention and as part of religious traditions. It is generally defined as a complete or partial restriction of food and/or water intake for periods ranging from 12 h to several weeks and can be classified into short-term, long-term, or intermittent regimens (Lessan and Ali, 2019). Among these, intermittent fasting (IF) refers to dietary patterns alternating between fasting and feeding periods (Harney et al., 2019). Notably, this regimen resembles the centuries-old Ramadan fasting tradition observed among Muslims, which typically involves daily fasting for 12-16 h over approximately one month (Ahmad and Chowdhury, 2019). The beneficial effects of intermittent fasting on health have been well documented in animal models (Baumeier et al., 2015), and growing evidence from human studies suggests that IF may reduce cancer risk and improve metabolic disorders such as obesity and type 2 diabetes (Horne et al., 2015). However, the precise molecular mechanisms underlying the health-promoting effects of fasting remain incompletely understood, highlighting the need to elucidate the metabolic regulatory processes involved (Ma et al., 2021). The liver plays a central role in systemic energy metabolism, particularly in regulating glucose and lipid homeostasis (Chao et al., 2019). Its metabolic activity is tightly regulated by nutrient availability and feeding status (Liu et al., 2022). As a central organ for metabolic regulation (Lawrence et al., 2019), the liver actively takes up glucose and synthesizes glycogen and triglycerides in the fed state. Conversely, during fasting, it maintains blood glucose levels via glycogenolysis and gluconeogenesis while initiating ketogenesis (Besse-Patin et al., 2019). Elevated glucagon levels during fasting further stimulate hepatic glucose production, ensuring a continuous energy supply to peripheral tissues (Moore et al., 2012). Therefore, investigating the effects of fasting on hepatic metabolism is essential for elucidating the molecular mechanisms by which nutritional states regulate energy balance, affect physiological functions, and influence the development or prevention of metabolic diseases (Chen et al., 2021b).

Existing evidence indicates that fasting-induced physiological remodeling exerts profound regulatory effects on metabolic processes. This intervention not only restores reproductive function, improves eggshell quality, reduces egg breakage, and enhances overall egg production in laying hens, but also optimizes flock composition by uniformly eliminating weak or low-producing individuals. As a central metabolic organ, the liver plays a key role in maintaining physiological homeostasis, and its functional state is closely linked to the health and productivity of laying hens. However, there remains a lack of systematic research on whether fatty liver syndrome can be alleviated and whether hepatic function undergoes adaptive remodeling during this fasting-induced physiological remodeling process. In this study, physiological remodeling refers to the systemic adaptive changes induced by fasting during molting, whereas induced molting describes the symbolic phenotypes used to demonstrate this remodeling process. Therefore, elucidating the patterns of hepatic lipid metabolism during this period is of great significance. Such insights will not only clarify the mechanisms through which fasting influences metabolism and mobilization of endogenous energy reserves but also provide theoretical foundations for understanding the physiological strategies animals employ to adapt to nutritional stress.

Materials and methods

Ethics statement

All procedures in this study were conducted in accordance with the ethical guidelines authorized by the Ethics Committee of Henan Agricultural University (Approval No. 11-0099). Special attention was paid to ensuring the welfare of animals and minimizing any potential distress.

Experimental animals and protocols

The experimental chickens were sourced from the Poultry Germplasm Resource Field of Henan Agricultural University. A total of 90 healthy chickens (Houdan), 500 days of age, with comparable body weights and an average egg production rate of approximately 60 %, were selected for the study. The chickens were randomly allocated to 9 replicates, each consisting of 10 individuals, and were housed individually in three-tier stepped cages under standardized rearing conditions.

Before the formal experiment commenced, the body weight and egg production rate of the flock were continuously recorded for more than 7 d. Individuals with uniform body weight and similar egg production performance were selected as experimental chickens. One week prior to transferring the chickens from the commercial laying house to the experimental facility, they were vaccinated against Newcastle disease, and the experimental house as well as its surrounding environment were thoroughly disinfected. Following transfer, routine disinfection was performed weekly with the chickens present, and deworming was conducted every two weeks. After relocation, the chickens were fed under normal conditions for 15 days to acclimate to the new environment. Subsequently, a fasting-induced physiological remodeling program was implemented according to the protocol described in Table 1. The fasting period was denoted as F, and the resume feeding period was denoted as R. Samples were collected at six time points: 1 day before fasting (F0), 3 days of fasting and water restriction (F3), 16 days of fasting (F16), 6 days after resume feeding (R6), 15 days after resume feeding (R15), and 32 days after resume feeding (R32). The selected time points represent early fasting (F3), peak fasting-induced regression (F16), and early recovery after refeeding (R6), based on previous molting studies and pilot observations of body weight and egg production changes.

Table 1.

Fasting-induced molting procedures.

Period Processing time(day) Feed Water Light(h/d)
Fasting (F) 1-3 fasting no-water 8
4-16 fasting water-free 10
Recover (R) 1-2 30g/d(per) water-free 10
3-4 60g/ d(per) water-free Increase by 0.5 h each day until it reaches 16 h
5 90g/ d(per) water-free
6-32 120g/ d(per) water-free
Second egg-laying period 32- culled 120g/ d(per) water-free 16
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Test materials

At each sampling session, one chicken was randomly selected from each replicate. All groups, including the pre-fasting (F0) group, were subjected to a standardized 12-hour feed withdrawal prior to sampling to minimize short-term feeding effects on biochemical and molecular parameters. Live body weight was measured and recorded before blood collection. Approximately 5 mL of blood was drawn from the brachial vein into coagulation tubes and centrifuged at 3500 rpm for 10 min. The resulting serum was transferred into 1.5 mL Eppendorf tubes and stored at −80°C until further analysis. After blood collection, chickens were euthanized by cervical exsanguination. The abdominal cavity was opened to expose the internal organs, and the liver was photographed, excised, and weighed. A liver sample measuring approximately 2 × 5 × 5 mm was rinsed with saline and fixed in 4 % paraformaldehyde for histological analysis. An additional liver fragment was rinsed with saline, placed into a 1.5 mL RNase-free Eppendorf tube, snap-frozen in liquid nitrogen, and stored at −80°C for subsequent molecular assays.

Oil Red O staining

Fresh liver tissues (approximately 10 mm) from five laying hens per group were embedded in Tissue-Tek OCT compound (Sakura, Tokyo, Japan) and sectioned into 8-10 μm frozen slices. Lipid droplets were visualized using a Modified Oil Red O Staining Kit (Nanjing, China) according to the manufacturer’s protocol. Briefly, liver samples were dehydrated in sucrose, embedded in OCT, and sectioned for Oil Red O staining. The distribution of lipid droplets in liver sections at different time points was examined using Case Viewer software under identical imaging conditions. The integrated optical density (IOD) and tissue area were quantified using ImageJ software, and the IOD/AREA ratio was used as an indicator of hepatic lipid droplet content.

Measurement of serum hormones

The determinations of E2, T3, and T4 were all performed strictly in accordance with the corresponding kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Measurement of liver oxidative indicators

Liver tissue samples were cut into small pieces, and then, add 9 times the volume of physiological saline according to the weight (g): volume (mL) ratio of 1:9, homogenize using a tissue homogenizer at 55 Hz for 90 s, remove and centrifuge at 2500 rpm for 10 min at 4°C, and collect the supernatant for analysis. The activities of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), in the serum and hepatic supernatants were assayed using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as per the instructions of the manufacturer.

Determination of serum and liver biochemical indicators

The activities of serum and liver triacylglycerol (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as the content of serum alkaline phosphatase (AKP) were analyzed using an automatic biochemical analyzer (HATICHI 7180, Tokyo, Japan).

Western blot analysis

Western blotting was conducted according to the methods described in our previous study (Wu et al., 2023). Protein samples (n = 3) were quantified and separated by SDS-PAGE, then transferred onto PVDF membranes. The membranes were blocked with 5 % nonfat milk prepared in TBST for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4°C, and immunoreactive bands were visualized using an Amersham Imager 600 system (GE Healthcare, Switzerland). The relative intensities of protein bands were analyzed using ImageJ software. The primary antibodies were rabbit anti-Bcl2 (polyclonal, 26593-1-AP, Proteintech, Wuhan, China, 1:1500) and anti- Beclin1 (polyclonal, 11306-1-AP, Proteintech, Wuhan, China, 1:1000), LC3B (polyclonal, 14600-1-AP, Proteintech, Wuhan, China, 1:5000), PI3K (polyclonal, 20584-1-AP, Proteintech, Wuhan, China, 1:300), mTOR (polyclonal, 28273-1-AP, Proteintech, Wuhan, China, 1:4000), GAPDH(polyclonal, 10494-1-AP, Proteintech, Wuhan, China, 1:20000). The second antibody was HRP-conjugated goat anti-rabbit (SA00001-2, Proteintech, Wuhan, China, 1: 2000). Following washing, the membranes were incubated with the appropriate secondary antibodies, after which the optical density (OD) of the target bands was analyzed using the AlphaEase FC software system (Alpha Innotech, San Leandro, CA, USA).

Quantitative real-time PCR (qRT-PCR)

Gene expression levels were determined by quantitative real-time PCR (qRT-PCR) following the procedures described in our previous study (Wu et al., 2024). Total RNA was isolated from the Chicken liver using Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. RNA concentration and purity were measured with a spectrophotometer. Complementary DNA (cDNA) was synthesized from total RNA using the First-Strand cDNA Synthesis SuperMix kit (Vazyme, Nanjing, China). qRT-PCR was then conducted on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, USA). Relative mRNA expression levels of target genes were normalized to β-actin and calculated using the 2^−ΔΔCt method. The primer sequences are shown in the Supplementary Table S1.

Statistical analysis

All statistical analyses were conducted using SPSS version 23.0. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was employed to assess differences among treatments. Data are expressed as means ± standard errors (SE). Statistical significance was defined as p < 0.05, and extreme significance as p ≤ 0.01; values of p > 0.05 were considered non-significant. GraphPad Prism 8.0 was utilized for figure preparation and data presentation.

Results

Changes in egg production rate during fasting-induced physiological remodeling

As shown in Fig. 1, egg production in hens remained low during the late laying stage. Following the initiation of fasting, egg production declined gradually during the first three days, then dropped sharply and completely ceased by day 7. Egg laying remained suspended until day 15 after refeeding. From day 16 onwards, egg production gradually resumed and increased steadily during the subsequent week. After day 24 of refeeding, the rate of increase in egg production accelerated markedly. Between days 26 and 35, egg production fluctuated slightly but remained at a relatively high overall level. By day 40, the production rate stabilized at a level comparable to the first peak, indicating the establishment of a second laying peak. By day 46 after refeeding, egg production exceeded 80 %, surpassing the first peak and indicating that the flock laying performance had been effectively restored and enhanced.

Fig. 1.

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Changes in egg production rate during fasting-induced physiological remodeling.

Changes in liver morphology and liver index during fasting-induced physiological remodeling

Fasting-induced alterations in hepatic morphology are presented in Fig. 2A. During the late laying stage, the livers were markedly enlarged, exhibiting substantial lipid accumulation, occasional hemorrhagic foci, and a friable, greasy texture characteristic features of fatty liver. During fasting (F3 and F16), hepatic size progressively decreased, and the color changed from yellow to red, indicating the mobilization and utilization of stored lipids. Following refeeding (R15 and R32), hepatic size increased again, accompanied by renewed lipid deposition. By R47, hepatic size had decreased again, with reduced lipid content and restoration of a normal reddish-brown color, without hemorrhage or other pathological abnormalities. Fig. 2B shows that the liver index (liver weight/pre-slaughter weight) was highest during the late laying stage, decreased during fasting, and showed a transient rebound followed by a gradual decline after refeeding (p < 0.05), consistent with the observed morphological alterations. These findings suggest that pronounced hepatic lipid accumulation functions as an adaptive energy reserve that prepares hens for the subsequent laying cycle.

Fig. 2.

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Changes of hepatic morphology (A) and liver index (B) during fasting induced physiological remodeling. Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD.

Changes in liver lipid content during fasting-induced physiological remodeling

To further investigate the effects of fasting on hepatic lipid metabolism in laying hens, Oil Red O staining was performed on liver tissues. As shown in Fig. 3A, extensive red staining was observed in hepatic tissues before fasting (F0). During fasting (F3 and F16), the red-stained regions remained prominent, indicating continuous lipid mobilization. After refeeding, the proportion of red-stained areas followed a distinct pattern-initially increasing (R6, R15, and R32) and subsequently decreasing (R47). Quantitative analysis of Oil Red O-stained sections from identical microscopic fields was conducted using ImageJ software, with the IOD/AREA ratio representing the relative hepatic lipid content. As shown in Fig. 3B, hepatic lipid levels were elevated before fasting, declined during fasting, and exhibited a transient rebound followed by a decrease after refeeding. The trend in hepatic triglyceride concentrations (Fig. 3C) closely paralleled these histological observations, confirming that hepatic fat reserves were mobilized during fasting, transiently reaccumulated after refeeding, and subsequently utilized during the restoration of egg production.

Fig. 3.

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Oil Red O staining (A), quantitative analysis of IOD/AREA ratio (B) and hepatic triglyceride content (C) during fasting-induced physiological remodeling. Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD.

Effects of fasting-induced physiological remodeling on serum hormones and liver biochemical indicators

The biochemical parameters of serum and liver were further analyzed to assess the effects of fasting-induced physiological remodeling in laying hens. As shown in Fig. 4A, estradiol (E2) concentrations were highest at F0. During fasting (F3 and F16) and the early phase of refeeding (R6), E2 concentrations decreased significantly (p < 0.05). Subsequently, E2 concentrations increased markedly at R15 and R32 relative to the end of fasting (p < 0.05), but remained significantly lower than those observed at F0 (p < 0.05). As shown in Figs. 4B and 4C, serum triiodothyronine (T3) and thyroxine (T4) concentrations increased significantly during fasting (F3 and F16) and the early refeeding phase (R6) (p < 0.05). With continued refeeding (R15 and R32), T3 and T4 concentrations declined compared with F16 and R6 (p < 0.05), yet remained significantly higher than those at F0 and F3 (p < 0.05). As shown in Fig. 4D, serum AKP activity progressively decreased during fasting, reaching a significant reduction at F16 (p < 0.05). After refeeding (R6, R15, and R32), serum AKP activity increased significantly (p < 0.05) and exceeded the pre-fasting level at R32 (p < 0.05). As shown in Fig. 4E, serum alanine aminotransferase (ALT) activity increased significantly during fasting (F3 and F16) (p < 0.05) and decreased markedly during refeeding (R6, R15, and R32) (p < 0.05). Conversely, serum aspartate aminotransferase (AST) activity decreased significantly at F16 and continued to decline during refeeding (R6, R15, and R32) (p < 0.05) (Fig. 4F). In hepatic tissues (Fig. 4G, H)), both ALT and AST activities declined during fasting, showing a significant reduction at F16 (p < 0.05), followed by a marked recovery after refeeding (p < 0.05). These findings indicate that fasting and subsequent refeeding significantly regulate endocrine function and hepatic enzyme activities in laying hens, reflecting a dynamic metabolic adaptation to nutritional stress and recovery.

Fig. 4.

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Effects of fasting-induced physiological remodeling on serum estradiol (E2, A), triiodothyronine (T3, B), thyroxine (T4, C), alkaline phosphatase (AKP, D), alanine aminotransferase (ALT, E), aspartate aminotransferase, (AST, F), hepatic ALT (G) and AST (H) activities. Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD.

Effects of fasting-induced physiological remodeling on liver antioxidant function

To investigate the effects of fasting and refeeding on hepatic oxidative stress and antioxidant capacity in laying hens, hepatic lipid peroxidation and antioxidant enzyme activities were measured. As shown in Fig. 5A, hepatic malondialdehyde (MDA) levels changed significantly during fasting-induced physiological remodeling. Hepatic MDA levels increased significantly after 3 days of fasting (F3) (p < 0.05) but decreased markedly after refeeding (p < 0.05). The activities of hepatic antioxidant enzymes during the remodeling process are presented in Figs. 5(B–D). The activities of SOD and CAT showed no significant changes (p > 0.05), whereas GSH-Px activity was significantly elevated during late fasting (F16) and the refeeding phase compared with the pre-fasting stage (F0) (p < 0.05). Figs. 5 (E–G) display the relative mRNA expression levels of hepatic antioxidant enzymes. The mRNA expression levels of SOD, CAT, and GSH-Px were significantly upregulated after fasting and refeeding compared with pre-fasting levels (p < 0.05). Overall, these findings suggest that fasting induces transient hepatic oxidative stress, whereas refeeding activates antioxidant defense mechanisms that help restore hepatic redox homeostasis during nutritional remodeling.

Fig. 5.

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Effects of fasting-induced physiological remodeling on hepatic antioxidant parameters. (A) Malondialdehyde (MDA) content, (B–D) superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities; (E–G) relative mRNA expression levels of SOD, CAT, and GSH-Px. Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD.

Enrichment analysis of differentially expressed genes in liver transcriptome sequencing during fasting-induced physiological remodeling

Genes associated with hepatic lipid metabolism and autophagy during fasting-induced physiological remodeling were integrated into a unified gene set and reanalyzed using KEGG pathway enrichment (Fig. 6A). The analysis revealed significant enrichment of pathways related to fatty-acid metabolism, fatty-acid degradation, autophagy (animals), and ketone body synthesis and degradation (p < 0.05). Subsequently, a protein–protein interaction (PPI) network was constructed for genes associated with lipid metabolism and autophagy (Fig. 6B). In this network, each node represents a gene, and each edge denotes a predicted functional association; the number of connections reflects the degree of protein–protein interaction. The PPI analysis revealed extensive functional crosstalk between autophagy and lipid-metabolism pathways. Core autophagy-related genes (BCL2, PDGFRA), apoptosis-related genes (CASP3, CASP7), and lipid metabolism-related genes (ACAT2, APOB) were identified from the network for further verification.

Fig. 6.

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Integrated analysis of hepatic lipid metabolism and autophagy during fasting-induced physiological remodeling. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (B) Protein–protein interaction (PPI) network of genes associated with lipid metabolism and autophagy.

Expression of genes related to apoptosis and lipid metabolism during fasting-induced physiological remodeling

The interaction between autophagy and apoptosis is critically involved in the regulation of cellular processes. KEGG enrichment analysis of differentially expressed genes in the transcriptome indicated a significant enrichment within the apoptosis pathway. Real-time quantitative PCR was performed to validate the expression of genes involved in this pathway, with results presented in Fig. 7. The pro-apoptotic genes CASP3, CASP7, and MDM2 were significantly downregulated during fasting (F3 and F16) (p < 0.05) and significantly upregulated after refeeding (R6, R15, and R32) (p < 0.05). Conversely, CDK1 and CCNB1 were significantly upregulated at F3 (p < 0.05) and downregulated after refeeding (p < 0.05). The expression of the lipid synthesis-related gene ACAT2 (Fig. 7F) was significantly decreased during fasting (F3 and F16) (p < 0.05) and significantly increased after refeeding (p < 0.05). Catabolism-related genes ACSBG2 and APOB (Figs. 7G and 7H) were significantly downregulated during fasting (p < 0.05) and subsequently upregulated following refeeding. In contrast, ACSL1 (Fig. 7I) was significantly upregulated during fasting and significantly downregulated after refeeding (p < 0.05). These findings suggest that fasting suppresses apoptosis and lipid synthesis while activating cell survival mechanisms, whereas refeeding reverses these effects, indicating a dynamic regulatory balance between apoptosis and lipid metabolism during hepatic remodeling in laying hens.

Fig. 7.

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Effects of fasting induced physiological remodeling on autophagy and apoptosis. The relative expression level of (A) CASP-3, (B) CASP-7, (C) MDM2, (D) CDK1, (E) CCNB1, (F) ACAT2, (G) ACSBG2, (H) APOB and (I) ACSL1. Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD.

Expression of key autophagy genes in fasting-induced physiological remodeling and analysis of their interaction with apoptosis and lipid metabolism

To validate differentially expressed autophagy-related genes associated with lipid metabolism, qRT-PCR analysis was performed (Fig. 8). The results showed that key autophagy-related genes—including BCL2, ERN1, PDGFRA, IGF1R, and ATG5-exhibited an increasing trend in expression during fasting, with all significantly upregulated at F16 (p < 0.05). PIK3CB expression was significantly elevated at both F3 and F16 (p < 0.05). Notably, ATG5 remained significantly higher at R6 compared with F0, and ERN1 and PDGFRA levels remained elevated at R32 (p < 0.05). Further analysis of classical autophagy marker genes revealed dynamic changes during fasting and refeeding. LC3I expression significantly decreased at F16 (p < 0.05) but rebounded after refeeding, whereas LC3II expression increased at F3 (p < 0.05). The LC3II/LC3I ratio was significantly elevated at F3, F16, and R6 (p < 0.05) and declined at R32. Beclin1 expression increased markedly at F3 and F16 (p < 0.05) and decreased following refeeding, while the autophagy inhibitor mTOR was significantly downregulated during fasting and early refeeding (p < 0.05). Western blot analysis further confirmed these transcriptional patterns: the protein expression of autophagy activators BCL2, Beclin1, LC3, and PI3K was significantly elevated during fasting and early refeeding (p < 0.05), whereas mTOR protein expression was significantly reduced (p < 0.05), indicating effective activation of autophagy in hepatic tissues. Correlation analysis demonstrated that autophagy-related genes were significantly positively correlated with their corresponding protein expression (p < 0.05) and negatively correlated with hepatic triglyceride (TG) content (p < 0.05). Pro-apoptotic genes were significantly negatively correlated with autophagy-activating genes (p < 0.05). Genes involved in lipid synthesis showed significant positive correlations with TG content (P < 0.05), while lipid catabolism–related genes exhibited variable correlations. Overall, these findings indicate that fasting activates hepatic autophagy through the PI3K–mTOR signaling pathway, which promotes lipid degradation, inhibits apoptosis, and contributes to the restoration of hepatic lipid homeostasis during refeeding.

Fig. 8.

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Changes in the relative mRNA expression levels of autophagy-related differential genes in the liver during fasting-induced molting (A and B), expression of autophagy key proteins during fasting induced physiological remodeling (C), and correlation heatmap of related indicators (D). Statistical analysis was performed by one-way ANOVA with Duncan’s multiple range test and the results are presented as means ± SD. Correlation analysis was performed using the Pearson correlation coefficient.

Discussion

At present, research on the application of induced physiological remodeling to improve eggshell quality in aged laying hens and stimulate a second egg-laying peak is well-established (Wang et al., 2023). The liver is a key organ for yolk synthesis and lipid mobilization, and its functional state directly affects egg-laying performance (Huang et al., 2022). Starting from the liver, this study systematically explored the effects of induced physiological remodeling on the liver by combining morphological observation, oxidation and antioxidant indicators, liver lipid content, and lipid metabolism-related gene and pathway analysis. The results showed that the liver volume of laying hens at the late laying stage of 500 days old increased, lipid accumulation was significant, and pathological features such as hemorrhage were present, indicating the presence of fatty liver. At the same time, the ovarian function of old laying hens declined, egg-laying performance decreased, and lipids used for yolk synthesis decreased. The experiment found that serum estrogen levels were high during the F0 period, and estrogen can promote liver fat synthesis and deposition; when the lipid synthesis rate remains unchanged and the transport to the periphery is reduced, a large amount of fat is retained in the liver, which becomes one of the important reasons for the high incidence of fatty liver in the late laying stage.

In the early stage of fasting (F0 to F3), the egg production rate of laying hens slowly decreased and then quickly dropped to zero. However, the liver's Oil Red O staining and triglyceride content at F3 showed that lipids were rapidly consumed, indicating that after the start of fasting, the liver not only provided energy to the body but also continued to provide lipids to the ovaries that had not yet completely stopped, resulting in a rapid decrease in liver fat (Longo and Mattson, 2014). There was no significant difference in liver morphology, liver index, triglyceride content, and Oil Red O staining results between F3 and F16, suggesting that in the early stages of fasting, energy supply not only relied on the liver but also came from other reserves such as abdominal fat and intramuscular fat, and finally mobilized protein (Bannister and Cleland, 1978). However, since the lipids required for egg production can only be supplied by the liver, the egg production rate slowly decreased as the liver fat was gradually consumed; once the liver fat was exhausted and could not support yolk synthesis, the flock quickly stopped laying eggs. After 15 days of resuming feeding (R15), the flock resumed laying eggs. At this time, liver lipids had reaccumulated, which is consistent with the report of J. Garlich et al. (Garlich et al., 1984), who pointed out that the liver lipid status of hens that did not lay eggs after fasting was similar to that of pullets that were about to lay eggs, and did not show fatty liver syndrome. By R32, liver lipid levels remained high, while egg production had rebounded significantly to approximately 60 %, indicating that liver lipid accumulation during this period served as a reserve for the second peak of egg production. When egg production returned to levels similar to those of the first peak (R47), Oil Red O staining and triglyceride analysis revealed renewed liver lipid mobilization. Notably, by R15 and R32, the livers had lost their hemorrhages, improved in texture, and were no longer greasy to the touch, indicating that the underlying fatty liver pathology had been effectively alleviated.

Thyroid hormones play a central role in regulating systemic substance and energy metabolism (Sinha and Yen, 2024). The liver, as a major target organ, is finely modulated by thyroid hormones under physiological conditions. Elevated thyroid hormone levels accelerate hepatocellular metabolism, thereby enhancing energy production and utilization efficiency. In the present study, concentrations of triiodothyronine (T3) and thyroxine (T4) increased significantly during fasting, which is consistent with previous reports on hormonal fluctuations during physiological remodeling (Zhang et al., 2023). Elevated plasma thyroid hormone levels are generally believed to compensate for heat loss caused by feather shedding and to stimulate hepatic lipid metabolism (Hoshino et al., 1988). Serum enzyme activity serves as an important indicator of tissue metabolic status and functional integrity. Alkaline phosphatase (AKP), primarily derived from the liver, is not only a critical parameter for evaluating hepatic function but is also closely related to calcium absorption, overall metabolic activity, and production performance. Previous studies have shown that livestock and poultry with higher serum AKP activity typically exhibit superior growth and productivity (Chen et al., 2021a). In this study, the egg production rate and serum AKP activity were both low prior to fasting (F0). During the fasting period, AKP activity decreased further; however, following refeeding, it rose markedly and surpassed pre-fasting levels. This increase was accompanied by a concurrent improvement in egg production rate, suggesting that liver function and productive performance were enhanced after physiological remodeling. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are enzymes predominantly localized in hepatocytes. Even mild hepatic injury can cause a marked increase in their serum activities (Dufour et al., 2000); hence, they are widely used as sensitive indicators of hepatocellular integrity and damage. In this study, serum ALT and AST activities were elevated before fasting (F0), indicating that the liver may have already sustained mild damage during the late laying stage. During fasting (F3, F16), hepatic enzyme activities declined, whereas serum activities continued to rise, suggesting that short-term fasting may have transiently aggravated hepatocyte membrane permeability. Nevertheless, after refeeding, serum ALT and AST activities significantly decreased and fell below pre-fasting levels, indicating that the liver underwent structural and functional recovery following fasting-induced stress. Taken together, the dynamic changes in key serum enzymes (AKP, ALT, and AST) indicate that although the fasting–refeeding remodeling process imposes transient stress on hepatic tissue, it ultimately enhances hepatic metabolic function and promotes tissue repair. This adaptive recovery provides a physiological foundation for the subsequent restoration and improvement of laying performance in hens. Beyond hepatic and reproductive benefits, fasting-induced physiological remodeling simultaneously improves meat quality in aged laying hens through phospholipid remodeling in pectoralis muscle, as evidenced by upregulated ACSL4/LPCAT expression and enhanced intramuscular fat deposition at R20 (Zhang et al., 2025).

This study focused on the dynamic changes in hepatic function in laying hens during the late laying period and its adaptive response mechanisms under fasting-induced physiological remodeling. During the late laying phase, the liver’s capacity to synthesize yolk precursors declines, and redox homeostasis becomes increasingly fragile, predisposing hepatocytes to steatosis and oxidative damage. Fasting, as a physiological stressor, further perturbs hepatic redox balance (Socha and Hrabia, 2019). Malondialdehyde (MDA), a major lipid peroxidation product, serves as a marker of oxidative injury, whereas the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) reflect the organism’s antioxidant defense capacity (Xing et al., 2021). In this study, hepatic MDA levels remained elevated before and during fasting, indicating pronounced oxidative stress in the late laying period and during nutrient deprivation. Although SOD and CAT activities did not change significantly during fasting, both exhibited an upward trend upon refeeding. Meanwhile, GSH-Px activity and the mRNA expression of SOD, CAT, and GSH-Px increased markedly in the late fasting and refeeding stages, surpassing pre-fasting levels. These findings suggest that the liver counteracted fasting-induced oxidative damage by upregulating its antioxidant defense system, highlighting the role of induced physiological remodeling in enhancing hepatic antioxidant capacity.

Based on our previous liver transcriptome analysis during fasting, differentially expressed genes were not only enriched in lipid metabolism pathways but also in those associated with autophagy and apoptosis (Zhang et al., 2022a). To further elucidate the interaction between lipid metabolism and autophagy, enrichment and protein–protein interaction (PPI) network analyses were performed using relevant genes. Key node genes identified from this network were validated by qRT-PCR, and the results were consistent with transcriptomic data, supporting the activation of the autophagy pathway during fasting. Under conditions of energy deficiency, hepatic lipid synthesis is downregulated, while stored triglycerides are hydrolyzed by lipases into free fatty acids to supply energy (Zhang et al., 2022b). Concurrently, lipid droplets can be degraded via selective autophagy (lipophagy), providing an alternative pathway for energy mobilization (van Zutphen et al., 2014). Autophagy plays a critical role in regulating gluconeogenesis, fatty acid β-oxidation, and ketone body production. During fasting, some of the fatty acids used for gluconeogenesis are derived from the autophagic degradation of lipid droplets (Meijer and Codogno, 2009). Histological examination and hepatic triglyceride quantification in this study confirmed that fasting induced the degradation of hepatic lipid droplets. Autophagy initiation is typically associated with suppression of the mTOR signaling pathway and activation of AMPK. Our results showed that mTOR expression was downregulated during fasting, whereas the expression of upstream autophagy-related genes (such as ERN1 and PDGFRA) increased, and conversion of the autophagy marker LC3 was enhanced. These changes collectively indicated that hepatic autophagy was robustly activated. Quantification of the LC3-II/internal control protein ratio further confirmed that autophagic activity increased significantly during fasting. Although the elevated LC3-II/LC3-I ratio and increased expression of Beclin1 and PI3K strongly suggest activation of the autophagy pathway, it should be noted that these markers primarily reflect autophagy-related changes and do not independently distinguish between enhanced autophagy initiation and reduced autophagosome degradation. Autophagic flux was not directly assessed in the present study, and future investigations incorporating lysosomal inhibition assays would further clarify the dynamic regulation of hepatic autophagy during fasting-induced remodeling.

Autophagy and apoptosis are closely interconnected cellular processes that often exhibit reciprocal regulatory patterns (Ploumi et al., 2022). During fasting, autophagy sustains cellular energy homeostasis by recycling intracellular substrates, thereby delaying apoptotic processes (Amelio et al., 2011). In the present study, activation of autophagy was accompanied by downregulation of pro-apoptotic genes (CASP3, CASP7, MDM2) and upregulation of genes associated with cell proliferation, suggesting that fasting-induced autophagy inhibited apoptosis and promoted hepatocyte survival under energy stress. Furthermore, the expression patterns of lipid catabolism-related genes showed divergent trends, with some being upregulated and others downregulated, indicating that the liver may employ multiple regulatory pathways to control lipid metabolism during fasting-among which lipid autophagy represents a key mechanism. From a management perspective, fasting-induced physiological remodeling may reduce metabolic disorders in laying hens during the later stages of production, alleviate fatty liver symptoms, reduce the rate of premature culling, and increase the egg-laying continuity throughout the entire production cycle. Such an operation procedure can enhance the consistency of the chicken flock, improve the health condition of the flock, effectively cope with fluctuations in market raw material prices and the prices of meat and egg products, and ultimately increase economic benefits in the commercial egg chicken production system.

Conclusion

In conclusion, fasting-induced physiological remodeling effectively counteracts the decline in egg production and the high prevalence of fatty liver disease in late-phase laying hens. By activating hepatic autophagy, this process alleviates metabolic liver dysfunction, reduces lipid deposition, and enhances hepatic function, thereby establishing the physiological foundation for the onset of a second laying cycle. These findings highlight autophagy as a key regulatory mechanism in mitigating fatty liver disease and offer important theoretical insights for future research on autophagy-mediated metabolic adaptation in laying hens.

CRediT authorship contribution statement

Yujie Gong: Writing – review & editing, Writing – original draft, Supervision, Formal analysis. Qingduo Zeng: Writing – review & editing, Writing – original draft, Visualization. Xiaoqing Geng: Validation, Investigation. Jun Zhang: Software, Data curation. Xiangtao Kang: Resources, Project administration, Funding acquisition, Conceptualization. Yadong Tian: Project administration, Funding acquisition, Formal analysis. Ruirui Jiang: Supervision, Resources, Project administration, Methodology, Conceptualization.

Disclosures

The authors declare no conflict of interest.

Acknowledgements

This research was financially supported by the Scientific Studio of Zhongyuan Scholars (NO. 30601985); and the Special Fund for Henan Agricuture Research System (HARS-22-18-S).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.106775.

Supplementary Table S1 Information of primers.

Appendix. Supplementary materials

Supplementary Table S1 Information of primers.

mmc1.docx (19.5KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table S1 Information of primers.

mmc1.docx (19.5KB, docx)

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