Multi-omics analysis of the gut-liver axis reveals the health hazards of embryonic egg exposure to zearalenone in chicks

Highlights

• •Embryonic ZEN exposure reduces hatching performance and chick growth.
• •ZEN induces intestinal inflammation and gut microbiota dysbiosis in chicks via embryonic exposure.
• •Hepatotoxicity and metabolic disorders in chicks emerge from embryonic ZEN exposure.
• •Multi-omics integration targets key pathways mediating gut-liver axis toxicity after embryonic ZEN exposure.

Abstract

Zearalenone (ZEN), a widespread Fusarium-derived mycotoxin, poses health risks to animals and humans through contaminated food chains. However, its transgenerational effects following embryonic egg exposure on avian development and chick health remain poorly characterized.This study employed embryonic egg injection and multi-omics approaches to evaluate the impacts of ZEN (0, 5, 50, or 500 μg/egg) administered to Hy-line Brown eggs on day 5 of incubation. ZEN exposure significantly decreased hatchability and initial chick weight. During the 20-day post-hatch period, it induced dose-dependent reductions in net weight, average daily gain, and average daily feed intake, while increasing the feed conversion ratio.Histopathology showed intestinal inflammation and structural damage, coupled with gut microbiota dysbiosis, notably an elevated abundance of pathogenic bacteria such as Escherichia coli. ZEN also caused liver injury, manifested by elevated serum biochemical indices, oxidative stress, and inflammatory responses. Hepatic metabolomics revealed pronounced alterations in metabolites and pathways associated with lipid metabolism, autophagy, and antioxidant defense.Correlation analyses indicated interconnections between gut microbiota dysbiosis and hepatic metabolic disturbances, suggesting mediation of ZEN developmental toxicity via the gut–liver axis. These results elucidate novel mechanisms of ZEN embryotoxicity and underscore the need to control ZEN contamination in poultry production.

Graphical abstract

Introduction

Zearalenone (ZEN), a mycotoxin produced by Fusarium species, is one of the most prevalent and potent contaminants in cereals and cereal-derived products. It not only incurs substantial economic losses in agriculture but also poses significant threats to animal and human health via the food chain (Rai et al., 2020). Studies have demonstrated that ZEN ranks among the top three mycotoxins in complete animal feeds, detected in 45% of all samples, and is also the most frequently occurring mycotoxin in soybean and soybean meal samples (Liu and Applegate, 2020). In China, surveillance data from 2018 to 2020 revealed that 81.5% of feed ingredients and 95.7% of complete feeds were co-contaminated with at least two mycotoxins among aflatoxin B1 (AFB1), deoxynivalenol (DON), and ZEN (Zhao et al., 2021) Notably, ZEN exhibits high physicochemical stability: it degrades only under extreme conditions (temperatures >175°C or alkaline environments) and is resistant to conventional chemical treatments such as acids and oxidizing agents. Additionally, physical adsorbents show poor specificity in binding to ZEN (Liu and Applegate, 2020; Wu et al., 2021), and processes including grinding, extrusion, storage, and heating fail to effectively remove it (Gromadzka et al., 2008).

Zearalenone (ZEN) and its metabolites (α-zearalenol, α-ZEL; β-zearalenol, β-ZEL) share a structural similarity to estradiol, endowing ZEN with reproductive toxicity. This represents one of the primary toxic effects of ZEN, which can induce reproductive system disorders such as miscarriage, ovarian dysfunction, and stillbirth (Lv et al., 2025; Zhang et al., 2025). Beyond reproductive organs, the intestine and liver are key target organs of ZEN. Previous studies have reported that ZEN exerts toxic effects on the intestine by inducing gut microbiota dysbiosis and impairing immune function (Jia et al., 2020; Zhang et al., 2025b). Meanwhile, it can also induce liver damage, including alterations in liver weight, immune impairment, and the induction of hepatic oxidative stress responses (Zhang et al., 2022; Yang et al., 2024). Furthermore, Yang et al. demonstrated that the presence of ZEN in feed induces blood toxicity to varying degrees (Yang et al., 2016). Additionally, ZEN exhibits immunotoxicity, genotoxicity, and carcinogenicity (Han et al., 2022).

The estrogenic effects of ZEN have increasingly drawn attention to the transgenerational toxic impacts of maternal ZEN exposure. However, studies on the effects of ZEN exposure during the egg stage on embryonic hatching rate and chick growth and development remain scarce. Previous studies in our laboratory have confirmed that ZEN exerts significant adverse effects on laying performance, egg quality, and ovarian function in laying hens (Yuan et al., 2022).To further explore the mechanism underlying the effects of ZEN on embryonic hatching rate and chick growth and health, the doses of ZEN used in this experiment (5, 50 and 500 μg/egg) were selected to cover a wide exposure range from low level exposure to frank toxic challenge and employed embryonic egg injection technology combined with multi-omics analytical approaches such as metagenomics and untargeted metabolomics to investigate the impacts of ZEN on embryonic hatching rate, post-hatching chick performance, and physiological health. Meanwhile, the potential mechanisms underlying the toxic effects of ZEN on chicks were preliminarily revealed by analyzing the intestinal microbiota, metabolites, and their correlations. This study systematically evaluated the effects of ZEN on embryonic hatching performance and its transgenerational toxic effects in chicks from multiple dimensions, thereby providing a novel theoretical perspective for elucidating the toxic mechanisms of ZEN during early avian developmental stages.

Materials and methods

Preparation of ZEN Injection

The ZEN standard (Pribolab) was prepared as a 100 mg/mL masterbatch with a methanol solution, and then the masterbatch was diluted stepwise with a 1% methanol solution to reach the desired concentration. The control group was a 0.1% methanol solution. The injection volume for each embryonic egg in each treatment group was 1 mL. The concentration of ZEN injected in each treatment group is presented in Table 1.

Table 1.

The ZEN treatment and grouping of embryonic eggs.

Abbreviations	Groups	ZEN injection volume
C	control group	0μg/egg
L	Low-dose ZEN	5 μg/egg
M	Medium-dose ZEN	50 μg/egg
H	High-dose ZEN	500 μg/egg

In ovo feeding and experimental design

The embryonic eggs were obtained from the Laboratory Animal Centre, Huazhong Agricultural University. The number for the use of laboratory animals of the centre was SYKXK (Hubei) 2020-0084. All experiments performed on the eggs and chicks were approved by the Animal Care and Use Committee of the Laboratory Animal Centre, Huazhong Agricultural University (HZAUCH-2025-0058).

The prepared ZEN injections of varying concentrations were filtered and sterilized with a 0.22 μm disposable sterile filter, resulting in the production of a sterile ZEN solution. A small electric drill was employed to create an opening in the air chamber of the egg, approximately 1 mm in diameter. Subsequently, 0.5 mL of the ZEN solution was administered into the amniotic cavity via a 1 mL syringe, reaching a depth of approximately 2.5 cm. Finally, the injection site was sealed with melted paraffin wax.

A total of 480 embryonic eggs of the Hy-line Brown variety were randomly divided into four treatment groups, with each group comprising six replicates of 20 eggs. On the fifth day of embryonic egg hatching, the corresponding concentration of ZEN injection was administered to each group of embryonic eggs. Following hatching, 60 chicks with uniform body condition and no obvious abnormalities were selected from each group. These were placed into 6 replicates of 10 chicks each, and the experiment was conducted for 20 days. The experimental diets for the chicks are detailed in Table 2. All chicks were reared in brooding cages with 10 chicks per cage in one replicate for a period of 20 days. The chicks were maintained for a period of 20 days. The room temperature was maintained at a range of 32 to 34 degrees Celsius for the initial three days, and then gradually decreased by 2 to 3 degrees Celsius each week. The chicks were provided with adequate lighting, free access to food and water, and were kept under controlled conditions. The chicks were weighed on a daily basis throughout the rearing period, and the resulting data were used to calculate their growth performance. Following a 12-hour fast at the conclusion of the experiment, one chick was randomly selected from each replicate of each group for slaughter. The live and liver weights of the chicks at the time of slaughter were recorded to analyze the liver index. The left tibia was collected for determination of bone strength, while the duodenum, proximal jejunum, ileum, and liver tissues were taken and fixed in 4% paraformaldehyde solution (Servicebio, China) for histological analysis. The remaining liver tissues and cecum contents were rapidly frozen in liquid nitrogen and subsequently stored at −80°C for subsequent analyses.

Table 2.

Basal diet composition and nutrient levels.

Ingredients	Percentage, %	Nutrition component	%
Maize	50.92	Crude protein	20
Flour	10	ME(MJ/kg)	12.34
Puffed soybean	6	Crude ash	5.47
Soybean meal	27.3	Ether extract	4.79
Soybean oil	1.34	Crude fiber	3.40
Limestone	1.16	Calcium	0.9
Calcium hydrogenphosphate	1.55	Phosphorus	0.61
Salt	0.29	Lys	1.30
Lys (70%)	0.59	Met	0.57
Met (98%)	0.28	Arg	1.27
Thr (98%)	0.14
Phytase	0.02
VL620a	0.22
M-Pb	0.2
Total	100

^aProvided per kilogram of complete diet: vitamin K, 3.5 mg; vitamin A, 10,000 IU; vitamin D3, 3,300 IU; vitamin E, 25 mg; vitamin B1, 2.2 mg; vitamin B2, 6.6 mg; vitamin B6, 4.5 mg; pantothenic acid, 10 mg; vitamin B12, 23 μg; folic acid, 1 mg; Mn, 90 mg;Zn, 85 mg;Fe, 30 mg;Cu, 15 mg;I, 1.5 mg;Se, 0.25 mg.

^bThe nutrient levels in the basal diets were quantified, with the exception of the metabolizable energy content, which was calculated.

Production performance

The weight of the chicks was recorded on a daily basis, and the average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR) were calculated for each treatment group replicate.

Serum hormone level and biochemical indicators analysis

Serum reproductive hormones, including estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4), were analyzed using the double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. The assays were conducted using corresponding kits from Jiangsu Enzyme Immunity Industry Co., Ltd. (Jiangsu, China) and the SpectraMax M5 enzyme marker from Molecular Devices (Wokingham, UK).

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total cholesterol (CHOL), and triglyceride (TG) were analyzed using a fully automated biochemical analyzer (Hitachi 7600-020, Beijing, China).

Histological analysis

The duodenum, proximal jejunum, ileum and liver tissues were fixed in 4% paraformaldehyde solution (Servicebio, China) for 24 h, washed with running water, and dehydrated with different concentrations of ethanol (70%, 80%, 90%, 95% and 100%), followed by treating with xylene, and embedding in paraffin wax. The buried samples were cut into 4 µm-thick sections. Sections were stained with hematoxylin and eosin (H&E) and observed under a microscope (LEICA DM3000, Wetzlar, Germany).

The length of the intestinal villi and the depth of the crypts were measured using Image J software, five sections were measured in each group, and five set of data were measured in each section, and the mean values were calculated.

Macrogenomic assay of cecum microorganisms

Total DNA from cecum contents was extracted using the QI-Aamp DNA fecal mini-kit (Qiagen, Hilden, Germany), and the extracted genomic DNA was examined by 1% agarose gel electrophoresis. DNA was fragmented into approximately 400 bp fragments using Covaris M220. After splice ligation of the fragments using the NEXTFLEX™ Rapid DNA-Seq Kit, the splice self-linking fragments were removed using magnetic bead screening, the library template was enriched using PCR amplification, and the PCR products were recovered using magnetic beads and the final library was obtained. The primer bases are complementary to one end of the library molecule, and after one round of amplification, the information of the template is fixed on the chip, and then another primer is complementary to the other end of the molecule fixed on the chip randomly and then fixed to form a “bridge”, and then PCR amplification is carried out to obtain the DNA clusters, and the DNA amplicon is linearized to make a single strand. The DNA amplicon is linearized to a single strand, and bases are synthesized by adding dNTP with four fluorescent markers and modified DNA polymerase. In this process, only one base is synthesized per cycle, and the reaction plate is scanned with a laser, and the “termination group” and “fluorescent group” are removed by reading the type of nucleotide that has been polymerized by the first round of the reaction for each template sequence. After reading the type of nucleotide polymerized in the first round of each template sequence, the “termination group” and “fluorescent group” are cut to restore the 3′ end stickiness, followed by the polymerization of the second nucleotide, and the fluorescent signals collected at each time are counted to obtain the sequence of the template DNA fragments.

Bioinformatic analysis

The cecum microbiota was analyzed for alpha diversity with indices sobs, chao, and ace reflecting community richness as well as indices shannon and simpson reflecting community diversity, and a comparative analysis of differences between the two groups was performed by the Wilcoxon rank-sum test to assess whether the diversity indices differed significantly between the different subgroups. On the basis of these analyses, species were analyzed at the species level and functionally by the size of the distance between samples for PCA analysis and PCoA analysis. Differences in abundance as well as function of each species between groups were analyzed comparatively using Student's t-test, and the results were visualized and displayed to mine the effective information in the data. LEfSe difference discriminant analysis was first used to test the differences in species/function abundance between groups using the nonparametric Kruskal-Wallis (KW) sum-rank test, to obtain significantly different species/functions, and then finally to estimate the magnitude of the effect of these different species/functions on the difference between groups using LDA linear discriminant analysis.

Liver index analysis

Liver index of chicks in all groups were calculated with the following formula: Liver index = liver weight *100%/chick weight

Assessment of inflammatory factors in livers

Interleukin-1β(IL-1β), interferon-γ (IFN-γ), interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) were measured with corresponding kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China)and assayed using a spectrophotometer (Thermofisher, Shanghai, China).

Assessment of antioxidant indices in livers

Malondialdehyde (MDA), catalase (CAT), total superoxide dismutase (T-SOD), peroxidase (POD), glutathione peroxidase (GSH-PX), and glutathione (GSH) were tested with corresponding kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Liver untargeted metabolomics assay

Briefly, Liver tissues were ground in a homogenizer and then 400 μL of methanol:water (v:v = 4:1) containing 0.02 mg/mL l-2-chlorophenylalanine was added, and the samples were extracted using cryo-sonication after grinding in a frozen tissue grinder, and then the samples were placed in a 20°C resting place for 30 min, centrifuged using a cryo-high speed centrifuge (4°C, 13,000 g) for 15 min, and the supernatants were taken and used in the LC-MS sample preparation.

The samples were then subjected to LC-MS analysis using an CBM-10A VP Plus instrument (Shimadzu, Kyoto, Japan) equipped with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) and GC-MS analysis using a UHPLC liquid chromatography system (Thermofisher, Shanghai, China). The raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment analysis, and finally a data matrix of retention times, mass-to-charge ratios, and peak intensities was obtained, and at the same time, the information of MS and MSMS mass spectra was matched with the metabolic public The MS and MSMS mass spectrometry information was also matched with the metabolic public databases HMDB and Metlin as well as self-constructed libraries to obtain the metabolite information. The results of the assays were analyzed using a cloud platform (cloud.majorbio.com), and the comparative analysis of chick liver metabolites was performed using the ropls package in R (Version 1.6.2) for PCA and PLS-DA analysis. Differential metabolite analyses were performed based on the Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) model, with variable weight values (VIP values), Student's t-tests, and p-values to identify the significantly different metabolites; metabolites with VIP values > 1, P < 0.05 were considered significantly different metabolites. Differential metabolites were analyzed by KEGG PATHWAY enrichment of metabolites using Fisher's exact test for enrichment analysis. Spearman’s correlation analysis was further analysed to uncover the relationship between cecum microbiota and liver metabolites.

Statistical analysis

All experimental data from this chapter of the trial were initially processed in Excel (Microsoft Office 2010, Seattle, WA, USA) and then analyzed for differences between groups by one-way ANOVA using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA) software. On the basis of ANOVA significance, means between groups were compared using Duncan's multiple ANOVA. Results are expressed as Mean ± SD, and P < 0.05 indicates significant differences.

Results

Effects of embryonic egg exposure to zearalenone on hatching performance and production performance

To investigate the effects of ZEN exposure on embryonic egg production performance, we examined hatchability of embryonic eggs and the initial body weight of hatched chicks. The results revealed that ZEN injection reduced the hatchability of embryonic eggs, with significant decreases observed in both the M and L groups (Fig. 1A, 1B). Additionally, ZEN exposure decreased the initial hatch weight of chicks; however, a significant reduction was only detected in the M group (Fig. 1C). Furthermore, we collected growth and development data from chicks hatched from eggs exposed to ZEN at 20 days of age. As shown in Fig. 1D and 1F, both the net weight and ADFI significantly decreased with increasing ZEN injection concentration. ADG was affected by medium and high ZEN levels, with significantly lower ADG in the M and H groups compared to the control group (Fig. 1E). Concurrently, high-level ZEN injection led to a significant increase in FCR (Fig. 1G). These findings indicate that embryo exposure to ZEN negatively impacts hatching performance and persists to affect post-hatch chicks. Notably, these adverse effects were more pronounced in high-dose ZEN treatment groups, confirming a dose-dependent impact of ZEN on both hatching performance and chick development.

Fig. 1.

Effects of embryonic egg exposure to zearalenone on hatching performance and production performance. (A) Zearalenone-treated embryonic egg strategy. (B) Hatching ratio. (C) Hatching weight. (D) Net weight. (E) Average daily gain. (F) Average daily feed intake. (G) Feed conversion ratio. Different letters indicate P < 0.05.

Effects of zearalenone on reproductive hormones in chicks

To assess the influence of ZEN exposure on the development of the chick reproductive system, we measured the serum levels of key reproductive hormones in chicks hatched from ZEN-exposed embryonic eggs. As presented in Fig. 2A, E2 levels in the L group and H group were significantly higher than those in the control and M groups. Although ZEN exposure did not cause significant changes in the serum levels of FSH and LH across all groups (Fig. 2B, 2C), a slight upward trend in FSH and LH levels was observed in ZEN-treated groups compared to the control group. In contrast, ZEN exposure significantly reduced the serum P4 content in chicks (Fig. 2D). These results demonstrate that embryonated egg exposure to ZEN affected the levels of reproductive hormones in the serum of chicks, where the effect of the high dose was more pronounced than that of the low and medium doses.

Fig. 2.

Effects of zearalenone on reproductive hormones in chicks. (A) E2 levels in serum of 20-day-old chicks. (B) FSH levels in serum of 20-day-old chicks. (C) LH levels in serum of 20-day-old chicks. (D) P₄ levels in serum of 20-day-old chicks. Different letters indicate P < 0.05.

ZEN exposure causes damage to intestinal structures and inflammation in chicks

H&E staining of duodenum, proximal jejunum, and ileum sections revealed distinct morphological changes in the intestines of chicks hatched from ZEN-exposed embryonic eggs. Specifically, inflammatory cell infiltration was observed in the mucosal layer of the duodenum and jejunum, and increased vacuolization of jejunal villi was detected in the M and H groups (Fig. 3A). In addition, ZEN exposure had no significant effect on the villus crypt ratio in the duodenum and jejunum of 20-day-old chicks (Fig. 3B, C). In contrast, a significant increase in the ileum villus-to-crypt length ratio was observed in the H group (Fig. 3D). The above results suggest that medium and high doses of ZEN treatment of embryonated eggs disrupted the morphology of the chick intestine and induces intestinal inflammatory responses.

Fig. 3.

Effects of zearalenone on intestinal health in chicks. (A) Histological morphology of gut was observed by H&E staining. The red arrows showed inflammatory infiltration. (B) The villus/crypt length of the duodenum. (C) The villus/crypt length of the jejunum. (D) The villus/crypt length of the ileum. Different letters indicate P < 0.05.

Effects of zearalenone on the composition of the intestinal microbiota in chicks

Combining the results of the effects of embryonic egg exposure to ZEN on the growth performance of chicks, we found that group H had the greatest effect on the phenotypic indexes of chicks, therefore, we selected the cecum microorganisms of group C and group H for macrogenomic sequencing analysis. To investigate the effect of embryonated egg exposure to ZEN on microbial diversity in the cecum of chicks, we analyzed the alpha diversity of microorganisms at the species level using Shannon and Simpson indices (Fig. 4A, B), which showed that there was no significant difference in bacterial diversity between the two groups. At the species level, the results of PCA analysis and PCoA analysis based on Bray-Curtis distance of the samples from both groups showed that there was a significant separation of the structure of the gut microbiota between the control and H groups, with no intersection on the axes (Fig. 4C, D). This shows that ZEN altered the structure and composition of the cecum microbiota of chicks. The cecum microbial community was profiled at the phylum level, and differential taxa were identified by two-tailed Student’s t-tests. Among the 10 most abundant species, Firmicutes dominated in both cohorts, followed by Bacteroidota and Proteobacteria (Fig. 4E). Proteobacteria exhibited a statistically significant enrichment in the H group relative to the C group, whereas Firmicutes and Bacteroidota showed no significant inter-group differences.

Fig. 4.

Effects of zearalenone on the composition of the intestinal microbiota in chicks. (A) Shannon index of cecum microbiota. (B) Simpson index of cecum microbiota. (C) PCA and (D) PCoA at species level of cecum microbiota. (E) Histogram of the community composition of cecum microbiota at the phylum level. (F) Phylogenetic trees at the phylum and species levels of cecum microbiota. Nodes of different colors in the figure represent microbial groups that are significantly enriched within their respective clusters and exert a significant influence on inter-group differences. Light yellow nodes indicate microbial groups that show no significant differences across clusters or exert no significant influence on inter-group variations. (G) LEfSE plots of cecum microbiota based on species level.

Furthermore, in the present study, species differences across multiple taxonomic levels were visualized using a cladogram (Fig. 4F). Combined with LEfSe discriminant analysis, significantly different dominant strains were identified in the cecum of chick from the control group and the ZEN-exposed group (Fig. 4G). Specifically, the control group was enriched in 5 commensal bacterial species, including Odoribacter splanchnicus, Clostridia bacterium, Candidatus Blautia faecipullorum, Candidatus Acutalibacter stercorigallinarum, and Candidatus Pullilachnospira gallistercoris. Conversely, 8 bacterial species were overrepresented in the H group, among which Escherichia coli and Clostridium spiroforme were the most prominently enriched. The marked expansion of Escherichia coli is indicative of the fact that ZEN-driven dysbiosis favors opportunistic pathogens. This microbiota shift holds the potential to exacerbate intestinal inflammation in chicks.

Effects of zearalenone on the gut microbiota function in chicks

In order to analyze the functional consequences of ZEN exposure on the cecum microbiota, metagenomic reads were annotated against the KEGG database. The analysis of β-diversity metrics computed at KEGG levels 1-3 revealed a clear separation between the control and ZEN-treated communities (PERMANOVA, P < 0.01) (Fig. 5A-F). This finding indicates that embryonic administration of ZEN induces significant reprogramming of the functional landscape of the chick cecum microbiota. Subsequently, LEfSe analysis (LDA > 3.0) was conducted at KEGG level 3 to identify functionally differential pathways between the two groups. A total of 12 pathways were enriched in the C group, including those involved in central carbon fixation, DNA replication, cell-cycle regulation, and cofactor biosynthesis modules that underpin homeostatic microbiota-host interactions (Fig. 5G). In contrast, seven pathways were significantly overrepresented in the H group, such as ABC transporters, ascorbate/aldarate metabolism, glutathione turnover, and biofilm-formation cascades characteristic of Escherichia coli and Vibrio cholerae. These results imply that ZEN-induced dysbiosis compromises microbial metabolic stability, thereby amplifying oxidative stress and pro-inflammatory circuits. This, in turn, poses a direct threat to avian intestinal health.

Fig. 5.

Effects of zearalenone on the gut microbiota function in chicks. PCA Analysis in groups C and H at (A) KEGG Level 1, (B) KEGG Level 2 and (C) KEGG Level 3. PCoA Analysis in groups C and H at (D) KEGG Level 1, (E) KEGG Level 2 and (F) KEGG Level 3. (G) LEfSE plot of cecum microbiota in groups C and H based on KEGG Level 3.

ZEN exposure induces liver damage in chicks

As shown in Figure 6A, ZEN had no significant effect on the liver indices of chicks. Subsequent histopathological examination revealed that inflammatory cell infiltration was detected in the liver tissue of all three experimental groups. Concretely, the livers exhibited significant structural abnormalities, including hepatocyte vacuolization and apoptosis. In addition, hepatic vascular congestion was observed only in H group (Fig. 6B). We then measured serum biochemical parameters to assess liver function. The results showed that the levels of ALT and TC were significantly elevated in group M, while ALP, TG, and TC levels were significantly increased in group H. However, no significant difference was observed in AST levels (Fig. 6C-G). Next, the results of the liver antioxidant marker analysis revealed that ZEN exposure significantly reduced CAT activity, markedly elevated MDA levels in the H group, and significantly decreased GSH-PX activity. No significant alterations were observed in the levels of T-SOD and GSH (Fig. 6H-L). In addition, the inflammatory factors IL-6 demonstrated a significant increase in both group L and group H, while TNF-α exhibited a significant increase only in group L. No significant changes were observed in the remaining inflammatory factors (Fig. 6M-Q).

Fig. 6.

Effects of zearalenone on the liver health in chicks. (A) Liver index. (B) H&E staining of liver. The red arrows showed inflammatory infiltration, the yellow arrows indicated vacuolation, and the black arrows indicated congestion. Serum levels of (C) ALT, (D) AST, (E) ALP, (F) TG, and (G) TC. Levels of (H) MDA, (I) CAT, (J) T-SOD, (K) GSH, and (L) GSH-PX in liver tissue.Inflammatory factors of (M) IL-1β, (N) TNF-γ, (O) IL-8, (P) IL-6, and (Q) TNF-α, in the tissues of liver. Different letters indicate P < 0.05.

ZEN exposure causes metabolic disorders in the livers of chicks

A non-targeted metabolomics analysis was performed on chick liver tissues to investigate the effects of ZEN exposure on hepatic metabolites and metabolic pathways in chicks. The results demonstrated a clear distinction between the samples from Groups C and H, with significant differences observed in both PCA and PLS-DA analyses (Fig. 7A). Compared with the control group, a total of 161 differentially expressed metabolites were detected in group H, including 69 significantly downregulated metabolites and 92 significantly upregulated metabolites (Fig. 7B). KEGG enrichment analysis was further conducted to explore the biological functions of these differential metabolites. At KEGG Level 1, the metabolites were enriched in multiple biological processes, including metabolism, organismal systems, human diseases, cellular processes, and environmental information processing. At KEGG Level 2, the dominant enriched biological processes were lipid metabolism, the nervous system, cancer, amino acid metabolism, and viral/bacterial infection (Fig. 7C). Moreover, KEGG enrichment analysis at level 3 primarily enriched pathways including Retrograde endocannabinoid signaling, Choline metabolism in cancer, Kaposi sarcoma-associated herpesvirus infection, GnRH signaling pathway, Fc gamma R-mediated phagocytosis, and Long-term depression (Fig. 7D). Based on these differential metabolic pathways, ZEN appears to influence lipid and amino acid metabolism, immune response and oxidative stress in the liver. Its hepatotoxic effects may render the liver more susceptible to viral or bacterial infections, potentially leading to liver inflammation, cancer, and other pathological conditions.

Fig. 7.

Effects of zearalenone on the liver metabolites in chicks. (A) Comparative analysis of liver metabolites. (B) Volcano plots of differential liver metabolites. (C) KEGG analysis of differential metabolites at the Level 1 and Level 2. (D) KEGG enrichment analysis of differential metabolites at the Level 3.

Correlation analysis between gut microbiota and metabolites enriched in shared functional pathways

To verify whether gut microbiota influences hepatic metabolism through the gut-liver axis, we integrated gut metagenomic and hepatic metabolomic data. KEGG functional prediction was performed on the differentially abundant bacteria identified by LEfSe discriminant analysis (Fig. 4G), resulting in the identification of 52 differential functional pathways (Supplementary Table 1). Concurrently, KEGG functional enrichment analysis of differential metabolites revealed 47 differential signaling pathways (Supplementary Table 2). Venn diagram analysis of both datasets revealed 5 signaling pathways that were enriched in both the differential microbiota and the differential metabolites. These pathways included ABC transporters, alpha-Linolenic acid metabolism, Autophagy–other, cAMP signaling pathway, and Glutathione metabolism, suggesting they may serve as key mediators through which ZEN affects gut and liver health in chicks via the gut-liver axis (Fig. 8A). Correlation analysis between the differential microbiota and key metabolites enriched in these five pathways (Supplementary Table 3) revealed that the dominant microbiota in the healthy group showed negative correlations with Succinic Acid in Glutathione metabolism, ABC transporters, and the cAMP signaling pathway, and positive correlations with rDG (20:4(8Z,11Z,14Z,17Z)/18:0/0:0) in the alpha-Linolenic acid metabolism, Autophagy-other, and cAMP signaling pathways. In contrast, in the H group, these correlations were completely reversed compared to the control group (Fig. 8B).

Fig. 8.

Correlation analysis between gut microbiota and metabolites enriched in shared functional pathways. (A) Venn diagram of differential signaling pathways in gut microbiota and liver metabolites. (B) Correlation analysis between differential microbiota and metabolic products of the five key pathways.

Discussion

Zearalenone (ZEN), as one of the mycotoxins threatening global livestock feed and food safety, is widely present in moldy corn, rice, wheat, barley, and oats, posing a potential risk to human and animal health (Jing et al., 2022). The estrogenic effect of ZEN is one of its most harmful characteristics (Jing et al., 2022). Therefore, the transgenerational toxic effects on offspring resulting from maternal exposure to ZEN warrant significant attention. However, the impacts of ZEN exposure during the embryonic egg stage on embryonic egg development, as well as on the subsequent growth, development, and health status of chicks remain unclear. In this study, we confirmed that ZEN exposure during the hatching egg stage significantly reduces chick hatchability and negatively impacts net weight,ADG,ADFI and FCR in 20-day-old broilers. Additionally, ZEN disrupts hormone levels in chicks. The study also indicates that ZEN treatment not only causes intestinal tissue damage and gut microbiota dysbiosis but is also accompanied by liver lipid metabolism disorders, elevated oxidative stress levels, and increased inflammatory factor levels. Furthermore, we found that exposure of embryonic eggs to ZEN led to increased abundance of harmful bacterial strains such as s_Escherichia_coli and s_Clostridium_spiroforme in the chick gut, along with alterations in metabolic products within the liver. Probably, these changes represent key factors contributing to lipid metabolism disorders, oxidative stress responses, and inflammatory reactions in chicks, thereby adversely affecting chick growth, development, and overall health.

The hatchability and hatch weight of embryonic eggs are key indicators reflecting the hatching performance of eggs (Kadam et al., 2013). Mycotoxin contamination in hatcheries is one of the primary factors contributing to low hatchability rates, reduced egg quality, and compromised chick immunity (Allen et al., 1983; Liu et al., 2022). Similarly, in this study, injection of 50 μg/egg and 500 μg/egg ZEN significantly reduced the hatching rate of fertilized eggs, while injection of 5 μg/egg ZEN only showed a trend toward decreased hatching rate. This suggests that the fertilized egg hatching rate exhibits a dose-response relationship with ZEN contamination levels. Furthermore, the nonlinear trend in hatching weight may be partially attributed to "survivorship bias." Given that the highest dose (H group) exerted significant embryotoxicity, resulting in higher embryonic mortality, it is plausible that the surviving embryos represented a cohort with greater physiological robustness. This selective survival likely masked the underlying detrimental effects of ZEN on development, leading to mean hatching weights that did not significantly differ from the control group (Minervini and Dell'Aquila, 2008). Interestingly, the hatch weight of chicks in Group M was higher than that in other groups. However, after 20 days of rearing, the net weight of chicks in the control group was significantly higher than that in the ZEN-treated group. This may be related to ZEN-induced lipid metabolism disorders and abnormal fat deposition in animals (Nagl et al., 2021). ZEN caused abnormal early fat accumulation in chicks, yet subsequent developmental impairment led to lower body weights compared to the control group. Additionally, ZEN exposure significantly reduced production performance in 20-day-old chicks and exhibited a dose-dependent effect, demonstrating that embryonic exposure to ZEN also negatively impacts chick growth performance, which is consistent with the findings of Chang et al. (Chang et al., 2020).

Serum reproductive hormone levels are directly correlated with the development of animal reproductive organs and also play a crucial regulatory role in maintaining normal reproductive performance (Kaprara and Huhtaniemi, 2018; Molefe and Mulunda, 2023). In our study, E2 and P4 levels were significantly increased in 20-day-old chicks after embryonic eggs were exposed to ZEN. However, E2 levels in group M were similar to those of the control group, FSH levels showed an increasing trend, and LH levels were not affected by ZEN. Gao et al. found that serum levels of E2 were reduced in female offspring born to ZEN-exposed mice, yet increased in male offspring, suggesting that the sex of the animal is one of the factors causing differences in the effects of ZEN on reproductive hormones (Gao et al., 2017, 2018). Therefore, the similar E2 levels of chicks in group M to those in the control group in this study may be related to the sex of the chicks. Unfortunately, the sex of the chicks in each group was not further confirmed. Wang et al. demonstrated that ZEN increased serum levels of E2 and LH in young sows and had no effect on FSH levels, suggesting that the estrogen-like effects of ZEN lead to precocious puberty in young animals (Wang et al., 2010). Furthermore, gilts fed a diet containing 1.04 mg/kg ZEN for 35 days exhibited vulvar swelling, accompanied by increased serum E2 and FSH levels (Wan et al., 2021). Song et al. reported that exposure to 1.5 mg/kg ZEN reduced serum P4 concentrations in gilts (Song et al., 2022). These results are consistent with the changes in serum hormone levels observed in chicks in our study. Overall, the alterations in reproductive hormone levels induced by ZEN treatment may lead to sexual precocity in chicks, which could further exert adverse effects on the development of their reproductive organs and their reproductive performance.One limitation of the present study is that sex determination was not performed in the hatched chicks, and therefore sex-specific responses to embryonic ZEN exposure could not be evaluated. Given the estrogenic activity of ZEN and the physiological differences in endocrine regulation between male and female chickens, variation in sex composition among treatment groups may have influenced the observed reproductive hormone profiles. Although chicks were randomly selected from each treatment group, the potential confounding effect of sex cannot be excluded. Future studies should incorporate sex identification and sex-stratified analyses to better elucidate the endocrine-disrupting effects and sex-dependent susceptibility associated with embryonic ZEN exposure.

When considered together, the changes in E2 levels and hatching weight indicate a non-monotonic dose-response pattern, suggesting the presence of dynamic compensatory and subsequent decompensatory processes in embryos exposed to exogenous estrogenic stress. It is well-documented that endocrine-disrupting chemicals (EDCs), such as Zearalenone (ZEN), often exhibit non-monotonic dose-response curves (NMDRC). This phenomenon is recognized as an inherent feature of receptor-mediated pathways, where low-dose exposure may induce hypersensitivity, whereas intermediate doses may trigger homeostatic adjustments, such as receptor downregulation or the upregulation of metabolic enzymes (e.g., hydroxysteroid dehydrogenases), thereby temporarily normalizing physiological parameters (Li et al., 2013).

Upon ingestion of contaminated food or feed, the gastrointestinal tract is the first to be affected by mycotoxins. For instance, mycotoxins can alter normal intestinal function and impair intestinal histomorphology (Liew and Mohd-Redzwan, 2018; Xia et al., 2024; Huangfu et al., 2025).In the present study, embryonic exposure to ZEN was found to exert adverse effects on intestinal function in chicks. Furthermore, the intestinal tract harborsbillions of microorganisms that participate in complex metabolic processes of the animal body. Baines et al. demonstrated that a mixture of aflatoxins and fumonisins increased the levels of Shiga toxin-producing Escherichia coli in cattle feces, which in turn enhanced cytotoxicity and triggered intestinal inflammation (Baines et al., 2013). Consistently, our results showed that ZEN treatment significantly increased the abundance of Shiga toxin-producing s_Escherichia_coli in the cecum of chicks, disrupting the intestinal microbial balance and inducing inflammatory cell infiltration in the intestinal mucosa. Additionally, s_Odoribacter_splanchnicus, the dominant bacterium in the control group, has been proven to exert immunomodulatory effects. Its reduced abundance is associated with the development of non-alcoholic fatty liver disease, cystic fibrosis, and inflammatory bowel disease (Hiippala et al., 2020). This suggests that the intestinal inflammation observed in chicks following embryonic ZEN exposure might be related to the decreased abundance of s_Odoribacter_splanchnicus. In studies investigating the effects of ZEN exposure on the gut microbiota of suckling offspring, it was found that after maternal mice were exposed to ZEN for 21 days, the abundance of gut microbiota in their offspring underwent significant changes, including Bacteroidetes, Proteobacteria, and Firmicutes. These microbial alterations led to changes in glutathione metabolism and the expression of antioxidant enzymes in the ovaries (Yan et al., 2022). Consistent with this finding, our study also revealed that ZEN-induced changes in the gut microbiota of chicks affected the glutathione metabolic pathway. Similarly, Bai et al. had previously proposed that mycotoxins may adversely affect the organism through their induced oxidative stress (Bai et al., 2022). However, no studies have reported the effects of embryonic ZEN exposure on intestinal health and gut microbiota in chicks. Whereas, the results of the present study demonstrated that embryonic ZEN exposure not only induces gut microbiota dysbiosis in chicks but also exerts adverse effects on their intestinal health, specifically triggering intestinal inflammatory responses and oxidative stress.

Simultaneously, ZEN-induced oxidative stress appears to be a primary driver of disrupted gut-liver axis integrity. By suppressing critical antioxidant pathways such as the Nrf2 signaling cascade, ZEN may exacerbate cellular damage and selectively trigger the release of specific pro-inflammatory cytokines. Previous research has demonstrated that Fusarium mycotoxins disrupt placental and epithelial barrier integrity, leading to a differential expression of pro-inflammatory markers such as IL-6, which in turn compromises metabolic homeostasis (So et al., 2014; Seyed Toutounchi et al., 2019).

In addition to the intestine, the liver is also one of the key organs for mycotoxin metabolism (Zhang et al., 2020). Consistent with the functional prediction results of the gut microbiota, chicks in the embryonic ZEN exposure group exhibited inflammatory cell infiltration and cell apoptosis in the liver, which caused liver damage. The increased levels of pro-inflammatory cytokines in the liver further indicated an inflammatory response in the chicks' livers. This is consistent with previous reports that ZEN can induce inflammatory lesions, immunosuppression, and even liver damage (Rajendran et al., 2020; Xu et al., 2021). Interestingly, our results also showed a significant increase in the levels of GLU, CHOL and TG, suggesting that ZEN further induces hepatic lipid metabolism disorders. Borutova et al. also showed a significant decrease in GSH-PX activity and an increase in MDA levels in the liver of broilers after 2 weeks of exposure to ZEN-contaminated feed (Borutova et al., 2008). Similarly, ZEN treatment resulted in a significant increase in MDA levels but a significant decrease in CAT and GSH-PX activity in chick liver in our study, which suggested that embryonic ZEN injection with ZEN induced the onset of oxidative stress in chick liver, as one of the reasons for ZEN-induced liver injury. Metabolomic results also indicated that ZEN significantly altered the hepatic metabolites and KEGG metabolic pathways in chicks, including pathways related to lipid metabolism, autophagy, inflammatory response, and antioxidant defense. Consistent with this, previous studies have reported that ZEN disrupts hepatic glycerophospholipid metabolism, and such lipid metabolism disorders can induce inflammation and cell apoptosis. Interestingly, we identified five pathways associated with lipid metabolism, autophagy, inflammatory response, and antioxidant defense that were significantly enriched in both the functions of differential gut microbiota and differential metabolites (Li et al., 2021; Wang et al., 2021). Spermine and spermidine, which are key metabolites in the glutathione metabolic pathway, exhibited a negative correlation with the dominant bacteria in the ZEN-treated group. The above results indicated that intestinal microbiota dysbiosis induced by embryonic ZEN exposure was also involved in the processes of liver damage, oxidative stress, and lipid metabolism disorders.

Conclusion

In conclusion, this study investigated the effects of different concentrations of ZEN on the growth and development, liver and intestinal health of embryonic eggs and the post-hatch chicks using the embryonic egg injection technique as well as a combined multi-omics analysis. The results demonstrated that embryonic ZEN exposure not only reduced the hatching performance of embryonic eggs but also impaired the production performance of chicks after hatching. Further mechanistic analysis revealed that ZEN could mediate the pathological damage process of liver and intestinal tissues by inducing intestinal microbiota dysbiosis, activating inflammatory signaling pathways in liver and intestinal tissues, disrupting the oxidative stress balance, and perturbing lipid metabolism homeostasis. These findings deepen the understanding of the potential hazards of ZEN exposure during the embryonic egg stage, and more importantly, provide a crucial theoretical basis and practical reference for formulating precise prevention and control strategies against ZEN contamination in breeder and commercial layer farming, as well as for safeguarding the safety of poultry farming and the quality of poultry products. Nevertheless,as this study employed a direct embryonic egg injection model, the exposure route differs from natural maternal transfer encountered in commercial production systems. Consqequently, caution should be exercised when extrapolating these findings directly to field conditions. Future studies should elucidate the causal relationships between embryonic ZEN-induced gut microbiota dysbiosis and subsequent hepatic metabolic disturbances induced, particularly by validating key signaling pathways involved in lipid metabolism, oxidative stress, and autophagy. Furthermore, longitudinal researches are warranted to assess the long-term and potential transgenerational effects of embryonic ZEN exposure, thereby characterizing the associated risks under practical poultry production scenarios.

Authorship Contribution

Tao Yuan and Luoyi Zhu designed the study, performed primary experiments, analyzed the data, and wrote the original manuscript draft. Kerui Zhou, Yifan Yuan, Han Jiang, and Sunli Zheng executed key experimental procedures and collected experimental data. Desheng Qi, Wei Zhang, and Shen Hu contributed to establishing experimental protocols and optimizing testing procedures. Niya Zhang conceptualized the overall research project, guided the experimental strategy, interpreted the data, and critically revised the manuscript for key intellectual content. All authors have read and approved the final manuscript.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data used and/or analyzed to support the findings of this study are available in the main paper or in the Supplementary Information. Sequence data and metabolomics data supporting the results of this study have been deposited in the Mendeley data(DOI:10.17632/ncdht53852.1, DOI:10.17632/rgd3rbzhrx.1, DOI:10.17632/4tn5nn6554.1, DOI:10.17632/6kb6p6htgz.1, DOI:10.17632/h5mwz3w58t.1, DOI:10.17632/z2c84cmk7x.1, DOI:10.17632/mnw9fv3bdd.1).