s40168-024-01966-y

Huang et al.
Microbiome (2024) 12:269 Microbiome

https://doi.org/10.1186/s40168-024-01966-y
RESEARCH Open Access
New insights into the combined effects

of aflatoxin B1 and Eimeria ovinoidalis on uterine
function by disrupting the gut–blood–
reproductive axis in sheep
Shu‑cheng Huang1†, Kai‑li Liu1†, Pan Chen1, Bo‑wen Xu1, Wen‑li Ding1, Tao‑jing Yue1, Ya‑nan Lu1, Sen‑yang Li1,
Jia‑kui Li2* and Fu‑chun Jian1*
Abstract
Background Sheep coccidiosis is an infectious parasitic disease that primarily causes diarrhea and growth retar-
dation in young animals, significantly hindering the development of the sheep breeding industry. Cereal grains
and animal feeds are frequently contaminated with mycotoxins worldwide, with aflatoxin B1 (AFB1) being the most
common form. AFB1 poses a serious threat to gastrointestinal health upon ingestion and affects the function of par-
enteral organs, thus endangering livestock health. However, the impact of the combined effects of coccidia and AFB1
on the reproductive system of sheep has not been reported. Therefore, this study utilized sheep as an animal model
to investigate the mechanisms underlying the reproductive toxicity induced by the individual or combined effects
of AFB1 and Eimeria ovinoidalis (E. ovinoidalis) on the gut–blood–reproductive axis.
Results The results showed that AFB1 and coccidia adversely affect the reproductive system defense of sheep
by altering uterine histopathology and hormone levels and triggering inflammation, which is associated with changes
in the gut microbiota and metabolites. Moreover, co-exposure to AFB1 and coccidia disrupted the intestinal struc-
ture of the colon, resulting in reduced crypt depth. The impaired barrier function of the colon manifests primarily
through the suppression of barrier protein expression, changes in the gut microbiome composition, and disrup-
tions in gut metabolism. Importantly, the levels of blood inflammatory factors (IL-6, IL-10, TNF-α, and LPS) increased,
suggesting that exposure to AFB1 and coccidia compromises the function of uterine organs in sheep by perturbing
the gut–blood–reproductive axis. Blood metabolomics analysis further revealed that the differential metabolites
predominantly concentrate in the amino acid pathway, particularly N-acetyl-L-phenylalanine. This metabolite is signifi-
cantly correlated with IL-6, TNF-α, LPS, ERα, and ERβ, and it influences hormone levels while inducing uterine damage
through the regulation of the downstream genes PI3K, AKT, and eNOS in the relaxin signaling pathway, as demon-
strated by RNA sequencing.
Conclusions These findings reveal for the first time that the combined effects of AFB1 and E. ovinoidalis on sheep
uterine function operate at the level of the gut–blood–reproductive axis. This suggests that regulating gut microbiota
†
Shu-cheng Huang and Kai-li Liu contributed equally to this work.
*Correspondence:
Jia‑kui Li
[email protected]
Fu‑chun Jian
[email protected]
Full list of author information is available at the end of the article
© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
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Huang et al. Microbiome (2024) 12:269 Page 2 of 21
and its metabolites may represent a potential therapeutic strategy for addressing mycotoxins and coccidia-co-
induced female reproductive toxicity.
Keywords Mycotoxin, Aflatoxin B1, Coccidia, E. ovinoidalis, Reproduction toxicity, Gut microbiota
Graphical Abstract
Introduction 5]. The colon, ileum, and cecum are the organs primar-
Sheep coccidiosis, caused by the genus Eimeria, is an ily affected by the pathological changes induced by E.
infectious disease that primarily affects young animals, ovinoidalis.
developing in the intestines [1]. The clinical infections, The contamination of mycotoxins is a significant chal-
characterized by diarrhea and subclinical infections, lenge faced by global agricultural production. Research
which result in stunted growth and development, caused indicates that approximately 75% of feed worldwide is
by this parasite lead to a deterioration in the quality of contaminated with mycotoxins, presenting serious health
meat, skin, wool, and other derived products. Under risks to livestock [6, 7]. Aflatoxin B1 (AFB1), a mycotoxin
intensive breeding conditions, accompanied by high ani- produced by Aspergillus flavus and Aspergillus parasiti-
mal density and productivity, the prevention and control cus, is a member of the aflatoxin family and is commonly
of sheep coccidiosis hold significant economic impor- found in cereals and animal feed all over the world,
tance [2]. Eimeria ovinoidalis (E. ovinoidalis), a type of together with other fungal toxins [8, 9]. The toxicity and
coccidian parasite, has been identified as the most patho- dangers of aflatoxins have gained considerable attention,
genic species among sheep worldwide [3]. In sheep, the making their detection and management a crucial aspect
primary clinical symptoms include diarrhea and catarrhal in the field of food safety. The Food and Drug Adminis-
enteritis, along with specific pathological findings [4, tration (FDA) of the USA has established the maximum
limits for total aflatoxins (B1, B2, G1, and G2) in all foods that sheep often experience co-exposure involving multi-
at 20 µg/kg, with a specific restriction of 100 µg/kg for ple toxins or various coccidia [25–27]. Therefore, it is of
peanut and corn feed products [10, 11]. Meanwhile, the great economic significance to investigate cases of AFB1
European Commission has established lower thresholds exposure in conjunction with coccidia infection. Further-
of 2 µg/kg for AFB1 and 4 µg/kg for total aflatoxins [12]. more, enhancing our understanding of the interactions
Notably, excessive exposure to AFB1 has been linked to between these two pathogenic factors within the gut–
some serious health issues in animals, including immu- blood–reproductive axis may improve feed efficiency and
notoxicity, hepatotoxicity, nephrotoxicity, carcinogenic- overall health in sheep farming, thereby promoting the
ity, and reproductive and developmental toxicity [13–15]. development of sustainable sheep production practices
Furthermore, AFB1 and its metabolites can persist in based on scientific knowledge. Consequently, this study
major body tissues, posing significant health risks to peo- aims to explore the main effects of the combined action
ple consuming contaminated meat [9], while most studies of AFB1 and coccidia on the uterus and intestines of
on the effects of AFB1 on animal tissues and organs have sheep, while also investigating the interactions between
primarily concentrated on the liver and kidney [16, 17]. these two pathogenic factors in the context of the gut–
It is essential to note that high doses of AFB1 can also blood–reproductive axis. This study will elucidate the
lead to reproductive toxicity in female animals, resulting underlying mechanisms and provide novel insights into
in reduced fertility and potential miscarriages [18, 19]. the combined toxic effects of AFB1 and coccidia.
Given the widespread prevalence of aflatoxins in grains
and feed, investigating their effects on reproductive tox- Materials and methods
icity is of paramount importance. Chemical reagents
The gastrointestinal tract is the initial organ in the AFB1 was purchased from Qingdao Pribolab Chemi-
body that interacts with harmful substances. Research cal Inc. (Shandong, China) and was dissolved in dime-
has shown that AFB1 and coccidia invasion can alter the thyl sulfoxide (DMSO) solution before use. DMSO was
abundance of the gut microbiota and its metabolites, sourced from Tianjin Fuyu Fine Chemical Co., Ltd. (Tian-
leading to the proliferation of pathogenic or opportunis- jin, China). The unsporulated oocysts of E. ovinoidalis
tic pathogens and compromising intestinal barrier func- were obtained from the Laboratory of Parasites at Henan
tion [20, 21]. As the first line of defense against harmful Agricultural University. The oocysts were treated with
substances, the intestine plays a crucial role in regulat- a 2.5% potassium dichromate solution facilitate sporu-
ing the immune system, the endocrine system, and the lation. Enzyme-linked immunosorbent assay (ELISA)
digestive process [13]. Following intestinal intake and kits for the following assays were supplied by Shang-
absorption, mycotoxins can cause direct damage through hai Enzyme-linked Biotechnology Co., Ltd. (Shanghai,
biotransformation and metabolism of target organs. They China): interleukin-6 (IL-6, #YJ622840), interleukin-10
can also induce changes in blood-related substances via (IL-10, #YJ507611), tumor necrosis factor-α (TNF-α,
the intestinal mucosa or red blood cell metastasis [22, #YJ220635), lipopolysaccharides (LPS, #YJ100325), estro-
23]. To date, it remains unclear whether AFB1-induced gen receptor α (ERα, #YJ155970), and estrogen receptor
reproductive toxicity is associated with changes in gut β (ERβ, #YJ155971).
microbiota-derived metabolites or blood metabolism
influenced by the synergistic effects of coccidia. There- Animal ethics
fore, gaining a deeper understanding of the interactions All animal experimental procedures were conducted fol-
among the host, microbiota-derived metabolites, and lowing the guidelines of the Animal Welfare and Ethics
blood metabolism could help elucidate potential mecha- Research Committee of the College of Veterinary Medi-
nisms underlying the reproductive toxicity induced by cine at Henan Agricultural University (ethical permission
the combined effects of AFB1 and coccidia. code: HNND2022030818) in Zhengzhou, China. There
Sheep are a significant source of meat, leather, wool, were no signs of pain or suffering exhibited by the ani-
and dairy products worldwide, making them vital for mals prior to their euthanasia during the experiment.
global food security. However, the sheep farming indus- Furthermore, all the possible measures were taken to
try faces considerable challenges due to the presence of ensure the welfare of the sheep throughout the study.
fungal toxins in feed. Moreover, the susceptibility and
resistance to sheep coccidiosis urgently require the atten- Experimental procedure
tion of researchers [24]. While numerous studies have The experiment was conducted at Luoyang Xinning Ani-
focused on animals with compromised AFB1 toxicity mal Husbandry Technology Co., Ltd. in Luoyang City,
and coccidia infection alone, the pervasive presence of Henan Province. One hundred healthy weaned ewes,
mycotoxins and the high infection rate of coccidia mean 35 days old, were selected and 1 mg/kg of Diclazuril
was administered for two consecutive days to eliminate paraformaldehyde for histopathological examination. The
potential coccidia infections. The ewes were housed in remaining colon and uterine tissues were rapidly frozen
newly constructed, flame-sterilized pens. On the 11th in liquid nitrogen and stored at − 80℃ for subsequent
day after medication, each ewe underwent rectal sam- molecular experiments.
pling and microscopic examination for 3 consecutive
days. Sixteen healthy female Hu sheep weighing approxi- Histopathological analysis and coccidia oocysts detection
mately 8–12 kg and free from coccidia, cestode, and The tissues from the uterus and colon were preserved in
trematode infections were selected and assigned to four 4% paraformaldehyde. All specimens were deparaffinized
groups: control (Con) (n = 4), AFB1 group (n = 4), E. ovi- in xylene, dehydrated in graded ethanol solutions, and
noidalis (E.o) group (n = 4), and AFB1 + E.o group (n = 4). embedded in paraffin using a fully enclosed tissue proces-
Each group was housed in separate new enclosures, with sor (ASP300S, Leica Biosystems, Buffalo Grove, IL, USA).
one enclosure left empty between two experimental The sections were stained with hematoxylin and eosin
enclosures to prevent mingling among the lambs. (HE) and then visualized using a Mito More Tan micro-
The experimental design is illustrated in Fig. 1a. The scope. Additionally, the crypt depth of the intestines and
Con group received daily oral administration of PBS the thickness of the uterine endometrium were evaluated
solution and DMSO. The AFB1 group received daily oral using Motic Images Advanced 3.2 software (Motic China
administration of AFB1 at a dosage of 60 μg/kg. The E.o Group, Co., China).
group received a single oral administration of 2 × 104
sporulated oocysts on day 0 only. The AFB1 + E.o group Hematology analysis
received a single oral administration of 2 × 104 sporu- The complete blood count parameters were analyzed by
lated oocysts on day 0 along with daily oral administra- the Zhengzhou Furunde Pet Hospital in Henan Prov-
tion of AFB1 at a dosage of 60 μg/kg. The experimental ince, using the Maestro BC-5000Vet analyzer. An ELISA
sheep were fed lamb starter pellets and roughage, con- method was employed to measure the levels of inflamma-
sisting of peanut vine and corn stalk, sourced from Lian- tory factors IL-6, IL-10, TNF-α, and LPS, as well as the
tai Biotechnology Co., Ltd. (Hebi City, Henan Province). expression of ERα and ERβ in plasma.
The lambs were raised in a free-range environment with
unlimited access to water and were fed a fixed amount of RNA extraction and RT‑qPCR
pellets feed and peanut vine daily at 8 am and 4 pm. The Trizol reagent and liquid nitrogen were utilized to extract
unconsumed feed was collected and weighed daily. Lamb total mRNA from the colon and uterus. The quality of
body weights were recorded throughout the 14-day the mRNA was assessed using a Nanodrop spectropho-
experimental period. tometer, followed by cDNA synthesis through reverse
transcription according to the manufacturer’s protocol.
Sample collection After cDNA synthesis was completed, real-time fluo-
Starting from day 7, rectal sampling of each lamb was rescence quantitative PCR (qPCR) was performed using
conducted daily, with 5 g of feces collected in CPE gloves. the ChamQ Universal SYBR qPCR Master Mix (Vazyme
Lamb identification and fecal consistency were recorded −ΔΔCT calculation method
Biotech Co., Ltd., China). The 2
and scored according to the fecal scoring criteria out- was employed to determine mRNA expression levels.
lined in Table S1. On both day 7 and day 14 of the experi- Specific primers for GAPDH mRNA and target gene
ment, 7 mL of blood was collected from the jugular vein sequences (including ERα, ERβ, IL-6, IL-10, TNF-α,
of each lamb before morning feeding. Of the 7 mL col- PI3K, AKT, eNOS, COL4A4, FOSB, COL11A1, GNG2,
lected, 2 mL of whole blood sample was placed in blood Claudin1, Claudin4, Occludin, and ZO-1) were designed
collection tubes, gently inverted to mix, and kept on dry using Primerbank from Shanghai Biotech Co., Ltd. and
ice within an insulated box before being immediately sent synthesized by Tsingke Biological Technology Co., Ltd.
for hematology analysis. The remaining 5 mL of blood The primers used in this study are listed in Table S2. The
was centrifuged at 4 °C and 3000 rpm for 15 min, with GAPDH gene served as an internal reference.
the supernatant stored at − 80 °C for further analysis.
Additionally, fresh feces were collected on day 14 and 16S rRNA sequence analysis
stored at 4 °C and − 80 °C for subsequent analysis. After Microbial genomic DNA samples were extracted from
14 days, the sheep were fasted for 12 h and euthanized feces using the QIAamp DNA kit (Qiagen, Hilden, Ger-
via exsanguination through the jugular vein. The uterus many), following the manufacturer’s instructions and
of each sheep was collected, washed with physiologi- stored at − 20 °C prior to further analysis. The quan-
cal saline, and weighed for record-keeping. The midsec- tity and quality of the extracted DNA were measured
tion of the colon and uterine tissues was fixed in 4% using a spectrophotometer and assessed via agarose
Fig. 1 The reproductive toxicity of sheep induced by AFB1 and coccidia exposure is associated with alterations in uterine hormones
and inflammation. a Experimental design flowchart. b Feed intake of sheep over 0–14 days (n = 4). c Body weight changes of sheep over 0–14
days (n = 4). d Number of oocysts per gram (OPG) in each group (n = 4). e H&E staining of uterine tissue (100×, 400×; n = 4). Red arrows indicate
endometrial epithelial cells, yellow arrows indicate inflammatory cells, green arrows denote nuclear shrinkage and dense staining, and blue arrows
indicate bleeding spots. f Measurement of endometrial thickness (n= 4). g Expression levels of ERα and ERβ mRNA in uterine tissue (n= 3). h mRNA
expression levels of IL-6, IL-10, and TNF-α in uterine tissue (n = 3). Statistical significance is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 (AFB1, E.o,
and AFB1+E.o vs Con); or #p < 0.05, ##p< 0.01, and ###p < 0.001 (AFB1+E.o vs AFB1 and E.o)
gel electrophoresis, respectively. PCR amplification of ACGGGAGGCAGCA-3’) and the reverse primer 806R
the bacterial 16S rRNA gene V3–V4 region was con- (5’-GGACTACHVGGGT WTCTAAT-3’). DNA librar-
ducted using the forward primer 338F (5’-ACT CCT ies were sequenced on the Illumina MiSeq 250 platform.
Sequence data analysis was primarily performed using Diego, CA, USA). The raw sequencing data underwent
QIIME2 with the dada2 plugin and the R software pack- quality control using the Fastp software for Q20 and Q30,
age (v3.2.0). Microbial community diversity and abun- resulting in clean reads that were subsequently visual-
dance were subsequently evaluated through α-diversity ized using the FastQC. The quality-controlled clean reads
and β-diversity analysis. The Gene Cloud tool (https:// were aligned to the corresponding species’ reference
www.genescloud.cn) was utilized for analyzing the 16S genome and gene structure annotation using the HISAT2
sequencing-related data. software. HTSeq v0.6.1 software was employed to count
the number of reads, and then the number of fragments
Metabolomic analysis of feces and blood per kilobase of transcript and per million mapped frag-
Fecal and blood samples were collected and added to a ments for each gene was calculated. To accurately reflect
pre-cooled methanol/acetonitrile/water solution (2:2:1, the expression level of transcripts, normalization was
v/v) after being slowly thawed at 4℃. Following vortexing performed based on the number of mapped reads in
and 30 min of low-temperature ultrasonication, the mix- the sample and the length of the transcripts. Fragments
ture was allowed to stand at − 20℃ for 10 min. After cen- per kilobase of transcript per million fragments mapped
trifugation at 14,000 g for 20 min at 4℃, the supernatant (FPKM) were used as the metric for gene expression lev-
was vacuum dried. For mass spectrometry analysis, 100 els. Differential gene expression analysis was performed
μL of an acetonitrile–water solution (1:1, v/v) was added using DESeq2 (with biological replicates)/edgeR (with-
for re-dissolution. The mixture was then vortexed and out biological replicates), with the filtering criteria for
centrifuged at 14,000 g for 15 min at 4℃. After filtration differentially expressed genes set to |log2FC|≥ 1 and
through a 0.22-μm membrane, 300 μL of the supernatant p-value < 0.05. Differential gene annotation informa-
was transferred to a detection vial for untargeted metab- tion and enrichment pathways were analyzed using the
olomics liquid chromatography-mass spectrometry (LC– KEGG database. The volcano plot was generated using
MS) analysis. the online tools from https://www.omicstudio.cn/tool.
The raw data were converted to mzXML format using The trend heat map and Sankey bubble map were created
ProteoWizard, followed by peak alignment, retention using the online tool http://bioinformatics.com.cn/.
time correction, and peak area extraction which were
performed using XCMS software. The data extracted by Statistical analysis
XCMS underwent metabolite structure identification and All data were analyzed using SPSS software (version
preprocessing, followed by an assessment of experimen- 26.0, SPSS, Inc., Chicago, IL, USA). A one-way analysis
tal data quality, and ultimately data analysis. All analyses of variance (ANOVA) was performed to assess the sig-
were based on an in-house database (Shanghai Applied nificance of differences among treatment groups, and a
Protein Technology). Metabolites with a log fold change two-way ANOVA was used to evaluate potential synergy
(log2FC) ≥ 1 and p-value < 0.05 were considered signifi- between the two factors. The data were subjected to the
cantly regulated metabolites. The identified metabolites Shapiro–Wilk normality test, and non-parametric analy-
were annotated, mapped to the Kyoto Encyclopedia of sis (Kruskal–Wallis test) was employed if the data did
Genes and Genomes (KEGG) pathway database, and not follow a normal distribution. GraphPad Prism 8.0.2
further subjected to metabolite set enrichment analysis. (GraphPad Software, La Jolla, CA, USA) was used for bar
Principal component analysis (PCA), orthogonal partial or line graph analysis. Pearson correlation test was con-
least squares discriminant analysis (OPLS-DA), and vol- ducted to analyze the relationships between metabolites,
cano plots were conducted using the online tools avail- core microorganisms, and related genes. All data are
able at https://www.omicstudio.cn/tool. And KEGG presented as mean ± standard error of the mean (SEM).
enrichment analysis was performed using https://www. p-value < 0.05 was considered statistically significant.
omicshare.com/tools/. The tool available at (https://www.
genescloud.cn) was utilized to analyze network diagrams Results
and network heat maps. The chord and heat maps were The establishment and validation of the AFB1 and E.o
generated using https://hiplot.com.cn/home/index.html. group
To verify whether the model of the AFB1 and E.o treat-
Uterine transcriptomics analysis ment groups was successfully established, we conducted
Total RNA was extracted from the uterus using the Tri- statistical analyses on the clinical performance, feed
zol reagent (Invitrogen, Carlsbad, CA, USA). Follow- intake, weight changes, and fecal score of the experimen-
ing the manufacturer’s protocol, RNA-seq libraries were tal sheep. The results indicated that, compared to the Con
prepared at BGI Genomics (Shenzhen, China) using the group, the feed intake of the AFB1, E.o, and AFB1 + E.o
Illumina TruSeq RNA Sample Prep Kit (Illumina, San treatment groups significantly decreased, particularly
on days 8–14 (p < 0.001, p = 0.003, and p < 0.001, respec- affects endometrial thickness. Nonetheless, the uneven
tively). Furthermore, the AFB1 group exhibited sig- arrangement of cells may explain the inconsistent obser-
nificantly lower feed intake compared to the AFB1 + E.o vations regarding endometrial thickness across different
group (p = 0.002) (Fig. 1b). Similarly, the trend in weight treatment groups. Overall, exposure to AFB1 and infec-
showed a comparable decline; all treatment groups tion with coccidia have caused significant pathological
(AFB1, E.o, and AFB1 + E.o) significantly decreased in damage to the uterus.
weight compared to the Con group (p = 0.016, p = 0.03, Next, we investigated whether AFB1 and coccidia treat-
and p = 0.004, respectively), with no significant differ- ment are accompanied by changes in uterine hormone
ence observed between the AFB1 and AFB1 + E.o group levels and trigger inflammation. Therefore, we detected
(Fig. 1c). Additionally, it was observed that the number the estrogen receptors (ER) α and β that are present in the
of oocysts per gram (OPG) in the E.o and AFB1 + E.o uterus and play a crucial role in reproduction. As shown
groups significantly increased after 12 days (Fig. 1d). in Fig. 1g, both AFB1 and E.o treatments significantly
Moreover, the fecal scores of the E.o and AFB1 + E.o downregulated the expression of the ERα (p = 0.016 and
treatment groups were significantly higher than that of p = 0.003, respectively) and ERβ genes (p = 0.008 and
the Con group (p < 0.001 and p < 0.001, respectively) (Fig. p = 0.021, respectively). The combination of AFB1 and E.o
S1a-b), while no difference in the AFB1 treatment group, treatment resulted in an even more pronounced decrease
indicating the successful establishment of the E. ovinoi- in ERα (p = 0.005) and ERβ (p = 0.004) mRNA expression
dalis infection model. Unfortunately, we did not observe levels compared to the individual AFB1 or E.o treatment
a synergistic effect between AFB1 and coccidia. groups. Additionally, the inflammatory response induced
by AFB1 or E.o treatment led to an upregulation of uter-
The combined effects of AFB1 and coccidia induce damage ine inflammatory factors, including IL-6 and TNF-α
to the uterus mRNA, accompanied by a reduction in IL-10 expres-
This study investigates the impact of consuming AFB1- sion. This inflammatory response was primarily driven
contaminated daily feed on the reproductive system of by AFB1 exposure (IL-6, p = 0.003; IL-10, p = 0.016; TNF-
sheep, with a focus on uterine damage under the coccidia α, p < 0.001). However, in the AFB1 + E.o group, it was
challenge. Observations revealed that the AFB1 treat- observed that E.o could inhibit the overexpression of
ment group displayed mild congestion and yellow stain- inflammatory factors rapidly induced by AFB1 exposure
ing during uterine morphology assessments. The uteri in (Fig. 1h). Taken together, these data suggest that the toxic
the AFB1 + E.o group exhibited obvious bleeding and yel- effects of AFB1 and coccidia treatment on the reproduc-
low staining, with uterine weight was significantly lower tive system are associated with alterations in uterine hor-
than that of the Con group (p = 0.023) (Fig. S2a-b). In mones and the induction of inflammation.
contrast, there were no significant differences in uterine
morphology between the E.o and Con groups. Addition- The combined effects of AFB1 and coccidia compromise
ally, histopathological findings indicated that endome- the integrity of the intestinal barrier
trial cells in the Con group were arranged consistently, Given that coccidia infection mainly affects the intes-
whereas the endometrial epithelial cells in the uteri of tine, we observed pathological changes in the colon. His-
the AFB1, E.o, and AFB1 + E.o groups were disorganized topathological examination of tissue sections revealed
and exhibited severe inflammatory lesions. Moreover, various stages of coccidia in both the E.o and AFB1 + E.o
the AFB1-treated group showed increased chromatin groups (Fig. S3). The intestinal structure in the E.o and
density and nuclear shrinkage, whereas bleeding lesions AFB1 + E.o groups was severely disrupted, characterized
were evident in the AFB1 + E.o group (Fig. 1e). Statisti- by significant inflammatory cell infiltration and conges-
cal analysis of uterine endometrial thickness revealed tion. Similar pathological changes were also observed
significant increases in the E.o and AFB1 + E.o groups in the AFB1 group, although to a lesser extent (Fig. 2a).
compared to the Con group, attributed to the disordered Moreover, coccidia infection significantly reduced the
arrangement of endometrial epithelial cells (p < 0.001 and depth of colonic crypts in both the E.o and AFB1 + E.o
p = 0.005, respectively). However, no significant changes groups compared to the Con group (p < 0.001 for both),
were observed in the AFB1 group (p = 0.499). Compared and there was a significant difference in crypt depth
to the AFB1 + E.o group, endometrial thickness was sig- between the AFB1 and AFB1 + E.o groups (p < 0.001).
nificantly reduced in the AFB1 group (p = 0.001) and sig- Although the difference was not statistically signifi-
nificantly increased in the E.o group (p = 0.019) (Fig. 1f ). cant, the reduction in crypt depth attributed to coc-
These results indicate that exposure to AFB1 and infec- cidia was notably more pronounced with the addition
tion with coccidia can lead to degeneration and disor- of AFB1 (Fig. 2b). These results indicate that both AFB1
ganization of endometrial epithelial cells, which in turn and coccidia treatment can disrupt intestinal structure,
Fig. 2 The intestinal barrier of sheep is compromised by AFB1 and coccidia treatment. a Histological examination of colon tissue via H&E staining
(200×, 400×; n = 4). Red arrows indicate intestinal epithelial cells, yellow arrows denote inflammatory cells, blue arrows represent bleeding points,
and green arrows represent coccidia oocysts. b Statistics on crypt depth (n = 6). c–f Expression levels of intestinal barrier-related genes Claudin1,
Claudin4, Occludin, and ZO-1 (n = 3). Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 (AFB1, E.o, and AFB1+E.o vs Con);
or #p < 0.05, ##p< 0.01, and ###p < 0.001 (AFB1+E.o vs AFB1 and E.o)
leading to impaired intestinal barrier function, with a alterations in the gut microbiota and metabolites, as
more pronounced effect in the E.o group. Therefore, the well as screening for potential biomarkers. We analyzed
expression levels of genes and proteins associated with the microbial composition in fecal samples from sheep
intestinal barrier function were examined next. The treated with AFB1 and coccidia using 16S rRNA gene
results demonstrated that AFB1 treatment significantly sequencing technology. Firstly, alpha diversity indica-
downregulated the mRNA expression levels of Claudin1 tors including Chao1, Shannon index, Simpson index,
(p < 0.001), Claudin4 (p = 0.001), Occludin (p < 0.001), and observed species were employed to assess the rich-
and ZO-1 (p = 0.016) (Fig. 2c, d). Coccidia infection sig- ness and diversity of individual taxa (Fig. 3a). The expo-
nificantly downregulated the mRNA expression levels of sure to both AFB1 and coccidia significantly affected the
Claudin1 (p < 0.001) and Occludin (p < 0.001). Further- diversity of the gut microbiota, as evidenced by lower
more, the combination of both treatments led to sig- microbial community abundances and diversities shown
nificant downregulation of mRNA expression levels of by the E.o and AFB1 + E.o groups compared to the Con
Claudin1 (p < 0.001), Claudin4 (p = 0.004), and Occludin group and the considerably larger trend in alpha diver-
(p < 0.001). Notably, the expression of Claudin1 mRNA sity indices shown by the AFB1 group compared to the
was influenced not only by AFB1 and coccidia treatments AFB1 + E.o group. Principal co-ordinates analysis (PCoA)
individually but also showed a synergistic effect between revealed differences in microbial structure among the
the two factors (F = 87.417, p < 0.001). groups (Fig. 3b). Additionally, the composition of species
at the phylum level was analyzed and high-abundance
The combined effects of AFB1 and coccidia induce phyla including Firmicutes, Bacteroidetes, and Proteo-
alterations in the composition and metabolites bacteria were found (Fig. 3c). Differential analysis of Fir-
of the intestinal microbiota micutes, Bacteroidetes, and the F/B ratio between groups
To elucidate the mechanism of gut damage caused by the showed a significant decrease in the relative abundance
combined effect of AFB1 and coccidia, we investigated of Firmicutes in the E.o group (p = 0.003) and an increase
in the relative abundance of Bacteroidetes. Moreover, as amino acid metabolism, lipid metabolism, and cofac-
the E.o group exhibited a significant decrease in the tor and vitamin metabolism (Fig. 4d). The AFB1 + E.o
F/B ratio compared to the AFB1 + E.o group (p = 0.005) group identified a total of 3974 differential metabolites,
(Fig. 3e). The results indicated that Firmicutes, Erysipel- including 3154 upregulated metabolites and 820 down-
otrichales, Erysipelotrichi in the Con group, Clostridia, regulated metabolites (Table S5). These metabolites were
Ruminococcaceae, Clostridiales in the AFB1 group, Bac- predominantly enriched in pathways such as amino acid
teroidetes, Bacteroidia, Bacteroidales in the E.o group, metabolism, cofactor and vitamin metabolism, and car-
and Lachnospiraceae, Clostridiaceae, Clostridium in the bohydrate metabolism (Fig. 4e).
AFB1 + E.o group possessed relatively high abundance According to the criteria of VIP > 1 and p-value < 0.05,
(Fig. 3f ). Moreover, we overlapped the top 20 genera we further screened for metabolites with significant dif-
with high abundance observed at the genus level and the ferences. Among them, 263, 99, and 359 differential
top 20 genera identified by random forest analysis using expression metabolites (DEMs) were identified in the
a Venn diagram, which revealed 8 genera shared among E.o, AFB1, and AFB1 + E.o groups, respectively. A Venn
the groups (Fig. 3d, g-h). We analyzed the expression pat- diagram analysis identified 14 common DEMs (Fig. 4f ).
terns of these shared genera among the groups. Among Additionally, the network graph demonstrated the inter-
these, the relative abundances of Bacteroides, Parabac- actions among these 14 DEMs (Fig. 4g). We then consid-
teroides, Epulopiscium, Succinivibrio, and Desulfovibrio ered these 14 common DEMs as potential biomarkers
increased in the E.o and AFB1 + E.o groups and dropped and performed a network heatmap analysis to correlate
in the AFB1 group. Conversely, the relative abundances of them with eight gut microbiota markers and intestinal
Blautia and [Eubacterium] dropped in the AFB1 and E.o barrier-related indicators. As shown in Fig. 4h, the com-
groups, but increased in the AFB1 + E.o group (Fig. 3i). mon DEMs were significantly positively correlated with
Fecal metabolomics was employed to further inves- Bacterioides, [Eubacterium], Epulopiscium, Claudin1,
tigate the potential mechanisms underlying intestinal Claudin4, and Occludin, while they exhibited negative
barrier damage induced by the interaction between correlations with Blautia, Campylobacter, Parabacteri-
AFB1 and coccidia. A total of 19,492 metabolites were oides, Succinivibrio, Desulfovibrio, and ZO-1. In conclu-
identified using both combined positive and negative sion, these results indicate that exposure to AFB1 and
ion modes. These metabolites exhibited a distinct distri- coccidia induces intestinal barrier dysfunction by altering
bution across different groups, as illustrated in Fig. 4a. gut microbiota composition and interfering with intesti-
Additionally, differential metabolites were screened nal metabolism.
based on |log2FC|≥ 1 and p-value < 0.05, with the five
most significantly different metabolites were highlighted The combined effects of AFB1 and coccidia induce blood
in Fig. 4b. Compared to the Con group, the AFB1 group inflammation and hormonal alterations, concurrently
identified a total of 786 differential metabolites, compris- affecting blood metabolism
ing 366 upregulated and 420 downregulated metabolites We have concluded that AFB1 and coccidia disrupt the
(Table S3). These differential metabolites were mainly intestinal barrier by perturbing the gut microbiota and
enriched in pathways such as lipid metabolism, metabo- metabolites. However, the relationship between uter-
lism of cofactors and vitamins, and amino acid metabo- ine damage caused by AFB1 and coccidia and intestinal
lism (Fig. 4c). Compared to the Con group, the E.o group impairment remains unclear. Thus, we hypothesized
identified a total of 3204 differential metabolites, includ- that the destruction of the intestinal barrier may permit
ing 2886 upregulated and 318 downregulated metabolites enterotoxins to enter the bloodstream and cause cir-
(Table S4), which were mainly enriched in pathways such culatory inflammation [22, 23]. The expression levels of
(See figure on next page.)

Fig. 3 AFB1 and coccidia exposure altered the intestinal microorganisms of sheep. a Alpha diversity index. Chao1 and Observed species indices
characterize richness, while diversity is assessed using Shannon and Simpson indices. b Principal coordinates analysis (PCoA) was performed
to calculate beta diversity on a distance matrix of Bray–Curtis indices. c Changes of intestinal microbial composition at the phylum level (TOP 10).
d Abundance analysis of the gut microbiota at the genus level (TOP 20). e Relative abundance of Bacteroidetes, Firmicutes, and F/B ratio. f Linear
discriminant analysis effect size (LEfSe) comparison analysis between the groups. g Random Forests analysis (top 20 at the genus level). h Venn
diagram analysis of differential genera. The differential genera screened by the top 20 genera and the importance top 20 genera from Random
Forest analysis are presented in a Venn diagram, with the coincidence part indicating the potential biomarkers. i Relative abundance analysis
of common differential genera. The groups were compared by the Kruskal–Wallis nonparametric test, and the two groups were compared
by Dunn’s test. *p < 0.05, **p< 0.01, and ***p < 0.001 (AFB1, E.o, and AFB1+E.o vs Con); or #p< 0.05, ##p < 0.01, and ###p < 0.001 (AFB1+E.o vs AFB1
and E.o)(n = 4)
Fig. 3 (See legend on previous page.)

Fig. 4 AFB1 and coccidia exposure altered intestinal metabolites in sheep. a The distribution of the metabolites was analyzed using principal
component analysis (PCA). b Differentially expressed metabolites (DEMs) selected based on |log2FC| ≥ 1, p-value < 0.05 are represented by volcanic
plots. c–e The KEGG enrichment pathways of the DEMs. f Overlapped differential metabolites between groups (VIP > 1, p-value< 0.05). g Network
correlation diagram between overlapping DEMs. Nodes represent the proportion of each metabolite in each group; line thickness indicates
the strength of correlation, while color denotes the nature of the correlation: green for positive and red for negative. h Correlation heatmap
of overlapping DEMs with differential bacteria and intestinal barrier indices (Claudin-1, Claudin-4, Occludin, and ZO-1). Pearson correlation analysis
and Mantel test were utilized to assess the correlation coefficients (r-values) and significance (p-values). The heatmap reflects correlation values,
with deeper colors and larger lattice sizes indicating greater absolute correlation values. A closer affinity to blue signifies a stronger positive
correlation, while a shift towards red indicates a stronger negative correlation. Asterisk denote significance levels. The network diagram illustrates
the relationships between overlapping DEMs, differential genera, and intestinal barrier indices. Line thickness represents correlation strength;
thicker lines indicate stronger correlations. Line color reflects significance, with red indicating p-values< 0.01 (n = 4)
inflammatory factors and hormones in the blood were TNF-α (r = 0.699, p = 0.014), LPS (r = 0.664, p = 0.022),
first assessed. Routine blood analysis revealed an increase ERα (r = 0.706, p = 0.013), and ERβ (r = 0.685, p = 0.017),
in inflammatory cells in the blood induced by AFB1 and while there was a significant negative correlation with
E.o treatment (Fig. S4). Then, plasma was subjected to IL-10 expression (r = − 0.650, p = 0.025). In summary,
ELISA and metabolomic assays (Fig. 5a). The levels of disrupted gut microbiota and metabolites are responsi-
inflammatory factors IL-6, IL-10, TNF-α, and LPS in ble for the blood inflammation and hormonal alterations
plasma were measured using ELISA (Fig. 5b). The levels induced by AFB1 and coccidia, with amino acid metabo-
of pro-inflammatory factors IL-6 (p < 0.001 and p = 0.007, lism potentially being a major contributing factor.
respectively), TNF-α (p < 0.001 and p = 0.001, respec-
tively), and LPS (p = 0.002 and p = 0.019, respectively) The combined effects of AFB1 and coccidia damage
were significantly elevated in the AFB1 and AFB1 + E.o the uterus by regulating the PI3K/AKT/eNOS pathway
treatment groups, while the expression level of the anti- Next, we explored the mechanisms of direct dam-
inflammatory factor IL-10 exhibited an opposite trend age caused by AFB1 and coccidia in the uterus through
(p < 0.001 and p = 0.002, respectively). Furthermore, we uterine transcriptome analysis. Differentially expressed
also measured the levels of estrogen receptors (ER) in genes (DEGs) were selected based on |log2FC|≥ 1 and
plasma. Compared to the Con group, the expression lev- p-value < 0.05, and these were illustrated using volcano
els of ERα (p < 0.001 and p < 0.001, respectively) and ERβ plots, with the top five significantly altered genes labeled
(p = 0.005 and p = 0.009, respectively) were significantly (Fig. 6a). Compared to the Con group, the AFB1 group
increased in the AFB1 and AFB1 + E.o groups (Fig. 5c). exhibited a total of 260 DEGs, with 97 upregulated and
Therefore, we speculate that uterine damage may result 163 downregulated (Table S9). The top five DEGs in this
from altered intestinal function, allowing endotoxins to group were COL1A2, NID2, LOC101120084, DNAJC22,
enter the bloodstream and leading to a series of meta- and LOC101104199. Similarly, there were 1442 DEGs,
bolic abnormalities. with 888 upregulated and 554 downregulated in the
Subsequently, changes in plasma metabolites were E.o group, and the top 5 DEGs were CCN1, HDAC11,
detected through comprehensive non-targeted metab- LOC101120084, ALDHIL1, and LOC105605766
olomics. The differences in metabolites between (Table S10). There were 884 DEGs, with 321 upregulated
groups were illustrated in the OPLS-DA score plot and 563 downregulated in the AFB1 + E.o group, and the
(Fig. 5d). DEMs were selected based on |log2FC|≥ 1 top 5 DEGs were LUM, SYNC, AQP1, MAMDC2, and
and p-value < 0.05, with the top five significantly dif- COL14A1 (Table S11). A Venn diagram was then used
ferent metabolites were labeled (Fig. S5). Compared to to illustrate the overlap of 84 common DEGs (Fig. 6b),
the Con group, the AFB1 group exhibited a total of 402 which were further categorized into different clusters and
DEMs, with 235 upregulated and 167 downregulated visually presented through a heatmap (Table S12; Fig. 6c).
(Table S6). The E.o group showed a total of 978 DEMs, Additionally, a Sankey bubble chart was used to display
with 325 upregulated and 653 downregulated (Table S7). the significant enrichment of 12 pathways along with the
The AFB1 + E.o group had a total of 1,211 DEMs, with corresponding DEGs enriched in each pathway (Fig. 6d).
874 upregulated and 337 downregulated (Table S8). In this study, we specifically focused on the relaxin sign-
Additionally, KEGG enrichment analysis revealed that aling pathway and validated the expression levels of four
the three comparison groups were mainly enriched in the enriched DEGs, COL11A1, COL4A4, FOSB, and GNG2
amino acid metabolism pathway (Fig. 5e). Therefore, we (Fig. 6e). Compared to the Con group and the AFB1 + E.o
selected metabolites enriched in this pathway and dem- group, exposure to AFB1 showed a significantly upregu-
onstrated their distribution among the groups (Fig. 5f ). lated in the expressions of COL11A1 (p = 0.002 and
Furthermore, the differential expression analysis of these p < 0.001, respectively) and COL4A4 (p = 0.044 and
metabolites between groups is shown in Fig. 5g. Com- p = 0.015, respectively). Furthermore, exposure to both
pared to the Con group, the levels of 3-dehydroquinic AFB1 and coccidia significantly increased the mRNA
acid, succinate, and N-acetyl-L-phenylalanine showed an level of GNG2 compared to the Con group (p = 0.002).
increasing trend in the AFB1, E.o, and AFB1 + E.o groups, However, the mRNA level of GNG2 was significantly
while the levels of indole, serine, L-methionine, O-acetyl- downregulated in the AFB1 and coccidia single expo-
L-serine, 3-hydroxyphenylacetic acid, and phenaceturic sure compared to the AFB1 + E.o group (p = 0.019 and
acid were significantly decreased. Next, we conducted p = 0.002, respectively). No significant change in the
a correlation analysis between these shared DEMs and expression of FOSB was observed following AFB1 and
blood inflammatory factors as well as hormone-related coccidia exposure. The KEGG map illustrates the impor-
indicators (Fig. 5h). N-acetyl-L-phenylalanine showed a tant downstream pathways regulated by the relaxin sign-
strong positive correlation with IL-6 (r = 0.580, p = 0.050), aling pathway, including the PI3K/AKT/eNOS pathway,
Fig. 5 Amino acid metabolism plays a role in blood inflammation and hormonal changes during the AFB1 and coccidia exposure. a Schematic
representation of the experimental design and program diagram. b Expression levels of inflammatory factors IL-6, IL-10, TNF-α and LPS. c Expression
levels of estrogen receptors ERα and ERβ (n = 3). d OPLS-DA score plots for the Con and AFB1, E.o and AFB1+E.o groups. e KEGG enrichment
pathway analysis of DEMs. f Expression of 10 metabolites enriched in the amino acid metabolic pathways in different groups. A, 3-dehydroquinic
acid. B, indole. C, serine. D, L-methionine. E, O-acetyl-l-serine. F, succinate. G, ephedrine. H, 3-hydroxyphenylacetic acid. I, N-acetyl-l-phenylalanine.
J, phenaceturic acid. g Differential analysis of the ten metabolites enriched in amino acid metabolic pathways. Groups were compared using
the Kruskal–Wallis nonparametric test, and the two groups were compared using Dunn’s test. h Association analysis of the ten metabolites
with inflammatory factors and blood hormones. Statistical significance was indicated by *p < 0.05, **p< 0.01, and ***p < 0.001 (AFB1, E.o,
and AFB1+E.o vs Con); or #p< 0.05, ##p < 0.01, and ###p < 0.001 (AFB1+E.o vs AFB1 and E.o)(n = 4)
which is known to regulate cell proliferation and differen- cells). Additionally, the metabolites themselves exert
tiation and is involved in the development of various dis- toxic effects on the reproductive system through their
eases (Fig S6; Fig. 6f ). The results indicated that exposure biotransformation reactions [22, 23]. Therefore, we con-
to AFB1 and coccidia activated the PI3K (Con vs AFB1, clude that co-exposure to AFB1 and coccidia compro-
p = 0.010; Con vs E.o, p = 0.002), AKT (Con vs AFB1, mises intestinal barrier function and triggers systemic
p = 0.002), and eNOS (Con vs AFB1, p < 0.001; Con vs inflammation through alterations in gut microbiota-
AFB1 + E.o, p < 0.001) genes in the uterus (Fig. 6g). These derived metabolites, thereby directly and/or indirectly
findings suggest that the occurrence of uterine pathologi- affecting reproductive system function.
cal inflammation is regulated by the PI3K/AKT/eNOS Due to the high prevalence of mycotoxins in grain feed
pathway, and changes in hormone levels (ERα and ERβ) and animal feed, investigating the reproductive toxic-
also contribute to uterine damage. ity caused by these toxins in animals is essential. Studies
have shown that high doses of mycotoxins consumed by
Discussion female animals can cause reproductive toxicity, leading to
In this study, we utilized a livestock infection model to reduced fertility and even miscarriage [18, 19]. In addi-
investigate the mechanisms of uterine injury caused by tion, under intensive breeding conditions characterized
individual and mixed infections of AFB1 and coccidia by high animal density and productivity, coccidiosis can
(see Fig. 7). The results showed that the toxicity induced become a significant economic concern in small rumi-
by AFB1 and coccidia initially disrupts the gut microbi- nants. Therefore, we utilized female sheep as an animal
ota and metabolites, leading to increased production of model to examine the effects of AFB1 and coccidia indi-
LPS due to the predominance of Gram-negative bacteria, vidually or in combination on the reproductive system.
including Bacterioides, Parabacterioides, Epulopiscium, Our clinical characterization data, changes in feed intake,
Succinivibrio, Desulfovibrio, Blautia, and Campylobacter. body weight, and uterine morphology confirmed the det-
This gut dysbiosis and altered metabolism compromise rimental effects of AFB1 and coccidia on reproductive
intestinal barrier function, which exacerbates systemic health. Subsequently, the mechanisms underlying the
inflammation in conjunction with elevated plasma LPS reproductive damage caused by AFB1 and coccidia were
levels and hormonal changes. Moreover, the systemic further elucidated through the application of fecal 16S
inflammation and hormonal alterations induced by AFB1 rRNA sequencing, fecal metabolomics, blood metabo-
and coccidia contribute to the development of inflamma- lomics, and uterine transcriptomics techniques, during
tory lesions in the reproductive system, while also trig- which several potential biomarkers were identified.
gering the relaxin signaling pathway and activating the First, the results of the 16S rRNA sequencing revealed
PI3K/AKT/eNOS pathway, resulting in the upregulation that under the influence of AFB1 and coccidia factors,
of relevant gene expression levels. In summary, AFB1 the relative abundance of Firmicutes, which is mainly
is initially absorbed and metabolized in the intestine (a influenced by coccidia factor, decreased under the com-
common route of exposure to this toxin), which is also bined effects of AFB1 and coccidia. In contrast, the
a target organ for coccidia parasitism. Damage to gut relative abundance of Bacteroides was increased. Firmi-
function and alterations in microorganisms and metabo- cutes are considered important metabolic players in the
lites (such as short-chain fatty acids, bile acids, etc.) are gut, as they can break down complex sugars, polysac-
directly translocated to reproductive organs through sys- charides, and fatty acids from feed, producing energy
temic circulation (intestinal mucosal cells or red blood and nutrients for absorption and utilization by the host
(See figure on next page.)

Fig. 6 AFB1 and coccidia exposure induced uterine damage through the relaxin signaling pathway. a Differentially expressed genes (DEGs)
were identified using criteria of |log2FC| ≥ 1 and p-value < 0.05, and are represented in volcano plots. b Overlapped DEGs between groups (VIP
> 1, p-value < 0.05). c A trend heatmap illustrates the expression status and trend of DEGs. The mfuzz algorithm was utilized to classify 84 DEGs
into different clusters, and then the heat map+each cluster line map was drawn to visually display the trend. d The Sankey bubble map displays
12 significantly different pathways and their enriched DEGs. The left side is the Sankey diagram, indicating the genes within each pathway. The
right side features the bubble diagram. The bubble size represents the number of genes that the pathway belongs to, and the bubble color
represents the p-value. e Expression levels of COL11A1, COL4A4, FOSB, and GNG2 genes (n= 4). f The activation of the PI3K/AKT/eNOS pathway
by relaxin signaling pathway. Relaxin signal binds to the receptor relaxin/insulin-like family peptide receptor 1 (RXFP1), which first interacts
with Gαs to activate adenylate cyclase (AC), resulting in increased cyclic adenosine monophosphate (cAMP) production, while also negatively
regulating through interaction with GαoB. Subsequently, RXFP1 associates with Gαi3 to activate the PI3K/AKT signaling pathway, leading to their
phosphorylation and subsequent activation of eNOS. g Gene expression levels of PI3K, AKT and eNOS (n = 3). Statistical significance is indicated
by *p < 0.05, **p< 0.01, and ***p < 0.001 (AFB1, E.o, and AFB1+E.o vs Con); or #p< 0.05, ##p < 0.01, and ###p < 0.001 (AFB1+E.o vs AFB1 and E.o)
Fig. 6 (See legend on previous page.)
[28]. Additionally, they play a crucial role in restor- considerably increases the abundance of the phylum
ing gut homeostasis as well as inhibiting or eradicat- Bacterioides, which can further damage intestinal epi-
ing gas-producing clostridia [29]. Coccidian infection thelial cells and enhance the invasion of other pathogens,
Fig. 7 A schematic diagram of the effects of AFB1 and coccidia exposure on the reproductive system of sheep through the gut–blood–
reproductive axis
thereby initiating or exacerbating enteritis [30]. There- opportunistic pathogens, which can induce or exacerbate
fore, we speculate that the decrease in Firmicutes and intestinal inflammation, and impair intestinal barrier
the increase in Bacterioides may be due to secondary function. This is supported by histopathological observa-
infections caused by coccidia. The microbiome, which tions of intestinal tissue and the expression of intestinal
includes bacteria, viruses, fungi, protozoa, and parasites, barrier-related genes. Additionally, both gut metabolites
plays a critical role in host health through symbiotic, and the microbiota play an important role in maintain-
pathogenic, or parasitic relationships [31, 32]. Numer- ing gut homeostasis, and disruption of intestinal barrier
ous studies have demonstrated complex interactions function is inevitably correlated with these metabolites.
between coccidia and other intestinal parasites, fungi, Through metabolomic screening, 14 common DEMs
or bacteria [33–36], which may result in more severe were identified under the individual or combined action
clinical manifestations in the host. Furthermore, fungal of AFB1 and coccidia. Correlation analysis with micro-
toxins have been proposed to negatively affect the cellu- biota markers and intestinal barrier-related indicators
lar immune response to parasites [34], and the reduced revealed significant positive correlations with Bacte-
microbial diversity observed under the combined action roides, [Eubacterium], Epulopiscium, Claudin1, Claudin4,
of AFB1 and coccidia may contribute to this possibility. and Occludin. These findings suggest that the disruption
Furthermore, we further screened out eight common of the intestinal barrier caused by AFB1 and coccidia is
genera, Bacteroides, Parabacteroides, Epulopiscium, Suc- mediated through the interference of microbial commu-
cinivibrio, Desulfovibrio, [Eubacterium], Blautia, and nities and metabolites.
Campylobact, most of them are gram-negative bacteria, Previous research has shown that gut microbial
which likely create favorable conditions for the produc- metabolites also affect various physiological functions
tion of LPS. These findings indicate that AFB1 and coc- of other visceral organs and participate in mediating
cidia, either alone or in combination, significantly alter the pathogenesis of different diseases in these organs
the gut microbiota, resulting in the enrichment of some [37–41]. After ingestion and absorption in the intestine,
mycotoxins mainly undergo biotransformation and enhanced amino acid metabolism in the blood and expo-
metabolism via bile, urine, plasma, liver, and uterus and sure to AFB1 and coccidia remains unclear. Consistent
may be transferred through the intestinal mucosa or with our findings, previous studies have demonstrated
red blood cells [22, 23]. Therefore, we speculate that in that exposure to AFB1 or co-exposure with other toxins
the case of a compromised intestinal barrier, microbial- can disrupt amino acid metabolism and affect cellular
derived LPS and their related molecules can enter the redox homeostasis [56–58]. Additionally, parasitic infec-
bloodstream and transfer to the reproductive system, tions have been demonstrated to induce the production
potentially leading to uterine damage and inflamma- of amino acid metabolites with antimicrobial and antivi-
tion. Increased levels of white blood cells, IL-6, TNF-α, ral properties, inhibiting the growth and reproduction of
and LPS expression in plasma indicate the presence of parasites [59]. Furthermore, amino acid metabolites can
inflammation in the body. Estrogens are known to be regulate the function of host immune cells and inflam-
regulators of immune function in the monocyte–mac- matory responses. For instance, L-arginine is a key amino
rophage system [42], playing a critical role in suppress- acid required for nitric oxide (NO)-mediated parasite
ing cytokine production [43–46]. Transcripts of estrogen killing and polyamine-mediated parasite reproduction
receptors ERα and ERβ have been identified in human within host target cells [60, 61]. It can activate phago-
macrophages and monocytes derived from original cytes to produce NO and oxidative intermediates, exhib-
primitive hematopoietic progenitor cells [47]. IL-6 is iting cytotoxic activity against intracellular pathogens. In
one of the main cytokines involved in monocyte func- combination with our findings, the alterations in blood
tion related to chronic inflammation. It is established that metabolism provide a fundamental mechanistic expla-
17β-estradiol (E2) can inhibit the expression of TNF-α, nation for the impairment of intestinal barrier function
IL-1, and IL-6 [48]. Additionally, in macrophages with induced by AFB1 and coccidia, with amino acid metabo-
ERα deficiency, the release of LPS-induced TNF-α is sig- lism being involved in systemic inflammation and disrup-
nificantly increased, suggesting that ERα plays a promi- tion of the reproductive system.
nent role in mediating the anti-inflammatory effects of We have elucidated the direct mechanisms underly-
estrogen [49]. Our results demonstrate that the expres- ing uterine injury induced by the separate or combined
sion of ERα and ERβ in plasma is significantly increased actions of AFB1 and coccidia through uterine tran-
under the dominant influence of AFB1, indicating a posi- scriptomics. KEGG enrichment analysis highlighted
tive role of these receptors ERα and ERβ in suppressing the relaxin signaling pathway and identified four DEGs
AFB1 and coccidia-induced blood inflammation. Addi- enriched in this pathway, namely COL11A1, COL4A4,
tionally, through blood metabolomics, we enriched the FOSB, and GNG2. COL11A1 and COL4A4 play sup-
amino acid metabolism pathway and identified N-acetyl- portive and connective roles in the extracellular matrix.
L-phenylalanine as a highly correlated metabolite in this Due to their low expression in normal tissues, changes
pathway. Amino acid metabolism is closely associated in COL11A1 expression are considered excellent prog-
with inflammation [50]. On the one hand, amino acid nostic markers for disease progression [62–64]. FOSB
metabolites are involved in regulating the body’s inflam- is involved in regulating gene transcription and is asso-
matory response, such as arginine can inhibit the activity ciated with the regulation of cell proliferation, differ-
of key enzymes in inflammatory cells, such as eNOS and entiation, apoptosis, and transformation [65]. GNG2
cyclooxygenase, thereby suppressing the synthesis and participates in multiple cell signaling pathways, playing a
release of inflammatory factors [51]. On the other hand, crucial role in cell proliferation and angiogenesis, and is
amino acid metabolism significantly impacts the func- a potential molecular target for the treatment of various
tion of immune cells. During inflammation, immune cells diseases [66–68]. Our results demonstrated that AFB1
such as macrophages and T cells have increased meta- exposure significantly upregulated the expression of the
bolic demands, requiring more amino acids for energy COL11A1 and COL4A4 genes in the uterus, while the
and synthesis. An insufficient supply of some essential combined action of AFB1 and E.o significantly upregu-
amino acids during the inflammatory state may impair lated the expression of the GNG2 gene, explaining the
various immune cell functions, including cell prolifera- activation of uterine relaxin signaling pathway primarily
tion, cell signaling, and cytotoxicity [52, 53]. Moreover, it driven by AFB1 exposure.
is interesting to note that the supplementation of dietary Furthermore, the downstream genes regulated by the
amino acids can activate unliganded ERα in the liver, PI3K/AKT/eNOS signaling pathway can regulate various
with this activation pathway relying on mTOR signal- biological processes, including cell survival, proliferation,
ing to regulate uterine growth [54, 55]. This may explain and differentiation. NO is an important signaling mol-
the correlation between amino acid metabolism and ecule produced by endothelial cells, which can induce
estrogen receptors. However, the relationship between smooth muscle relaxation. NO activates eNOS to
convert L-arginine to L-cyclic guanosine monophos- demonstrated that the effects were not solely driven by
phate (cGMP), promoting vasodilation and relaxation individual pathogenic factors, and our objective was to
of blood vessels [69]. In addition, the PI3K/AKT path- observe the impact of both pathogenic factors on the
way, when activated, can enhance the production of NO reproductive system. We observed a synergistic effect
in endothelial cells, leading to the phosphorylation and of AFB1 and coccidia on the expression levels of uterine
activation of eNOS, regulating vasodilation and hemo- IL-10, TNF-α, AKT, and COL11A1, as well as intestinal
dynamics [70, 71]. Specifically, PI3K activates the AKT Claudin1 and Occludin gene levels, along with IL-6 and
signaling molecule, which phosphorylates serine1177 of TNF-α levels in plasma. Moreover, it is undeniable that
eNOS, enhancing its enzymatic activity and maintain- AFB1 and coccidia as two pathogenic factors can cause
ing the anti-adhesive and anti-inflammatory proper- significant damage to animal health. Exploring the inter-
ties of endothelial cells [72]. Moreover, the PI3K/AKT action between these two factors was a focus of our study
pathway is implicated in AFB1-induced animal toxic- before its initiation. Unfortunately, the findings indicated
ity, as AFB1 activates the PI3K/AKT/mTOR signaling that the interaction between AFB1 and coccidia was
pathway to induce testicular oxidative stress in mice not optimal, possibly due to substantial individual dif-
[73, 74]. Furthermore, the activation of the PI3K/AKT ferences among large animals. In addition, our research
signaling pathway has been demonstrated to inhibit the results cannot conclusively determine the causal contri-
increased apoptosis of cecal epithelial cells caused by E. butions of the gut microbiota and their corresponding
tenella infection [75]. Additionally, proteomic analysis metabolites in relation to AFB1 and coccidia. Therefore,
has revealed that differential protein expression in cecal potential biomarkers identified require further validation
epithelial cells infected with E. tenella is also associated through more direct experimental evidence, such as gut
with the PI3K/AKT pathway [76]. Our data are consist- microbiota transplantation studies and intervention stud-
ent with the aforementioned findings, as the PI3K/AKT ies on differential metabolites. In summary, AFB1 and
pathway is activated in response to AFB1 and coccidia coccidia exhibit toxic effects on the reproductive system
treatments, enhancing the enzymatic activity of eNOS in sheep, mediated by the disruption of intestinal barrier
and regulating the proliferation of endometrial epithelial function, alterations in gut microbiota-derived metabo-
cells. The inflammatory damage to the uterus caused by lites, and the induction of systemic inflammation, all of
AFB1 and coccidia, as demonstrated in pathological sec- which directly and/or indirectly interfere with reproduc-
tions of uterine tissue and the expression of inflamma- tive function.
tory factors IL-6, IL-10, and TNF-α, is regulated by the
PI3K/AKT/eNOS pathway, as currently discovered. Con-
sidering the reproductive toxicity of AFB1 and coccidia, Conclusions
one potential endocrine-disrupting effect of their metab- In summary, our research findings highlight a potential
olites may be related to the disruption of pituitary hor- mechanism linking gut microbiota-derived metabolites,
mone production [77]. AFB1 can induce oxidative stress, such as LPS, with compromised intestinal–reproduc-
apoptosis, and interfere with testicular function in sheep, tive axis defense induced by AFB1 and coccidia through
damaging the hypothalamic–pituitary–gonadal axis and systemic inflammation. Thus, this comprehensive study
causing hormonal imbalance in rats [13, 78–80]. ERα and reveals a novel toxic mechanism of action for AFB1 and
ERβ are two subtypes of estrogen receptors that regulate coccidia in the gut–blood–reproductive axis and suggests
reproductive activities in female animals. ERα is mainly that regulating gut microbiota and its metabolites could
involved in the regulation of endometrial proliferation be a promising strategy to mitigate potential threats to
and angiogenesis, while ERβ is responsible for endome- farm animals and humans through dietary modifications.
trial differentiation and regeneration [81]. In this study,
the downregulation of ERα and ERβ expression under the Supplementary Information
combined influence of AFB1 and E.o demonstrates the The online version contains supplementary material available at https://doi.
role of estrogen receptors in mediating uterine damage. org/10.1186/s40168-024-01966-y.
Furthermore, supplementation with L-arginine has been
Supplementary Material 1.
shown to increase ERα expression in the endometrium
of non-pregnant sheep, highlighting the crucial role of
amino acid metabolism in the regulation of steroid hor- Supplementary Material 3.
mone receptor expression in the endometrium [82]. Supplementary Material 4.

This study investigated the individual and combined Supplementary Material 5.
effects of AFB1 and coccidia on the reproductive sys- Supplementary Material 6.
tem using a two-factor analysis of variance. The results Supplementary Material 7.
7. Peng WX, Marchal JLM, van der Poel AFB. Strategies to prevent and
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Ethics approval and consent to participate and cell apoptosis in mice kidney by curcumin. Antioxidants (Basel).
All sheep experiments adhered to the animal welfare guidelines of Henan 2022;11(6):1082.
Agricultural University (ethical permission code: HNND2022030818) in Zheng- 18. Hu LL, Chen S, Shen MY, Huang QY, Li HG, Sun SC, Luo XQ, et al. Aflatoxin
zhou, China. B1 impairs porcine oocyte quality via disturbing intracellular membrane
system and ATP production. Ecotoxicol Environ Saf. 2023;263:115213.
Consent for publication 19. Yaacobi-Artzi S, Kalo D, Roth Z. Effect of the aflatoxins B1 and M1 on
Not applicable. bovine oocyte developmental competence and embryo morphokinetics.
Reprod Toxicol. 2023;120:108437.
Competing interests 20. Zeng Y, Zeng D, Zhang Y, Ni XQ, Wang J, Jian P, Jing B, et al. Lactobacillus
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exposed to aflatoxin B1. J Anim Physiol Anim Nutr. 2018;102(1):e449–59.
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Huang et al.

Microbiome (2024) 12:269 Microbiome

RESEARCH Open Access

New insights into the combined effects

(See figure on next page.)

Fig. 3 (See legend on previous page.)

(See figure on next page.)

Fig. 6 (See legend on previous page.)

mone receptor expression in the endometrium [82]. Supplementary Material 4.

and the effectiveness of Celmanax®/Dairyman’s Choice™ applications to

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