Magnolol mitigates polystyrene microplastic-induced oviductal toxicity in laying hens via immunomodulation and microbiota regulation

Abstract

Environmental contamination with polystyrene microplastics (PS) has been shown to trigger inflammation and impair reproductive function in animals. Magnolol (MAG) is a natural compound with anti-inflammatory properties. However, the protective role of MAG against PS-induced oviductal damage remains unclear. This study aimed to investigate the protective effects and underlying mechanisms of dietary MAG supplementation in laying hens exposed to PS. A total of 270 Hy-Line white hens were randomly assigned to three groups: control group (CK), PS-exposed group (PS), and MAG protective group against PS exposure (PM). Hens in the PM group were fed a MAG-supplemented diet, while hens in the CK and PS groups received a basal diet. After eight weeks of dietary treatment, hens in PS and PM groups were treated with 1 mg/mL PS by gavage, and the CK group received phosphate-buffered saline for four weeks. The results revealed that PS exposure significantly reduced albumen quality and eggshell strength, which was associated with decreased abundance of the beneficial bacterium Lactobacillus crispatus, disrupted mucosal barrier integrity, enhanced oxidative stress, and increased inflammation and apoptosis in the oviduct. MAG supplementation alleviated these adverse effects by improving antioxidant status, reducing mucosal inflammation and apoptosis, enhancing epithelial barrier markers, and restoring the microbial balance in the oviduct. In vitro cell culture experiments also confirmed MAG could inhibit macrophage M1-type polarization via suppressing NF-κB signaling pathway activation. Consequently, MAG reduced mucosal inflammation and apoptosis, enhanced the expression of epithelial barrier markers (Claudin and MUC-2), and restored the microbial balance in the oviduct. These findings demonstrate that dietary MAG mitigates PS-induced oviductal damage through immunomodulation and microbiota regulation, ultimately improving reproductive performance in laying hens. This study highlights MAG as a promising dietary strategy to counteract microplastic-induced reproductive toxicity in poultry.

Introduction

Polystyrene microplastics (PS) are among the most prevalent types of plastic materials. PS can be absorbed and accumulated in food plants and livestock, posing numerous environmental and biological risks (Auneer Ahmad, 2023; Aziz et al., 2021; Matsumoto et al., 2023; Senathirajah et al., 2021). Accumulating evidence further indicates that PS particles can induce multi-organ toxicity through oxidative stress- and inflammation-related mechanisms. In chickens, PS-MP exposure has been reported to induce pulmonary inflammation through endoplasmic reticulum stress-associated NLRP3 inflammasome activation (Lu, H. et al., 2024). In aquatic cells, PS-MPs can promote ROS accumulation, leading to cell cycle arrest, apoptosis, and autophagy(Lu, Hongmin et al., 2024). Moreover, PS-MPs have been shown to promote liver inflammation by inducing macrophage extracellular trap formation(Yin et al., 2023a). These findings suggest that oxidative stress, inflammatory activation, and immune-cell dysfunction are central pathological events in PS-induced toxicity. Crucially, throughout the egg production cycle, laying hens are continuously exposed to various PS-derived materials, including feed bags, cages, and water pipes, which serve as persistent sources of microplastic contamination (Wu et al., 2021).There were studies found that microplastics could cause significant damage to testicular tissue and induce oxidative stress and inflammatory cell infiltration in chickens. However, research on female poultry—particularly regarding the effects of PS on oviductal structure, mucosal immunity, and egg production—remains severely limited, highlighting an urgent need to evaluate these impacts in laying hens.

The reproductive tract of female poultry is not the site for embryo development but the formation of each compartment of eggs, including egg yolk (ovary), albumen (magnum), eggshell membrane (isthmus), and eggshell (uterine) (Wen et al., 2021).To maintain oviductal health and ensure high-quality egg production, an intact mucosal barrier—comprising tight junctions, mucus, immune cells, and normal bacterial flora—is essential (Wang et al., 2016).It has been suggested that the microbiota of the reproductive tract has a greater impact on egg production than the microbiota of the digestive tract of laying hens (Su et al., 2021b). Concurrently, macrophages are important helper cells for reproductive function and are found in abundance in the reproductive tract of both males and females (Cohen et al., 1999).Under environmental stress, PS particles can be internalized by macrophages, accumulating in lysosomes and triggering oxidative and inflammatory stress (Das, 2023; Jasinski et al., 2023).This disruption promotes classical macrophage reprogramming (M1 polarization), leading to the massive secretion of pro-inflammatory cytokines (such as IL-1β, IL-18, TNF-α, and IL-6) via the NF-κB and MAPK pathways (Hegarty et al., 2023; Liu et al., 2014; Pan et al., 2022). In chronic salpingitis, these infiltrating M1 macrophages exacerbate mucosal barrier damage and tissue remodeling. Therefore, regulating oviductal microbiota composition and modulating macrophage polarization are vital strategies to prevent PS-induced mucosal damage (Wang et al., 2020); (Liao et al., 2024).

Magnolol (MAG) is a hydroxybenzene derivative obtained through extraction from the roots and bark of Magnolia officinalis trees (Borgonetti et al., 2021), which is utilized as a dietary supplement due to its extensive range of pharmacological effects (Sarrica et al., 2018) and potent anti-inflammatory activities (Wijesuriya and Lappas, 2018). In vitro studies have proven that MAG can inhibit the nuclear translocation and phosphorylation of the NF-κB p65 subunit, thereby inhibiting the activation of the NF-κB signaling pathway and exerting anti-inflammatory effects (Tse et al., 2007). Moreover, in vivo studies have shown that MAG regulates macrophage polarization by activating peroxisome proliferator-activated receptor γ and modulates the expression of M1 marker genes to alleviate diet-induced non-alcoholic steatohepatitis (Zhong and Liu, 2018). In poultry production, dietary supplementation is the most practical delivery route for such functional additives. Our recent in vivo dose-optimization study evaluated a gradient of dietary MAG (200, 400, and 600 mg/kg) in laying hens (Lv et al., 2024). The results demonstrated that supplementation at 400 mg/kg provided the optimal balance of efficacy in ameliorating mucosal barrier damage and modulating local immune responses without any adverse effects.

Building upon its well-documented mucosal-protective properties and established safe dosage profiles, we hypothesized that supplementing MAG at this optimized dose (400 mg/kg) would similarly exert protective effects on the oviductal mucosa against microplastic-induced toxicity. Therefore, the present study aimed to systematically investigate the effects and underlying mechanisms of dietary MAG supplementation on PS-induced oviductal damage, laying performance decline, and inflammatory responses in laying hens, with a specific focus on the modulation of macrophage polarization and oviductal microbiota.

Materials and methods

Materials

The polystyrene microplastics (PS) were purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China; cat. no. 9003-53-6). The stock suspension had a concentration of 2.5% w/v. To ensure the accuracy of the experimental material, the morphological characteristics and particle size were verified using scanning electron microscopy (SEM) (Fig. S1A). The particles exhibited a spherical morphology with high monodispersity, and the average particle diameter was 75.50 ± 5.38 nm (N = 65). Based on their nanoscale size, the PS particles used in this study are referred to as nano-sized PS particles where appropriate. Magnolol (MAG) was purchased from Huawei Ruike Chemical Technology Co. Ltd. (Beijing, China), which was obtained from the bark of Magnolia officinalis with 80% purity (Reagent Trademark Note).

Cell culture

The avian macrophage cell line HD11 was obtained from Procell Life Science & Technology Co., Ltd. The cell line was authenticated using Short Tandem Repeat (STR) profiling to confirm its identity and rule out cross-contamination. Furthermore, the cells were routinely screened and confirmed to be negative for mycoplasma contamination prior to all in vitro experiments. The HD11 chicken macrophage cells were grown in RPMI1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C in a humidified incubator with 5% CO₂. Cell passaging was performed before reaching 80-90% confluence.

For in vitro treatments, HD11 cells were seeded and treated when the cell density reached approximately 60% confluence. Cells were divided into three groups: control, PS, and MAG+PS. The PS group was treated with 200 μg/mL PS for 12 h to establish a stable inflammatory activation model, while the MAG+PS group was treated with 50 μM MAG together with 200 μg/mL PS for 12 h. MAG was used at 50 μM based on previous macrophage-related anti-inflammatory studies and our preliminary observations showing no obvious cytotoxicity under this condition. These concentrations were selected to evaluate whether MAG could directly modulate PS-induced macrophage inflammatory activation. For in vitro cellular experiments, Magnolol was initially dissolved in dimethyl sulfoxide (DMSO) to create a stock solution and subsequently diluted with the complete culture medium to the target working concentrations. The final concentration of DMSO in the culture medium was strictly maintained below 0.1% (v/v) to avoid any solvent-induced cytotoxicity. Control cells were treated with an equivalent volume of the DMSO vehicle (Miao et al., 2024).

Experimental design and diet

The Institutional Animal Care and Use Committee of Zhejiang University authorized the animal study protocols (No. ZJU20220310). A total of 270 Hy-Line white laying hens (37 weeks old) were obtained from a commercial farm and randomly allocated into three experimental groups (Table S1): the control group (CK), the polystyrene-exposed group (PS), and the Magnolol-protected group (PM). Each group consisted of 6 replicates with 15 hens per replicate (Zeng et al., 2026). Hens in the CK and PS groups were fed a basal diet, while hens in the PM group were fed a basal diet supplemented 400 mg/kg MAG throughout the entire 12-week experimental period. To ensure the homogeneity of the diet, MAG was incorporated into the feed using a stepwise expansion method (geometric dilution technique). Briefly, the MAG powder was first pre-mixed with a small amount of corn starch carrier, then blended with the premix, and finally mixed thoroughly with the basal diet using a vertical mixer. The optimal feeding dose has been confirmed by our previous study (Lv et al., 2024). In addition, an independent MAG-only feeding experiment was included in the revised Supplementary Material to support the safety and dose selection of dietary MAG supplementation (Supplementary Fig. S2). In that experiment, 37-week-old Hy-Line White laying hens were assigned to CK, MAG200, MAG400, and MAG600 groups, with 15 hens per group. Hens were fed basal diets supplemented with 0, 200, 400, or 600 mg/kg MAG for 12 weeks, respectively. At the end of the experiment, 6 hens per group were sampled for liver morphology and serum biochemical analyses. The formulated basal diet (Table S2) either met or surpassed the nutritional requirements recommended by the National Research Council (NRC, 1994). After the first 8 weeks of dietary treatment, hens in the PS and PM groups were orally gavaged with 1 mg/mL PS suspension at 5 mL/day for an additional 4 weeks, whereas hens in the CK group received an equal volume of PBS. Therefore, MAG supplementation was maintained during the PS exposure period in the PM group. The exposure dose of PS was chosen based on previous toxicological studies of microplastics in avian animals (Chen et al., 2023; Guo et al., 2024; Yin et al., 2023b). Environmental conditions within the cages were precisely monitored, providing the hens with constant access to water and dietary intake.

Collections and preparation of samples

At the conclusion of the 12-week trial, six hens with representative body weights (near the treatment average) were selected per group and humanely euthanized via cervical dislocation. Oviductal tissues, specifically the magnum and uterus, were collected; representative segments were fixed in either 4% formaldehyde or 2.5% glutaraldehyde, while the remaining portions were snap-frozen and stored for subsequent molecular analysis (Wang et al., 2022).

Determination of egg internal and external qualities

During the 12-week experimental period, egg production was recorded daily along with egg breakage rate for each group. Egg breakage rate (%) = (number of broken eggs / total eggs) × 100%. Egg quality parameters were assessed at the end of the 12-week trial period to determine egg quality as previously described (Liu et al., 2021b). To evaluate egg quality, 36 freshly laid eggs per group (six eggs per replicate) were harvested and analyzed within a 24-hour window. A digital vernier caliper was employed to measure the dimensions required for calculating the egg shape index. Whole weight, eggshell strength, albumen height, Haugh unit, and yolk color were determined using an egg quality tester (DET-6000; Nabel Co., Ltd., Kyoto, Japan). Eggshell thickness (without shell membrane) was measured at 3 points (tip, blunt end, and equator) using an Egg Shell Thickness Gauge (ESTG-1, Orka Food Technology Ltd., Ramat Hasharon, Israel) and was estimated for each egg based on the average of the 3 points was estimated (Fu et al., 2024).

Scanning electron microscope (SEM)

Oviductal uterine segments were rinsed with chilled saline and immersion-fixed in 2.5% glutaraldehyde (4°C). Following a graded ethanol dehydration series, samples underwent critical point drying and gold sputter-coating before observation under a Hitachi S-4800 field emission SEM. For shell ultrastructure, equatorial fragments were examined; morphological parameters, including mammillary thickness (MT), effective thickness (ET: palisade, vertical crystal layer, and cuticle), and mammillary width (MW), were quantified using the SEM’s integrated measurement software(Cao et al., 2019; Chen et al., 2025).

Transmission electron microscopy (TEM)

The tissue of the uterine portion of the oviduct in laying hens was cut into small fragments (1 mm × 1 mm × 1 mm). Fixed uterine portions were dehydrated using a graded ethanol series and cut into ultrathin sections. The sections were then stained with uranyl acetate and lead citrate. Micrographs were subsequently obtained using transmission electron microscopy (JEOL-JEM-1200EX, Peabody, Massachusetts, USA), as previously described (Gou et al., 2024; Liu et al., 2021a).

Magnum and uterine microbiota analysis

Genomic DNA was isolated from the samples with a MagBeads FastDNA Kit for Soil (MP Biomedicals, USA) according to the prescribed protocols, then cryopreserved at −20°C. The Pacific Biosciences (PacBio) Sequel platform was employed for Single Molecule Real Time (SMRT) sequencing, conducted by Shanghai Personal Biotechnology Co., Ltd. Subsequent data processing and visualization were completed via the Personalbio cloud infrastructure (https://www.genescloud.cn/home) (Cao et al., 2019; Chen et al., 2025; Gong et al., 2020).

Histological and morphological analysis

The magnum and uterine portion of the oviducts fixed in paraformaldehyde embedded in paraffin were cut into thin slices and stained with hematoxylin and eosin (H&E) and alcian blue periodate-schiff (AB-PAS) for morphometric analysis, as we previously described (Ge et al., 2024; Liu et al., 2023; Zhou et al., 2021).

TUNEL assay

Fixed paraffin sections were first deparaffinized and placed sequentially in xylene, anhydrous ethanol, and gradient alcohol, and washed using distilled water to ensure that the sections were ready for subsequent processing. Next, after shaking the sections dry, proteinase K working solution was added dropwise and incubated at 37°C for 20 minutes, followed by washing in PBS to remove excess liquid. The sections were then incubated in 3% hydrogen peroxide solution protected from light and washed again. Then, the sections were equilibrated with buffer at room temperature and a proportional mixture of the reaction solution was added for labeling reaction, followed by signal enhancement with Streptavidin-HRP reaction solution and DAB color development. After color development, the nuclei were stained with hematoxylin, and finally, the sections were dehydrated and sealed to ensure that they were well preserved and ready for observation under a light microscope (Hu et al., 2025; Lin et al., 2025).

Immunofluorescence analysis

Paraffin sections were deparaffinized in water and subjected to antigen retrieval. After applying a fluorescence quencher and blocking with serum, the sections were incubated overnight at 4°C with the following primary antibodies: anti-Claudin (1:1000; Servicebio, Wuhan, GB152543-50), anti-MUC-2 (1:1000; Servicebio, Wuhan, GB15344-100), anti-TNF-α (1:100; Servicebio, Wuhan, GB153968-100), anti-F4/80 (1:500; Servicebio, Wuhan, GB113373-100), and anti-NF-κB p65 (1:1000; Servicebio, Wuhan, GB11997-100) (Lin et al., 2025). Subsequently, the sections were washed and incubated with species-matched secondary antibodies. After DAPI staining and a second round of autofluorescence quenching, the slides were mounted with an anti-fluorescence quenching medium. Finally, images were captured using a fluorescence microscope (Lv et al., 2024).

RNA extraction and quantitative real-time PCR (qRT-PCR)

Following the manufacturer’s instructions, RNA from the magnum and uterus was extracted via FreeZol Reagent (Vazyme, R711-01). The resulting RNA was reverse-transcribed into cDNA using the HiScript III Kit (Vazyme, R312-01), which included a gDNA removal step. Transcript levels were subsequently quantified through qRT-PCR using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) on a Bio-Rad CFX Connect™ platform (Gong et al., 2020). The specific primers utilized are provided in Table S3. Target gene expression was normalized to β-actin mRNA using the 2^-ΔΔCt method (Li et al., 2018).

Determination of oxidative stress indicators

The magnum and uterine portions of the oviduct were collected for the determination of oxidative stress-related indicators. Briefly, the tissue samples were homogenized in ice-cold physiological saline and centrifuged to obtain the supernatants. The levels of malondialdehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) were measured using commercial assay kits according to the manufacturer’s instructions. The assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), including the MDA assay kit (A003-2), T-SOD assay kit (A001-1), GSH-Px assay kit (A005-1-1), and T-AOC assay kit (A015-2-1). Protein concentrations in the tissue homogenates were determined using a commercial protein assay kit, and all oxidative stress indicators were normalized to protein concentration. MDA levels were expressed as nmol/mg protein, while T-SOD, GSH-Px, and T-AOC activities were expressed as U/mg protein.

Statistical analysis

Statistical evaluations were conducted using the one-way ANOVA module within IBM SPSS Statistics 26.0 (SPSS Inc., Chicago, IL), with treatment differences identified via Tukey’s post-hoc analysis. Significance was predefined at P < 0.05. All experimental data are presented as mean ± standard deviation (SD). For figure generation and image processing, GraphPad Prism 9.5 (GraphPad Software Inc., San Diego, CA) was utilized (Li et al., 2022).

Results

Analysis of egg quality

After an 8-week pretreatment with MAG and a subsequent 4-week PS exposure, the laying performance was shown in Table S4 and the egg quality was examined and shown in Fig. 1. PS exposure significantly increased the broken egg rate and decreased the albumin height and Haugh unit as compared with the CK group (P < 0.05, Fig. 1a, f, and h). However, MAG could partially mitigate the unfavorable effects on broken egg rate, albumin height Haugh unit, and eggshell strength (P < 0.05, Fig. 1a, c, f, and h). There was no significant difference in egg weight, eggshell weight/egg weight (%), egg-shaped index, and yolk weight percentage among groups (P > 0.05, Fig. 1b, d, e, and g). The results of eggshell microstructure showed that the PS exposure significantly reduced (P < 0.05) the effective thickness and total thickness of the eggshell as compared to the CK group. The PM group had a trend to alleviate the reduction in the effective thickness and total thickness of the eggshell caused by PS exposure, but the difference was not significant (P > 0.05). However, no significant effects were observed (P > 0.05) on eggshell mammillary width and mammillary thickness (Fig. 1i and j).

Fig. 1.

MAG improved the egg quality and ultrastructure of eggshell of hens exposed to PS. (A) Broken egg rate during the feed trials; (B-H) The indicators of egg quality measured at the end of the experiment; (I) The ultrastructure of eggshell measured by scanning electron microscopy, Scale Bar = 50 μm and 200 μm; (J) Quantitative data on eggshell ultrastructure, including total thickness, effective thickness of eggshells, mammillary thickness, and mammillary width of the mastoid layer. Abbreviations: TT: total thickness of the eggshell; ET: effective thickness of eggshells; MT: mammillary thickness of the mastoid layer; MW: mammillary width of the mastoid layer. Data were shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Morphological analysis of the oviduct magnum

The morphology and secretion function of the oviduct magnum were demonstrated in Fig. 2a. The H&E staining showed that PS exposure resulted in inflammatory cell infiltration, glandular epithelial cell necrosis, and detachment. However, the MAG supplementation alleviated the adverse effects caused by PS exposure (Fig. 2b). The AB-PAS staining results revealed that the PS group exhibited severe goblet cell dysfunction impaired goblet cell maturation, and destruction of the mucus layer. Conversely, in the PM groups, there was an improvement in goblet cell consumption and relatively preserved integrity of the mucus layer (Fig. 2c).

Fig. 2.

MAG alleviated the damage of oviduct magnum induced by PS exposure. (A) Gross appearance of oviduct morphology of laying hens; (B) The representative image of H&E staining of the oviduct magnum and a partial magnification. The blue arrow indicates epithelial cell degeneration, necrosis, or detachment, Scale Bar = 1.2 mm and 200 μm; (C) AB-PAS staining of the oviduct magnum, Scale Bar = 1.2 mm and 200 μm.

Morphological analysis of the oviduct uterine

The morphology of the oviduct uterine in laying hens was shown in Fig. 3a. H&E staining analysis revealed that PS exposure markedly reduced (P < 0.05) the area of uterine mucosal folds, villus length, and width of uterine mucosal folds. However, MAG pretreatment was found to alleviate uterine mucosal damage, as confirmed by a significant increase (P < 0.05) in the area and width of uterine mucosal folds (Fig. 3b and e). The ultrastructure of uterine was further investigated and is shown in Fig. 3C. The uterine structure of hens in CK group exhibited complete integrity with a smooth surface and densely arranged villi, whereas in the PS group, there was a significant reduction in uterine villi accompanied by coarseness. However, in the PM group, both the decrease in uterine villi and the roughness of villi were mitigated. The TEM results revealed that the exposure to PS led to a decrease in tight junction protein expression compared to CK hens. However, MAG mitigated the decline in tight junction protein expression caused by PS exposure (Fig. 3d).

Fig. 3.

MAG alleviated the damage of oviduct uterine induced by PS exposure. (A) Gross appearance of oviduct morphology; (B) The representative image shows H&E staining of the oviduct uterus with partial magnification. Morphology parameters included villus length (L), height (H), width (W), and area (S) of mucosal folds, Scale Bar = 2.5 mm and 625 μm; (C) SEM findings of villi in the uterine portion of the oviduct, with localized magnified images, Scale Bar = 30 μm and 10 μm; (D) Transmission electron microscopy findings and local magnification of the uterine portion of the oviduct Scale Bar = 2 μm; (E) The quantitative data for the aforementioned morphological parameters are illustrated. Data were shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Analysis of the oviduct magnum and uterine microbiota

Alpha-diversity encompassed both diversity (assessed by Shannon and Simpson indices) and richness (measured by Chao1 indices) of the oviduct microbiota. In this study, no significant effects were observed (P > 0.05) on alpha-diversity among groups (Fig. 4a and e). Beta-diversity were analyzed by principal coordinate analysis (PCoA) at the ASV level visualized using the Bray-Curtis distance. The samples in the PM group were clustered together, which was different from those in the CON group and the PS group, indicating that there were significant differencesin the oviductal microbiota structure after MAG feeding (Fig. 4b and f). PS exposure and MAG treatment altered the relative abundance of oviduct microbiota at the species level. As shown in Fig. 4c and g, Lactobacillus crispatus is the most abundant bacterium in both the magnum and uterine areas. It is seen that under PS exposure, the content of Lactobacillus crispatus decreased, while after supplementation with MAG, its relative abundance was significantly increased in the MAG group. To further characterize the oviduct magnum and uterine microbiota associated with MAG treatment and PS exposure, LEfSe analysis was performed to identify genera with significant differences across the three experimental groups. Oviduct magnum analysis (Fig. 4d) revealed 12 taxonomic biomarkers (LDA score >2). Notably, s_Lactobacillus_crispatus exhibited higher abundance in the CK group, while s_Sanguibacter_inulinus, s_Flavobacterium_sp._HDG3, and s_Clostridium_perfringens were also identified as discriminative biomarkers. Uterine microbiota analysis (Fig. 4h) identified 8 taxonomic biomarkers. The CK group showed elevated levels of s_Desulforamulus_aeronauticus, s_Paenibacillus_peoriae, and s_Haemophilus_haemolyticus. Additionally, s_Nocardioides_sp. and s_Streptomyces_sp._HB-3 were highlighted as biomarkers in other groups.

Fig. 4.

MAG improved the PS-induced microbiota disorder of oviduct magnum and uterine. (A and E) Chao1 index, Simpson index, and Shannon index of the oviduct magnum and uterine microbiota; (B and F) The principal coordinate analysis (PCoA) plot of the oviduct magnum and uterine microbiota composition at the ASV level; (C and G) Percent of community abundance of the oviduct magnum and uterine microbiota community at the species level; (D and H) LDA effect size (LEfSe) analyzed (LDA score 2) from the species level. Data were showed as mean ± SD, n = 6 hens per group.

Analysis of the oviduct uterine barrier function

To assess the protective effect of MAG on the barrier function of the oviduct uterine, the tight junction (TJ) proteins in oviduct uterine were measured by immunofluorescence assays and qRT-PCR (Fig. 5). Immunofluorescence results demonstrated that the mean fluorescence intensity of Claudin and MUC-2 in PS group was significantly decreased (P < 0.05) compared with CK group. However, the PM prevention group significantly improved the decline (P < 0.05) in Claudin fluorescence intensity caused by PS exposure (Fig. 5a, c, and d). The results of qRT-PCR showed that PS exposure obviously reduced (P < 0.05) the mRNA levels of Occludin, MUC-2, and Claudin-1 in the uterine, while those changes caused by PS were reversed in the PM group (Fig. 5e). The PAS staining was performed to visualize the impact of different treatments on goblet cell mucin secretion. The results revealed that PS exposure led to a reduction in mucin secretion, while the PM group effectively reversed the decline caused by PS exposure (Fig. 5b).

Fig. 5.

MAG improved the oviduct uterine barrier function of laying hens exposed to PS. (A) Immunofluorescence staining of Claudin protein expression in the uterine portion of the oviduct; zoom refers to the magnification of a portion of these typical results, Scale Bar = 1 mm and 0.1 mm; (C) Immunofluorescence staining of MUC-2 protein expression in the uterine portion of the oviduct, Scale Bar = 1 mm and 0.1 mm; (B) Results of PAS special staining in the uterine part of the oviduct; (D) Quantitative results for the two above mentioned immunofluorescence stains (Claudin and MUC-2); (E) Expression of genes associated with tight junctions in the uterine portion of the oviduct. Data were shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Analysis of cell apoptosis and oxidative stress in the oviduct magnum and uterus

To investigate the impact of PS exposure and MAG treatment on uterine and magnum cell apoptosis in laying hens, immunohistochemical staining and qRT-PCR analyses were conducted (Fig. 6). The results demonstrated a significant increase (P < 0.05) in the TUNEL-positive area (Fig. 6a, b, c, and e) and in the relative mRNA expression levels of Bax (Bcl-2-associated X), Caspase3, and Caspase8 in both the magnum and uterine portions following PS exposure. However, compared with the PS group, the above apoptosis-related indexes were significantly reduced (P < 0.05) in the PM group (Fig. 6d and f). Notably, there were no significant differences (P > 0.05) observed in the relative mRNA expression levels of Caspase 9 in the three treatment groups of laying hens’ oviducts.

Fig. 6.

MAG suppressed the cell apoptosis of magnum and uterine of laying hens exposed to PS. (A) Representative images of TUNEL staining in the magnum portion of the oviduct. The zoom panels show magnified views of the indicated regions. (B) TUNEL results in the uterine portion of the oviduct; (C and E) The quantitative results for IHC; (D) The mRNA levels of apoptosis-related genes in the magnum; (F) The mRNA levels of apoptosis-related genes in the uterine. Data were shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

To further determine whether oxidative stress contributed to PS-induced oviductal injury, oxidative stress-related indicators were measured in the magnum and uterine portions of the oviduct (Fig. 7). In the magnum, PS exposure significantly increased MDA levels and decreased T-SOD, GSH-Px, and T-AOC activities compared with the CK group. MAG supplementation significantly reduced MDA levels and restored T-SOD and GSH-Px activities, while T-AOC showed a recovery trend. In the uterine portion, PS exposure significantly increased MDA levels and reduced T-SOD, GSH-Px, and T-AOC activities, and these changes were significantly reversed by MAG supplementation. These results indicate that PS exposure induced oxidative stress in the oviduct, whereas dietary MAG supplementation improved the antioxidant status, especially in the uterine portion of the oviduct.

Fig. 7.

MAG attenuated PS-induced oxidative stress in the oviduct of laying hens. (A) Oxidative stress-related indicators in the magnum portion of the oviduct, including MDA, T-SOD, GSH-Px, and T-AOC. (B) Oxidative stress-related indicators in the uterine portion of the oviduct, including MDA, T-SOD, GSH-Px, and T-AOC. Data are presented as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Analysis of the inflammation response in oviduct magnum and uterine

To further investigate whether oviduct damage caused by PS exposure is due to inflammatory factors as well as macrophage polarization. We probed the fluorescent labeling of macrophage markers as well as the expression of inflammation-related genes in the expanded oviduct and uterine sections, respectively. The immunofluorescence staining of macrophage markers in the oviduct magnum and uterine of laying hens revealed a significant increase (P < 0.05) in the mean fluorescence intensity of F4/80 and TNF-α in the PS group compared to the control group. Conversely, the PM group exhibited a significant down-regulation (P < 0.05) in the mean fluorescence intensity of F4/80 and TNF-α when compared to the PS group (Fig. 8a, b, d and 9a, b, d). To further investigate the impact of PS exposure on the inflammatory response of the oviduct magnum and uterine, the relative expression levels of TNF-α, IL-1β, IL-10, and inducible nitric oxide synthase (iNOS) were detected by qRT-PCR. The results demonstrated that PS exposure significantly up-regulated the inflammatory cytokines TNF-α and IL-1β (P < 0.05) but down-regulated the relative expression levels of IL-10 when compared to the CK group in the oviduct magnum (Fig. 8e). Importantly, co-treatment with PS and MAG effectively suppressed this upregulation (P < 0.05). Notably, there were no significant difference (P > 0.05) observed in the expression of IL-1β and iNOS among the three treatment groups (Fig. 9e). To identify potential links between changes in egg quality and inflammatory responses in the magnum portion of the oviduct. Correlation analysis (Fig. 8c) revealed a clear clustering pattern where elevated inflammatory cytokines in the magnum were negatively correlated with albumen quality indicators (Haugh unit and Albumen height). This suggests that local inflammation in the magnum directly compromises albumen secretion. Consistently, a similar pattern was observed in the uterus (Fig. 9), where pro-inflammatory markers showed a strong negative correlation with eggshell quality traits. This parallelism between Fig. 8, Fig. 9 confirms that PS-induced inflammation is a consistent driver of functional impairment across different oviduct segments.

Fig. 8.

MAG suppressed the inflammation response in oviduct magnum of laying hens exposed to PS. (A) Immunofluorescence results for the macrophage marker F4/80 in the magnum portion of the oviduct, and zoom refers to the magnification of a portion of these typical results, Scale Bar = 0.1 mm and 20 μm; (B) Immunofluorescence results of the inflammatory factor TNF-α in the magnum portion of the oviduct, Scale Bar = 0.1 mm and 20 μm; (C) Correlation analysis of egg quality indices characterizing oviduct magnum function and their inflammatory factors; (D) Quantitative results for the two above mentioned immunofluorescence stains (F4/80 and TNF-α); (E) Relative mRNA expression of genes related to the inflammatory response. Data was shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Fig. 9.

MAG suppressed the inflammation response in oviduct uterine of laying hens exposed to PS. (A) Immunofluorescence results for the macrophage marker F4/80 in the uterine portion of the oviduct, and Zoom refers to the magnification of a portion of these typical results, Scale Bar = 1 mm and 0.1 mm; (B) Immunofluorescence results of the inflammatory factor TNF-α in the uterine portion of the oviduct, Scale Bar = 1 mm and 0.1 mm; (C) Correlation analysis of eggshell quality indicators characterizing the function of the uterine part of the oviduct and their levels of inflammatory factors; (D) Quantitative results for the two above mentioned immunofluorescence stains (F4/80 and TNF-α); (E) Relative mRNA expression of genes related to inflammatory response in the uterine. Data were shown as mean ± SD. n = 6 hens per group. *P < 0.05, **P < 0.01.

Analysis of the inflammatory reaction in HD11 cells

The results demonstrated that, in comparison to the CK group, PS treatment significantly augmented (P < 0.05) the mean fluorescence intensity of NF-κB and TNF-α (Fig. 10). Furthermore, a notable decrease (P < 0.05) in the mean fluorescence intensity of TNF-α was observed in the PM group when compared to the PS group (Fig. 10a, b, and c). The mRNA expression of IL-12, IL-1β, TNF-α, CD86 (T-lymphocyte activation antigen), and IL-6 has been proposed as indicative of M1 activation, while the expression of IL-10, CD163 (scavenger receptor), TGF-β, and mannose receptor (CD206) has been suggested as indicative of M2 activation. The results demonstrated that, in comparison to the CK group, PS treatment significantly increased (P < 0.05) the expression levels of IL-12, IL-1β, TNF-α, CD86, and IL-6; meanwhile, it significantly decreased (P < 0.05) the expression levels of IL-10, CD163, TGF-β, and CD206. However, when compared to the PS group, these indicators exhibited opposite trends in the PM group (Fig. 10d and e).

Fig. 10.

MAG regulated the polarization trend of HD11 macrophages. (A) Immunofluorescence results of NF-κB in HDII cells, Scale Bar = 20 μm; (B) Immunofluorescence results of TNF-α in HDII cells, Scale Bar = 20 μm; (C) Quantitative results for the two above mentioned immunofluorescence stains (NF-κB and TNF-α); (D) The mRNA expression of M1 polarization-related marker genes; (D) The mRNA expression of M2 polarization-related marker genes. Data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

Due to the wide range of applications of plastics in agricultural and livestock production involving feed packaging and plastic water pipes (Ijaz et al., 2024), microplastic contamination is seriously threatening the health of animals. According to the chemical composition, microplastics can be divided into different polymer types (Allouzi et al., 2021), research has shown that PS is one of the largest polymer toxicities of placenta enzyme (Zhong et al., 2021). The available evidence suggests that PS has toxic effects on the liver and colon in poultry, accompanied by a disturbance of the gut microbiota (Yin et al., 2023b). However, its effects on the reproductive system of laying hens, especially on the oviduct, remain unclear. The results of this study indicate that the reproductive toxicity induced by PS can cause disorders of the oviduct microbiota and inflammatory infiltration in laying hens, resulting in damage to the uterine mucosal barrier and a decline in egg quality. In addition, MAG, an extract of Magnolia officinalis, increases the abundance of beneficial bacteria by effectively regulating the microbial composition, restores mucosal barrier damage, and improves egg quality by inhibiting the polarization of M1 macrophages and preventing PS-induced inflammatory infiltration. It also has a protective effect on the physiological toxicity induced by PS, indicating the potential application value of MAG in PS-related preventive strategies.

It has been established that PS causes negative effects on growth performance. Exposure to PS affected the growth rate of Japanese quail (Monclús et al., 2022). Microplastic exposure affected wet weight and body length of shell-less quail at hatching (Li et al., 2021). Consistent with these results, the laying performance and egg quality was affected by PS exposure, mainly reflected in the increase in egg breaking rate and the decrease in egg quality. The oviduct is a lengthy tubular structure comprising five distinct functional sections: the infundibulum for fertilization, magnum for egg white production, isthmus for soft shell-membrane formation, shell gland or uterine for calcified eggshell formation, and vagina for oviposition. The magnum, being the longest segment of the oviduct, contributes egg white protein to the developing egg during its traversal of this region spanning 3 hours (Sah and Mishra, 2019). Albumen quality, typically gauged by albumen height and HU, serves as a standard metric for evaluating the internal quality of eggs. The HU value is determined through a calculation that incorporates the height of the thick albumen and the egg's weight (Wang et al., 2018). The results showed that when contaminated with PS, both the protein height and HU of eggs decreased significantly compared with the control, and this trend was effectively alleviated when MAG was added to the diet. Endometrial morphology changes with the growth of the age and the hen, resulting in abnormal shell forms, thus resulting in a decline in eggshell quality (Park and Sohn, 2018). Eggshell quality is a major issue in the production of high-quality eggs, which has a significant impact on the commercial egg industry (Xiao et al., 2014). Eggshell ultrastructure consists of papillary, palisade, and vertical crystal layers. Any defect in eggshell structure will result in decreased eggshell quality. From the SEM results, PS contamination leads to a reduction in eggshell thickness, with a significant reduction in the effective thickness compared to the control. It is worth noting that while our study focused on the physical indicators (e.g., Haugh unit and eggshell strength) that directly reflect the secretory function of the oviduct, the potential impacts of PS exposure on the sensory flavor and detailed nutritional profile (such as amino acid composition or micronutrient deposition) of eggs were not evaluated in the current study. Given that microplastics may interfere with the accumulation of flavor compounds through metabolic disruption, future studies are warranted to investigate these multi-dimensional effects on egg quality, thereby providing a more comprehensive assessment of the food safety risks posed by environmental pollutants.

The microbiota composition of the reproductive tract of poultry is similar to that of the intestine, due to the physiology of the chicken (Shterzer et al., 2020). Reproductive tract microbiota plays a key role in the health and productivity of laying hens (Su et al., 2021a). A study characterizing the microbial composition of reproductive tract segments in laying hens with egg quality and quantity phenotypes found that the microbial communities differed between individual oviduct segments (Wen et al., 2021). Thus, it can be seen that the composition and diversity of the microbiota in the magnum and uterine part were negatively affected after PS exposure; however, with MAG supplementation, adverse effects can be mitigated. Among them, Lactobacillus crispatus has been reported to maintain mucosal barrier health through lactic acid production, maintenance of low pH and anti-inflammatory immunomodulation (Alizhan et al., 2025; Decout et al., 2024). The results showed that the relative abundance of Lactobacillus crispatus was reduced under PS exposure and effectively enhanced after MAG supplementation. In addition, the principles that determine the innate immune response of the vaginal microbiota remain unclear, however the activation of the innate immune receptor-mediated transcription factor NF-κB in immune cells and epithelial cells is a central feature (Doerflinger et al., 2014). Therefore, we focused on NF-κB as a central regulator of microplastic-induced inflammation.

Eggshell formation occurs mainly in the uterine (or eggshell gland) of the hen and is the longest stage in the egg formation process (Cheng and Ning, 2023). It has been noted that several key components related to chemical barriers have been identified in the oviductal uterine of laying hens, including avian β-defensin and MUC-2 (Elhamouly et al., 2019; Yoshimura, 2015). In addition to this, Claudins can protect tissues from toxic substances and pathogens by forming a paracellular physical barrier, and in the oviduct mucosa of laying hens, the expression levels of Claudin-1 and Claudin-3 can be used as an indicator of mechanical barrier function (Ariyadi et al., 2013). Therefore, after AB-PAS staining, the results showed decreased mucin secretion in the PS-exposed group, and the immunofluorescence MUC-2 labeling signal was weakened, which was consistent with the decreased expression of secretory cell marker genes (MUC-2); whereas, the SEM results showed that the degree of damage to microvilli in the uterine part of the uterine section was elevated after PS exposure, and the results of TEM showed a widening of the tight junction gap, an elevation of microvilli detachment, and a weakening of immunofluorescent Claudin labeling signals, which was consistent with the decreased expression of tight junction-associated markers of mRNAs (Claudin-1, Occludin, and ZO-1). In addition, we found that the adverse effects of PS contamination on the oviductal mucosal barrier were effectively alleviated when the diet was supplemented with MAG, which is consistent with a previous study by Xi Fan et al., who demonstrated that the intestinal inflammatory infiltrate and barrier damage in mice with colitis were effectively restored after feeding MAG to the mice due to its excellent anti-inflammatory effect (Fan et al., 2023).

There is growing evidence that PS activates inflammatory responses in multiple organs, including the liver, peripheral immune system, and colon (Huang et al., 2022). In addition, inflammation is increasingly recognized as a contributing factor to reproductive dysfunction (Negishi et al., 2020). Current findings suggest that prolonged exposure to PS induces uterine inflammation characterized by abnormal expression of inflammatory cytokines (Qin et al., 2024). The HE results of the magnum and the uterine of the oviduct showed that after PS exposure, there was an inflammatory infiltration, which was reduced by the addition of MAG.

TNF-α is a pro-inflammatory cytokine that plays a key role in mediating apoptosis as well as inflammation and immunity. Correspondingly, immunofluorescence labeling of the oviduct bulk and uterine TNF-α signals were enhanced, and analysis of the macrophage marker F4/80 showed an increase in signal intensity in both PS-exposed groups compared to the CK group. This coincided with the up-regulation of gene expression of pro-inflammatory related factors (TNF-α, IL-6) in the oviducts and down-regulation of mRNA levels of the anti-inflammatory factor IL-10, suggesting that the oviducts of laying hens were damaged by exposure to PS contamination, in which significant inflammatory effects were observed. It has been demonstrated that the release of inflammatory factors TNF-α and IL-6 could be promoted in the PS-treated group through the activation of the NF-κB pathway. The activation of the NF-κB pathway leads to inflammation, which inhibits the expression of Bcl-2 (B-cell lymphoma-2) in the apoptotic pathway and increases the expression of Bax, leading to apoptosis (Cao et al., 2023). The TUNEL results and the expression of apoptosis-related genes were consistent with this, showing that apoptosis was significantly increased after PS treatment. However, this phenomenon was alleviated when the diet was supplemented with MAG, confirming that MAG can inhibit apoptosis through the anti-inflammatory pathway. Oxidative stress is considered an important upstream event in PS-induced tissue injury. Excessive oxidative stress can promote lipid peroxidation, impair epithelial barrier integrity, and amplify inflammatory signaling, thereby contributing to apoptosis and tissue dysfunction. In the present study, PS exposure increased MDA levels and reduced T-SOD, GSH-Px, and T-AOC activities in the oviduct, indicating that PS exposure induced oxidative injury in both the magnum and uterine portions. Notably, dietary MAG supplementation reduced MDA accumulation and restored antioxidant enzyme activities, particularly in the uterine portion of the oviduct. These findings suggest that the protective effect of MAG against PS-induced oviductal injury is associated not only with immunomodulation but also with improved oxidative stress status.

Different types of immune cells are impacted by PS. Smaller particles, especially those in the nanometer size range, can passively cross cell membranes (Xiang et al., 2026). Lymphocytes (Sarma et al., 2022), neutrophils (Chi et al., 2022), and macrophages (Tang et al., 2022) can ingest nanosized plastic particles. Although mouse macrophages can phagocytose PS with a particle size of 10 μm, leading to induced metabolism to glycolysis and reduced mitochondrial respiration (Merkley et al., 2021). Zhao et al. reported that Lactobacillus plantarum induced M1 polarization and inhibited Salmonella-induced oviduct inflammation and atrophy by triggering TLR2/NF-κB (Zhao et al., 2022). Macrophage polarization is the process by which macrophages adopt different functional programs in response to signals from their microenvironment. When stimulated by exogenous substances, macrophages may be activated and polarized into two distinct phenotypes: the pro-inflammatory M1 (classically activated macrophages) and the anti-inflammatory M2 (alternatively activated macrophages) (Murray et al., 2014). Studies have shown that M1 macrophages can secrete the pro-inflammatory cytokines TNF-α and IL-6 and that stimulation of RAW264.7 cells with PS resulted in the secretion of TNF-α and IL-6, suggesting that PS induces macrophage polarization to M1 phenotype (Wang et al., 2023), which is consistent with previous in vivo studies showing that PS promotes an increase in the proportion of M1 in tissues. It is thus known that PS may lead to an inflammatory response by inducing macrophage polarization to M1 and the release of inflammatory cytokines (TNF-α, IL-6, and IL-10).

Macrophage polarization is tightly regulated by intracellular signaling networks, among which the NF-κB pathway plays a pivotal role in driving the M1 phenotype. Previous studies have established that the activation and nuclear translocation of NF-κB p65 initiate the transcription of key M1-associated genes, including TNF-α, IL-1β, and iNOS (Chunlian et al., 2014). Consistent with this, our in vitro results demonstrated that PS exposure triggered significant NF-κB activation, concurrent with a shift towards M1-like inflammatory polarization. Notably, MAG treatment effectively suppressed NF-κB signaling, which helps explain the observed reduction in M1 markers and the partial restoration of the M1/M2 balance (Li et al., 2010). Although the in vitro HD11 cell model provided supportive evidence that MAG directly suppressed PS-induced NF-κB activation and M1-like inflammatory polarization, the concentrations used in vitro may not fully reproduce the local exposure level in the oviduct. Future studies using broader dose-response designs and physiologically relevant exposure models are needed. It should be noted that PS-induced inflammatory injury is likely mediated by a multi-pathway network rather than by NF-κB alone. Previous studies have reported that PS exposure may activate TLR2/NF-κB signaling, ROS/NLRP3 inflammasome activation, and MAPK pathways. In the present study, we focused on NF-κB because of its central role in macrophage inflammatory activation and M1-like polarization. Therefore, our findings suggest that MAG alleviates PS-induced oviductal inflammation at least partly through suppression of NF-κB-mediated macrophage activation. Whether ROS/NLRP3 and MAPK pathways also participate in this process requires further investigation.

Furthermore, it is noteworthy that the PS-induced depletion of Lactobacillus crispatus coincided with M1 macrophage polarization in the oviduct. Emerging evidence suggests a critical microbiota-macrophage crosstalk, where Lactobacillus spp. and their metabolites can inhibit NF-κB signaling and promote the anti-inflammatory M2 phenotype (Zhang et al., 2023). Therefore, we postulate that the observed M1 polarization is driven not only by direct PS toxicity but also by the loss of L. crispatus-mediated immunomodulation. Biologically, the enrichment of Clostridium perfringens, a well-known opportunistic poultry pathogen, following PS exposure strongly suggests that mucosal barrier impairment facilitates pathogen colonization, thereby exacerbating local oviductal inflammation. Conversely, MAG modulated the abundance of Paenibacillus, a genus with probiotic potential known to produce antimicrobial peptides, further highlighting MAG's efficacy in restoring mucosal microecological homeostasis. MAG supplementation likely restores this immune homeostasis by simultaneously reshaping the microbiota and directly suppressing NF-κB activation. Although direct co-culture evidence is limited in the current study, these findings highlight the microbiota-immune axis as a potential therapeutic target, which warrants further investigation.

Based on our in vivo and in vitro findings, we propose a comprehensive mode of action for MAG. Mechanistically, MAG acts as a potent NF-κB inhibitor. By blocking the phosphorylation and nuclear translocation of the p65 subunit, Magnolol suppresses the transcription of downstream pro-inflammatory mediators (TNF-α, IL-1β), thereby inhibiting M1 macrophage polarization. Furthermore, this immunomodulatory effect creates a favorable microenvironment that promotes the restoration of beneficial Lactobacillus crispatus, reinforcing the mucosal barrier. Together, these findings suggest that the combined regulation of NF-κB-mediated inflammatory activation, oxidative stress status, and oviductal microbiota composition may represent an important mechanism by which MAG alleviates PS-induced reproductive toxicity.

Conclusions

PS exposure compromised the reproductive health of laying hens by disrupting oviductal microbiota, depleting Lactobacillus crispatus, inducing oxidative stress, and triggering inflammation and apoptosis. These alterations impaired mucosal barrier function, leading to decreased albumen and eggshell quality. MAG supplementation counteracted these effects by improving antioxidant status, enriching Lactobacillus crispatus populations, and suppressing NF-κB-mediated macrophage inflammatory activation. The immunomodulatory activities of MAG were further supported by in vitro experiments. Overall, MAG supplementation restored oviductal mucosal homeostasis by modulating oxidative stress, local inflammation, apoptosis, and microbiota composition, thereby improving albumen secretion and eggshell formation (Fig. 11). Our findings elucidate important pathological features of PS-induced reproductive damage and propose MAG-mediated mucosal protection as a potential dietary strategy against plastic particle-associated reproductive toxicity in poultry.

Fig. 11.

Schematic diagram of the regulatory network for the mitigating effects and mechanisms of MAG on oviduct injury in PS-exposed laying hens.

Availability of data and materials

Datasets used or analyzed in this study are available by reasonable request from the corresponding author.

CRediT authorship contribution statement

Yujie Lv: Writing – original draft, Methodology, Investigation, Data curation. Weichen Huang: Investigation, Data curation. Chaoyue Ge: Investigation. Lianchi Wu: Investigation. Zhaoying Hu: Investigation. Shenao Zhan: Investigation. Xinyu Shen: Investigation. Dongyou Yu: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Bing Liu: Writing – review & editing, Supervision, Investigation, Formal analysis.

Disclosures

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper. We have no conflict of interest for the manuscript being submitted for publication to the journal Poultry Science.