Journal of Animal Science and Biotechnology
Research

Zinc glycine chelate ameliorates DSS-induced intestinal barrier dysfunction via attenuating TLR4/NF-κB pathway in meat ducks

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Abstract

Background

Zinc glycine chelate (Zn-Gly) has anti-inflammation and growth-promoting properties; however, the mechanism of Zn-Gly contribution to gut barrier function in Cherry Valley ducks during intestinal inflammation is unknown. Three-hundred 1-day-old ducks were divided into 5 groups (6 replicates and 10 ducks per replicate) in a completely randomized design: the control and dextran sulfate sodium (DSS) groups were fed a corn-soybean meal basal diet, and experimental groups received supplements of 70, 120 or 170 mg/kg Zn in form of Zn-Gly. The DSS and treatment groups were given 2 mL of 0.45 g/mL DSS daily during d 15–21, and the control group received normal saline. The experiment lasted 21 d.

Results

Compared with DSS group, 70, 120 and 170 mg/kg Zn significantly increased body weight (BW), villus height and the ratio of villus to crypt, and significantly decreased the crypt depth of jejunum at 21 d. The number of goblet cells in jejunal villi in the Zn-Gly group was significantly increased by periodic acid-Schiff staining. Compared with control, the content of intestinal permeability marker D-lactic acid (D-LA) and fluxes of fluorescein isothiocyanate (FITC-D) in plasma of DSS group significantly increased, and 170 mg/kg Zn supplementation significantly decreased the D-LA content and FITC-D fluxes. Compared with control, contents of plasma, jejunum endotoxin and jejunum pro-inflammatory factors IL-1β, IL-6 and TNF-α were significantly increased in DSS group, and were significantly decreased by 170 mg/kg Zn supplementation. Dietary Zn significantly increased the contents of anti-inflammatory factors IL-10, IL-22 and sIgA and IgG in jejunum. Real-time PCR and Western blot results showed that 170 mg/kg Zn supplementation significantly increased mRNA expression levels ofCLDN-1and expression of OCLN protein in jejunum, and decreased gene and protein expression of CLDN-2 compared with DSS group. The 120 mg/kg Zn significantly promoted the expressions ofIL-22andIgA. Dietary Zn-Gly supplementation significantly decreased pro-inflammatory genesIL-8andTNF-αexpression levels and TNF-α protein expression in jejunum. Additionally, Zn significantly reduced the gene and protein expression of TLR4, MYD88 and NF-κB p65.

Conclusions

Zn-Gly improved duck BW and alleviated intestinal injury by regulating intestinal morphology, barrier function and gut inflammation-related signal pathways TLR4/MYD88/NF-κB p65.

Background

Ducks are reared worldwide due to their extensive adaptability and rapid growth [1]. However, in the context of antibiotic-free farming, a variety of environmental factors in commercial production can trigger intestinal inflammation, including high animal densities, intestinal pathogens (such as Escherichia coli), mycotoxins in feed and changes in feed formulation, leading to ducks suffering stress and bacterial endotoxin (ET) invasion, resulting in increased intestinal permeability and bacterial translocation [2, 3]. The resulting intestinal health problems lead to annual global economic losses of more than $6 billion [4]. Lipopolysaccharide (LPS) is one of the most common ETs in the gut, which can trigger the aggregation of macrophages by activating toll-like receptor 4 (TLR4) [5]. It also activates myeloid differentiation factor 88 (MYD88), which in turn activates nuclear factor κB (NF-κB) and tumor necrosis factor (TNF) receptor-related factor 6. These factors interact to stimulate the secretion of cytokines and chemokines, thereby accelerating occurrence of intestinal inflammation [6]. Therefore, there is an urgent need to find effective nutrients to promote intestinal barrier function and relieve intestinal inflammation of meat ducks.

The trace element zinc (Zn) plays an indispensable role in the maintenance of physiological functions such as metabolism, immune function, signal transduction and cell growth and differentiation [7]. As a structural factor, Zn regulates many protein functions, including transcription factors, enzymes, receptors and growth factors [8]. Organic and inorganic forms of Zn are used in the feed industry, and organic Zn is more easily absorbed than inorganic Zn. Studies showed that Zn glycine chelate (Zn-Gly) alleviated intestinal inflammation by modulating intestinal immunoglobulin gene expression [9]. In recent years, the view that Zn maintains the integrity of intestinal barrier function (physical, chemical, microbial and immune barriers) has been gradually confirmed. Sarkar et al. [10] found that Zn can regulate the phosphorylation of tight junction (TJ) proteins, and thus improve the intestinal barrier function. Shao et al. [11] showed that Zn enhanced the barrier function of intestinal epithelium by promoting the differentiation of Caco-2 cells and increasing expression of zonula occludens-1 (ZO-1). Levkut et al. [12] reported that addition of low-dose Zn significantly increased the expression of mucin 2 (MUC2) in the jejunum of broilers, and promoted the secretion of intestinal immunoglobulin A (IgA). He et al. [13] showed that Zn reduced the expression of interleukin (IL)-8 in intestinal cells of broilers suffering from necrotizing enteropathy, thus reducing the inflammatory response and improving intestinal barrier function. In addition, Zn is important for the maintenance and development of the innate and adaptive immune systems. Deficiency of Zn can lead to thymus atrophy, lymphocytopenia, impaired cellular and antibody-mediated immune responses, and even death in humans and animals [14, 15]. The Zn regulates the secretion of pro-inflammatory cytokines by activating or inhibiting NF-κB [16]. The underlying molecular pathways are based on TLR4/MYD88 signaling and are regulated by Zn signaling in monocytes [17]. Additionally, dextran sulfate sodium (DSS)-mediated, chemically-induced intestinal inflammation has been replicated in broiler chickens using oral gavage [18]. Increased intestinal permeability caused by DSS has been found in studies of broilers, as evidenced by leakage of fluorescein isothiocyanate dextran (FITC-D) into serum [18, 19]. In addition, elevated serum D-lactic acid (D-LA) levels were also observed in broilers fed DSS, suggesting damage to intestinal epithelial homeostasis caused by the toxic chemical DSS [20]. To the best of our knowledge, few studies have investigated the effects and mechanisms of Zn-Gly against intestinal inflammation of meat ducks.

Here, we selected DSS to establish the gut inflammation model of meat ducks in this study. Moreover, we studied the intestinal permeability and intestinal morphology in jejunum and measured inflammatory response and TJ protein related gene and protein levels following Zn-Gly supplementation and DSS challenge. Finally, we aim to investigate the beneficial effects and the underlying mechanisms of Zn-Gly action on the intestinal barrier function and inflammatory response when gut inflammation occurs in meat ducks.

Materials and methods

Ethics statement

Animals, diets and sampling procedures were approved by the Animal Welfare Committee of Sichuan Agricultural University (No. 20180718), Sichuan, China.

Animal, diets and experimental design

Three-hundred 1-day-old male Cherry Valley ducks were randomly assigned to 5 treatments (each with 6 replicates and 10 ducks per replicate): non-challenged group (CON), DSS-challenged group (DSS) and DSS-challenged groups with 70, 120 or 170 mg/kg of Zn from Zn-Gly, respectively. The selected Zn doses were based on our previous results [21, 22]. The experiment lasted 21 d, and ducks were administered intragastrically with 2 mL of 0.45 g/mL DSS (molecular weight: 36,000–50,000, Cat: CD4421; Coolaber, Beijing, China) or the same amount of saline during d 15–21. The basal diet was formulated according to National Research Council guidelines [23], and the composition and nutrient levels are shown in Table 1. All birds were reared in cages (1 m × 0.75 m × 0.75 m) and allowed pellet feed and water ad libitum with adequate ventilation. In the first week, room temperature was maintained at 33 ± 1 °C, and then decreased by 3 °C every week. From the third weekend, the temperature was maintained at 24 ± 1 °C, relative humidity was 65%–75% and light was continuous. The Zn-Gly used in the experiment was provided by Chelota Group (Guanghan, China), with a Zn content of 21% and purity of 98.5%. The ducks for the experiment were purchased from Sichuan Mianying Breeding Duck Co., Ltd. (China).

Table 1 Ingredients and compositions of the basic diets, % (dry matter basis)
Full size table

Sample collection

At 21 days of the experiment, one duck of the average pen body weight (BW) was selected from each replicate and 30 ducks were anesthetized by intravenous injection with sodium pentobarbital (30 mg/kg BW) and slaughtered. The middle parts of duodenum and jejunum were collected 1–2 cm and placed in 4% paraformaldehyde solution for pathological section determination. In addition, jejunum tissues were collected in 2-mL cryo-storage tubes, immediately placed in liquid nitrogen and transferred to a −80 °C refrigerator for storage, for the detection of indicators related to intestinal inflammation and to intestinal barrier function.

At 21 days of the experiment, the ducks closest to the average BW in each treatment group were selected, and were administered as oral gavage with FITC-D (molecular weight: 3–5 kDa, Sigma-Aldrich, St. Louis, MO, USA) at 4.16 mg/kg BW. After 2 h, blood samples of ducks with FITC-D were collected.

Measurements

Growth performance

On 1, 14 and 21 days of the experiment, fasting weight for each replicate was determined on the experimental ducks, and the feeding amount and residual amount were recorded. Then average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G) were calculated according to the obtained data.

Morphological analysis

Fixed duodenum and jejunum tissues were dehydrated by ethanol gradient and paraffin-embedded. These sections were stained with hematoxylin and eosin (HE) according to standard protocol (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Intestinal sections stained with periodic acid-Schiff (PAS) were observed under an optical microscope (40× magnification). Goblet cells (purple) and neutral mucins were selected according to each visual field, and their numbers determined, and then organizational observations were made.

Plasma FITC-D and D-LA analysis

Plasma samples were centrifuged at 500 × g for 15 min, and then stored in a −20 °C refrigerator for later determination of intestinal permeability markers D-LA and FITC-D. Fluorescence levels of diluted plasma were analyzed as described by Vicuna et al. [24]. The detailed procedure for plasma FITC-D determination is shown in Fig. S1. The D-LA content was measured using an enzyme-linked immunosorbent assay (ELISA) (MM-91646O1; Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China).

Measurements of inflammation factors and immunoglobulins in plasma and jejunum

Plasma samples were collected into sterile enzyme-free centrifuge tubes, and then the contents of ET (MM-1626O1), IL-1β (MM-925452O1), IL-6 (MM-91624O1), TNF-α (MM-32774O1) and D-LA (MM-91646O1) in plasma and jejunum and the concentrations of secretory immunoglobulin A (sIgA, MM-91217O1), immunoglobulin G (IgG, MM-91025O1), IL-10 (MM-33637O1), IL-22 (MM-925346O1) and MUC2 (MM-1790O1) in jejunum were measured by ELISA using a commercial kit (Jiangsu Meimian Industrial Co., Ltd.).

Quantitative real-time (qRT)-PCR analysis

The expression of TJ protein genes [claudin1 (CLDN-1), CLDN-2, ZO-1, ZO-2 and occludin (OCLN)], genes associated with chemical barriers MUC2, Notch1 and Notch2 and immune-barrier-related genes IgA in the jejunum were analyzed using qRT-PCR according to Chang et al. [25]. Also, expression levels of inflammation-related genes were measured: IL-8, TNF-α, IL-22, IgA, TLR4, MYD88, NF-κB and NLRP3. Table 2 shows the sequence, product length and accession number of the primers used in this analysis. Briefly, RNA was extracted using RNA-easyTM Isolation Reagent (Vazyme, Nanjing, China) and reverse transcription, and qRT-PCR were conducted according to the protocol of commercial kits (Vazyme). The reference gene was β-actin and gene expression levels were calculated using the 2–ΔΔCt method [26].

Table 2 Primer sequences for qRT-PCR
Full size table

Immunofluorescence analysis

The expression of NF-κB p65 protein of jejunum was measured using immunofluorescence. After sectioning, tissue slides incubated with rabbit anti-NF-κB p65 (1:500, Cat: ET1603-12, HUABIO) antibody at 4 °C overnight. After washing three times for 5 min each with PBS, the slides were incubated with HRP goat anti-rabbit IgG (H + L) (1:300, ABclonal, AS014) secondary antibody for 60 min in darkness. Then, we use 4′-6-diamidino-2-phenylindole (Servicebio; G1012) for staining the nuclei.

Western blot (WB) analysis

Jejunal protein was extracted using radio immunoprecipitation assay lysis buffer (P0013B; Beyotime, Shanghai, China), and sample protein concentrations were determined by protein quantitative reagent kit-BCA method (23225; Thermo Fisher Scientific, Shanghai, China). Then the protein was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto 0.45-μm polyvinylidene fluoride (PVDF) membrane (Millipore, Eschborn, Germany) and blocked with Tris-buffered saline tween containing 5% nonfat milk at room temperature for 2 h. Then the PVDF membranes were incubated with primary antibodies overnight at 4 °C. The details for the primary antibodies are as follows: β-actin (1:10,000, Cat: AC026; ABclonal, Wuhan, China), anti-OCLN (1:500, Cat: 27260-1-AP; Proteintech Biotechnology, Chicago, IL, USA), TNF-α (1:500, Cat: 60291-1-Ig; Proteintech Biotechnology), anti-CLDN-1 (1:500, Cat: ER1906-37; HUABIO, Hangzhou, China), anti-CLDN-2 (1:500, Cat: 14085; ABclonal), IL-1β (1:1,000, Cat: 511369; ZenBio, Chengdu, China), TLR4 (1:1,000, Cat: 350146; ZenBio), MYD88 (1:500, Cat: 340629; ZenBio), NF-κB p65 (1:500, Cat: ET1603-12; HUABIO) and anti-phospho-NF-κB p65 (S529) (1:500, Cat: ET1604-27; HUABIO). Then, the PVDF membranes were incubated with secondary antibody HRP goat anti-rabbit IgG (H + L) (1:5,000; ABclonal; AS014) at room temperature for 2 h, and then BeyoECL Moon (P0018FS; Beyotime) reagent was used to observe the protein bands. Image Lab software (National Institutes of Health, DC, USA) was used to analyze the protein expression levels.

Statistical analysis

The experimental data were preliminarily analyzed using Excel 2019, and the data were checked for normal distribution and equal variance using the Shapiro–Wilk and Levene’s tests before analysis, respectively. One-way analysis of variance was performed using SPSS 24.0 (SPSS Inc., Chicago, IL, USA) statistical software followed by Tukey’s post hoc test to determine statistical significance. Results were expressed as mean ± SEM, with P < 0.05 considered significant. GraphPad Prism 8.0 (GraphPad Inc., La Jolla, CA, USA) was used for data processing and graphics. In this study, indicators were measured by WB in 4 replicates, and other indicators were measured from 6 replicates.

Results

Effects of Zn-Gly on growth performance of ducks with DSS-induced intestinal inflammation

Table S1 shows the impact of Zn-Gly on growth performance of meat ducks at 14 d. There was no significant difference (P = 0.409) in the initial average BW of meat ducks among the five groups. Also, there were no significant differences in ADG and ADFI (P > 0.05), while the F/G of the 120 and 170 mg/kg Zn groups was significantly lower than the DSS group at 14 d (P < 0.05). Compared with the CON group, the BW and ADG of ducks in DSS group were significantly decreased (P < 0.05) at 21 d. Compared with the DSS group, dietary 70, 120 and 170 mg/kg Zn supplementation significantly increased BW at 21 d (P < 0.05), but there was no significant effect on ADFI during 15–21 d (P > 0.05). And compared with the CON group, DSS group significantly increased F/G of ducks. While the F/G of the 70, 120 and 170 mg/kg Zn group was significantly decreased during 15–21 d, compared with DSS group (Table 3).

Table 3 Effects of Zn-Gly on growth performance of meat ducks from 15 to 21 d
Full size table

Effects of Zn-Gly on plasma ET and inflammatory cytokines in ducks with DSS-induced intestinal inflammation

After DSS treatment, plasma levels of ET and pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were significantly increased (P < 0.05, Fig. 1B–E). Different Zn-Gly concentrations were used to relieve the symptoms of enteritis in ducks, and levels of pro-inflammatory cytokines were significantly decreased by feeding Zn-Gly (P < 0.05).

Fig. 1
figure 1

Establishment of intestinal inflammation model with DSS in meat ducks and effects of Zn-Gly on plasma ET and proinflammatory cytokines. A Establishment of DSS model of enteritis in duck. B–E The ET, IL-1β, IL-6 and TNF-α contents of plasma in ducks by the ELISA method. Mean ± SEM are shown (n = 6). *P < 0.05

Effect of Zn-Gly on intestinal morphology of ducks with DSS-induced intestinal inflammation

Intestinal HE staining of duodenum and jejunum showed that DSS and Zn levels had no significant effect on duodenal villus height, crypt depth and villus height/crypt depth (V/C) (P > 0.05, Fig. 2). For the jejunum, the villus height, crypt depth and V/C in DSS group did not significantly differ from those in the CON group (P > 0.05), but villus height was significantly increased in the 70 mg/kg Zn group (P < 0.05).

Fig. 2
figure 2

Effect of Zn-Gly on intestinal morphology of ducks with DSS-induced intestinal inflammation. A HE stained intestinal sections of duodenum and jejunum. B Villus height, crypt depth and V/C of duodenum. C Villus height, crypt depth and V/C of jejunum. Mean ± SEM are shown (n = 6). *P < 0.05

Effect of Zn-Gly on intestinal inflammatory cytokines in ducks with DSS-induced intestinal inflammation

The DSS stimulation increased the levels of ET, IL-1β, IL-6 and TNF-α in jejunum tissue (P < 0.05), while Zn alleviated the increase of these pro-inflammatory cytokines (P < 0.05, Fig. 3A–E). Accordingly, results showed that protein expressions of IL-1β and TNF-α increased in jejunum of ducks treated with DSS, while Zn treatment significantly reverse their expression levels (P < 0.05, Fig. 3F and G). The levels of IL-10 and sIgA in the DSS group were significantly decreased (P < 0.05), while Zn supplementation reversed these indicators; notably, the sIgA level in the 70 mg/kg Zn group was significantly increased.

Fig. 3
figure 3

Effect of Zn-Gly on intestinal inflammatory cytokines in ducks with DSS-induced intestinal inflammation. A–D The ET, IL-1β, IL-6 and TNF-α contents of jejunum in ducks by the ELISA method (n = 6). E–G The relative protein expression of IL-1β and TNF-α in ducks (n = 4). H–K Contents of anti-inflammatory factors and immunoglobulins in jejunum of meat ducks (n = 6). L and M Relative expression of immune barrier related genes in jejunum of meat ducks (n = 6). Mean ± SEM are shown. *P < 0.05

Effects of Zn-Gly on intestinal barrier function in ducks with DSS-induced intestinal inflammation

Intestinal permeability

The plasma FITC-D flux and D-LA content in the DSS group were significantly higher than those in the CON group (P < 0.05, Fig. 4A and B); however, both levels significantly decreased after Zn treatment (P < 0.05). Therefore, Zn may maintain the integrity of the intestinal barrier by regulating intestinal permeability.

Fig. 4
figure 4

Effects of Zn-Gly on intestinal permeability and physical barrier function in ducks with DSS-induced intestinal inflammation. A and B The FITC-D and D-LA content in plasma of ducks (n = 6). C and D The relative expression of physical barrier-related genes (n = 6). E–H The relative protein expression of OCLN, CLDN-1 and CLDN-2 in ducks (n = 4). Mean ± SEM are shown. *P < 0.05

Physical barrier

The TJ proteins play an important role in gut integrity, including ZO-1, OCLN, CLDN-1 and CLDN-2 [27]. Expression of genes ZO-1 and CLDN-2 were significantly affected in the DSS group (P < 0.05, Fig. 4C–G). The OCLN protein decreased significantly after DSS treatment, and expression levels of OCLN and CLDN-1 proteins were significantly higher after feeding Zn (P < 0.05). In addition, the mRNA levels of CLDN-2 in jejunum tissues of DSS group were significantly increased, but significantly down-regulated gene and protein levels of CLDN-2 by Zn (P < 0.05).

Mucus barrier

Generated by goblet cells, MUC2 is the major constituent of intestinal mucus layer [28]. Inhibition of the Notch pathway can result in a conversion of epithelial cells to goblet cells [29, 30]. Compared with CON, the number of goblet cells, and MUC2 mRNA expression in jejunum of ducks with DSS-induced intestinal inflammation did not significantly differ (P > 0.05, Fig. 5A, B and D), but the concentrations of MUC2 was significantly decreased (P < 0.05, Fig. 5C). The above indicators were significantly improved after Zn-Gly treatment (P < 0.05). In addition, the expression level of Notch1 mRNA associated with intestinal goblet cell differentiation was significantly increased after DSS treatment (Fig. 5E), while 70 mg/kg Zn significantly decreased the gene expression level (P < 0.05). The Zn-Gly treatment significantly decreased the gene expression level of Notch2 in intestinal tissues (P < 0.05, Fig. 5F).

Fig. 5
figure 5

Effects of Zn-Gly on the number of goblet cells and chemical barrier function in ducks with DSS-induced intestinal inflammation. A PAS staining of jejunum. B The number of goblet cells per villus of jejunum in ducks. C Relative PAS-positive content of jejunum. D The MUC2 content of jejunum in ducks. E–G The relative gene expression of MUC2, Notch1 and Notch2 of jejunum in ducks. Mean ± SEM are shown (n = 6). *P < 0.05

Immune barrier

Activation of TLR4/NF-κB signaling pathway is associated with the pathogenesis of inflammation [31]. Therefore, we further analyzed the impact of Zn-Gly on TLR4/NF-κB pathway (Fig. 6A–H). First, expression levels of genes TLR4, MYD88 and NLRP3 were significantly increased in the DSS group, while the mRNA levels of NF-κB, MYD88 and NLRP3 were significantly decreased by adding Zn-Gly (P < 0.05). Compared with CON, DSS treatment significantly increased TLR4, NF-κB p65 and phosphorylated NF-κB p65 (p-NF-κB p65) protein levels. The Zn significantly down-regulated the TLR4, MYD88, NF-κB p65 and p-NF-κB p65 proteins in jejunum treated with DSS (P < 0.05). These data suggested that Zn-Gly could improve intestinal barrier functions, possibly by inhibiting the TLR4/NF-κB signaling pathway.

Fig. 6
figure 6

Effects of Zn-Gly on immune barrier function in ducks with DSS-induced intestinal inflammation. A and B The relative gene expression of TLR4, NF-κB p65, MYD88, NLRP3 of jejunum in ducks (n = 6). C Immunofluorescence section of NF-κB p65 in jejunum. D–H The relative protein expression levels of TLR4, MYD88, p-NF-κB p65, NF-κB p65 of jejunum in ducks (n = 4). Mean ± SEM are shown. *P < 0.05

Discussion

In recent years, there has been considerable interest in exploring alternatives to antibiotics in animal production. There is ample evidence that trace element Zn can alleviate inflammatory response and promote intestinal barrier function [22, 32, 33]. The DSS impairs the intestinal epithelial barrier via stimulation of the secretion of pro-inflammatory cytokines that directly disrupt epithelial tight junctions. Firstly, clinical signs in murine models given oral DSS are a loss of BW along with intestinal histological lesions [34]. Vicuna et al. [24] also demonstrated that groups received DSS (0.75% in water for 5 d) had an increase in gut permeability associated with the serum levels of FITC-D. Furthermore, Zou et al. [20] explored the mechanism and revealed that increased inflammatory cytokine profiles measured in chickens with DSS could induce intestinal inflammation. These studies showed that the gut inflammation model measured by BW, intestinal morphology, FITC-D and inflammatory cytokine could be effective to study enteritis symptoms. In the current study, we measured the villus height, crypt depth and V/C of the jejunum, which in the DSS group did not significantly differ from those in the CON group; however, villus height and V/C had a decreasing trend, indicating that the DSS dose did not cause intestinal necrosis, but caused a significant decrease in BW and FITC-D and an increase in the content of pro-inflammatory cytokines, which can be used as a model of intestinal inflammation for further study. In this study, we analyzed the effect of organic Zn-Gly on a DSS-induced enteritis model of ducks and used the above indicators. These findings indicate that oral gavage with DSS caused non-necrotic enteritis, which could be developed as gut inflammation model in meat ducks.

In modern intensive feeding, ducks need a balanced nutrition strategy, especially the addition of trace elements. Although the amount of trace elements required by ducks is low, they play an important role in the enzyme system, physiology, metabolism, reproduction and growth of animals [35]. Among trace elements (copper, iron, manganese and Zn), Zn is important in stimulating piglet growth [36]. In poultry diets, Zn has long been used as a growth promoter and is used frequently due to its high bioavailability. In this study, there was significantly increased BW and ADG in meat ducks after Zn-Gly supplementation at 21 d, especially when 170 mg/kg Zn was added. Organic Zn may improve mineral absorption and reduce its binding to anti-nutritional factors such as phytic acid. However, we observed no effect of Zn-Gly on ADFI. In fact, in this study, DSS significantly reduced BW at 21 d and ADG during 15–21 d in meat ducks, and addition of Zn mitigated the loss of BW and ADG, with 170 mg/kg of Zn having the best effect. The ADFI of the DSS group was lower than that of other groups, but the difference was not significant. This showed that DSS treatment did not affect the feed intake of meat ducks. It is noteworthy that F/G of the DSS group was significantly higher than that of other treatment groups, demonstrating that the enteritis model was successfully constructed in the DSS group, and the feed conversion efficiency of meat ducks was affected. There was no significant difference in F/G of meat ducks between the control and the Zn supplementation groups, indicating that Zn may have a certain alleviating effect on enteritis of ducks. Therefore, Zn-Gly could reduce jejunum damage caused by DSS, restore intestinal barrier function and reduce gut permeability.

The effects of the organic Zn source Zn-Gly on intestinal health [37], tissue Zn deposition [38], antioxidant status [39], carcass traits [40] and body immune state [41] have been studied, suggesting its potential anti-inflammatory effect to promote intestinal health in animals. This study provided evidence of the protective effect of Zn-Gly on enteritis and explores the possible mechanism of its effect. A recent study demonstrated that Zn-Gly supplementation activated cellular and humoral immune responses in poultry, helping to maintain the balance between Th1 and Th2 responses and enhancing resistance to infection [41]. Studies have shown that IL-1β, IL-6 and TNF-α lead to intestinal barrier dysfunction and cell apoptosis [42]. In addition, Zn was reported to inhibit the production of IL-1β, IL-6 and TNF-α during LPS-induced damage to duck intestinal epithelial cells [43]. Therefore, we selected IL-1β, IL-6 and TNF-α as pro-inflammatory cytokines to further investigate the effect of DSS stimulation on inflammation. In the current study, Zn-Gly inhibited DSS-induced the production of pro-inflammatory IL-1β, IL-6 and TNF-α in plasma of meat ducks, suggesting that Zn-Gly alleviates intestinal inflammation via modulating inflammatory cytokine contents. In the meantime, contents of intestinal pro-inflammatory cytokines decreased following Zn-Gly supplementation, and the anti-inflammatory cytokines were increased. The results might indicate that the protective effects of Zn-Gly on intestinal inflammation were closely related to inhibiting production of pro-inflammatory cytokines and promoting production of anti-inflammatory cytokines.

Intact intestinal epithelial cells are the structural basis for maintaining normal barrier function. Overproduction of inflammatory cytokines erodes intestinal epithelial mucosa by destroying TJ proteins [44]. We found that Zn-Gly could reverse the decrease of OCLN and CLDN-1 genes and protein expression induced by DSS, while the Zn-Gly group significantly decreased the CLDN-2 gene and protein expression, suggesting that Zn-Gly directly protected the physical barrier of jejunum of ducks. Goblet cells protect against intestinal invasion by bacteria and pathogens by producing mucin [45]. Generated by goblet cells, MUC2 is the major macromolecular constituent of the intestinal mucus layer and is responsible for host defense [28]. An appropriate number of goblet cells are essential for MUC2 synthesis and maintaining the mucus layer. In contrast, dysfunctions in goblet cells promote inflammatory bowel disease [46, 47]. Studies have shown that MUC2 deficiency exacerbates colon inflammatory responses and spontaneous colitis occurs in MUC2 knockout mice [46]. In this study, PAS staining showed Zn-Gly restored goblet cell numbers and reversed DSS-induced downregulation of MUC2. Meanwhile, the Notch signaling pathway plays an important role in cell differentiation in the intestine. Inhibition of the Notch pathway in the intestinal epithelium results in a rapid and complete conversion of all epithelial cells to secretory cells such as goblet cells [29, 30]; consistent with this previous study, we found that 70 mg/kg Zn significantly inhibited Notch1 and Notch2 gene expression compared with the DSS group. In addition, enteritis caused by DSS also increased intestinal permeability, allowing inflammatory substances in the intestinal cavity to enter the circulation. Consistent with this, DSS ingestion resulted in significantly higher plasma ET, D-LA and FITC-D levels; and Zn-Gly reduced the D-LA and ET levels. In conclusion, Zn-Gly alleviated DSS-induced epithelial cell damage by enhancing the TJs, reducing intestinal permeability, decreasing intestinal inflammation and restoring intestinal barrier function.

Zhao et al. [48] showed that DSS-induced enteritis was associated with elevated IL-6, IL-1β and TNF-α levels as well as decreased levels of anti-inflammatory IL-10 in the gut. The Zn-Gly reduces intestinal inflammation response by regulating inflammatory cytokines. As previously noted, the DSS-induced increase in intestinal permeability allowed harmful substances to trigger an inflammatory response, activating the TLR4/NF-κB pathway [7]. In inflammatory responses, NF-κB is believed to be a key transcription factor that is activated and induces transcription of pro-inflammatory mediators. The cytokines trigger positive feedback regulation in inflammatory activation, ultimately damaging intestinal tissue [49]. In this study, Zn-Gly treatment reduced the protein expression of TLR4, MYD88, NF-κB p65 and p-NF-κB p65 in jejunum tissue, suggesting that Zn-Gly restore intestinal barrier function by reducing inflammation and inhibition the TLR4/ NF-κB p65 pathway.

Conclusions

The Zn-Gly alleviated DSS-induced enteritis in ducks and restored intestinal barrier function by relieving inflammatory response and gut permeability. The underlying mechanism for the effect of Zn-Gly on the intestinal barrier protection may be regulated by repressing the TLR4/MYD88/NF-κB p65 signaling pathway. The present work provides a scientific foundation for Zn-Gly application in ducks.

Data Availability

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Abbreviations

  • ADFI:: Average daily feed intake
  • ADG:: Average daily gain
  • BW:: Body weight
  • CLDN1:: Claudin1
  • CLDN2:: Claudin2
  • D-LA:: D-lactic acid
  • DSS:: Dextran sulfate sodium
  • ET:: Endotoxin
  • F/G:: Feed to gain ratio
  • FITC-D:: Fluorescein isothiocyanate
  • HE:: Hematoxylin and eosin
  • IgA:: Immunoglobulin A
  • IL-8:: Interleukin 8
  • LPS:: Lipopolysaccharide
  • MUC2:: Mucin2
  • MYD88:: Myeloid differentiation factor 88
  • NF-κB:: Nuclear factor κB
  • OCLN:: Occludin
  • PAS:: Periodic acid-Schiff
  • p-NF-κB p65:: Phosphorylated NF-κB p65
  • sIgA:: Secretory immunoglobulin A
  • TJ:: Tight junction
  • TLR4:: Toll-like receptor
  • TNF-α:: Tumor necrosis factor α
  • V/C:: Villus height/crypt depth
  • Zn-Gly:: Zinc glycine chelate
  • ZO-1:: Zonula occludens-1

References

  1. 1.Ding S, Li G, Chen S, Zhu F, Hao J, Yang F, et al. Comparison of carcass and meat quality traits between lean and fat Pekin ducks. Anim Biosci. 2021;34(7).(2021)1193–201.: 1193.
    10.5713/ajas.19.0612
    Article|CAS|PubMed|Google Scholar
  2. 2.Kogut MH, Genovese KJ, Swaggerty CL, He H, Broom L. Inflammatory phenotypes in the intestine of poultry.(2018)not all inflammation is created equal.Poult Sci.: 2339.
    10.3382/ps/pey087
    Article|CAS|PubMed|Google Scholar
  3. 3.Dal Pont GC, Farnell M, Farnell Y, Kogut MH. Dietary factors as triggers of low-grade chronic intestinal inflammation in poultry. Microorganisms. 2020;8(1).(2020)139.: 139.
    10.3390/microorganisms8010139
    Article|CAS|PubMed|Google Scholar
  4. 4.Anthony K, Wade B. The true cost of necrotic enteritis. World Poult. 2015;31(7).(2015)16–7.: 16.
    Article|CAS|PubMed|Google Scholar
  5. 5.Breitkopf K, Nagy LE, Beier JI, Mueller S, Weng H, Dooley S. Current experimental perspectives on the clinical progression of alcoholic liver disease. Alcohol Clin Exp Res. 2009;33(10).(2009)1647–55.: 1647.
    10.1111/j.1530-0277.2009.01015.x
    Article|CAS|PubMed|Google Scholar
  6. 6.Cani PD, Jordan BF. Gut microbiota-mediated inflammation in obesity.(2018)a link with gastrointestinal cancer.Nat Rev Gastroenterol Hepatol.: 671.
    10.1038/s41575-018-0025-6
    Article|CAS|PubMed|Google Scholar
  7. 7.Maares M, Haase H. Zinc and immunity.(2016)An essential interrelation.Arch Biochem Biophys.: 58.
    10.1016/j.abb.2016.03.022
    Article|CAS|PubMed|Google Scholar
  8. 8.Krezel A, Maret W. The biological inorganic chemistry of zinc ions. Arch Biochem Biophys. 2016;611.(2016)3–19.: 3.
    10.1016/j.abb.2016.04.010
    Article|CAS|PubMed|Google Scholar
  9. 9.Jarosz A, Marek A, Gradzki Z, Kwiecien M, Zylinska B, Kaczmarek B. Effect of feed supplementation with zinc glycine chelate and zinc sulfate on cytokine and immunoglobulin gene expression profiles in chicken intestinal tissue. Poul Sci. 2017;96(12).(2017)4224–35.: 4224.
    10.3382/ps/pex253
    Article|CAS|PubMed|Google Scholar
  10. 10.Sarkar P, Saha T, Sheikh IA, Chakraborty S, Aoun J, Chakrabarti MK, et al. Zinc ameliorates intestinal barrier dysfunctions in shigellosis by reinstating claudin-2 and -4 on the membranes. Am J Physiol Gastrointest Liver Physiol. 2018;316(2).(2018)G229–46.
    10.1152/ajpgi.00092.2018
    Article|CAS|PubMed|Google Scholar
  11. 11.Shao Y, Zhao L, Wolf PG, Gao Y, Guo Y, Zhang B. Zinc supplementation, via GPR39, upregulates PKCζ to protect intestinal barrier integrity in Caco-2 cells challenged by salmonella enterica serovar typhimurium. J Nutr. 2017;147(7).(2017)1282–9.: 1282.
    10.3945/jn.116.243238
    Article|CAS|PubMed|Google Scholar
  12. 12.Levkut M, Husáková E, Bobíková K, Karaffová V, Levkutová M, Ivanišinová O, et al. Inorganic or organic zinc and MUC-2, IgA, IL-17, TGF-β4 gene expression and sIgA secretion in broiler chickens. Food Agr Immunol. 2017;28(5).(2017)801–11.: 801.
    10.1080/09540105.2017.1313202
    Article|CAS|PubMed|Google Scholar
  13. 13.He B, Bortoluzzi C, King WD, Graugnard D, Dawson KA, Applegate TJ. Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maxima and Clostridium perfringens. Poul Sci. 2018;98(3).(2018)1146–52.: 1146.
    10.3382/ps/pey484
    Article|CAS|PubMed|Google Scholar
  14. 14.Prasad AS, Meftah S, Abdallah J, Kaplan J, Dardenne M. Serum thymulin in zinc deficiency. J Clin Invest. 1988;82(4).(1988)1202–10.: 1202.
    10.1172/JCI113717
    Article|CAS|PubMed|Google Scholar
  15. 15.King LE, Frentzel JW, Mann JJ, Fraker PJ. Chronic zinc deficiency in mice disrupted T cell lymphopoiesis and erythropoiesis while B cell lymphopoiesis and myelopoiesis were maintained. J Am Coll Nutr. 2005;24(6).(2005)494–502.: 494.
    10.1080/07315724.2005.10719495
    Article|CAS|PubMed|Google Scholar
  16. 16.Von Bulow V, Dubben S, Engelhardt G, Hebel S, Plumakers B, Heine H, et al. Zinc-dependent suppression of TNF-α production is mediated by protein kinase A-induced inhibition of Raf-1, IκB Kinase β, and NF-κB. J Immunol. 2007;179(6).(2007)4180–6.: 4180.
    10.4049/jimmunol.179.6.4180
    Article|CAS|PubMed|Google Scholar
  17. 17.Haase H, Ober-Blöbaum JL, Engelhardt G, Hebel S, Heit A, Heine H, et al. Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes. J Immunol. 2008;181(9).(2008)6491–502.: 6491.
    10.4049/jimmunol.181.9.6491
    Article|CAS|PubMed|Google Scholar
  18. 18.Kuttappan VA, Berghman LR, Vicuna EA, Latorre JD, Menconi A, Wolchok JD, et al. Poultry enteric inflammation model with dextran sodium sulfate mediated chemical induction and feed restriction in broilers. Poult Sci. 2015;94(6).(2015)1220–6.: 1220.
    10.3382/ps/pev114
    Article|CAS|PubMed|Google Scholar
  19. 19.Kuttappan VA, Vicuna EA, Faulkner OB, Huff GR, Freeman KA, Latorre JD, et al. Evaluation of changes in serum chemistry in association with feed withdrawal or high dose oral gavage with dextran sodium sulfate- (DSS-) induced gut leakage in broiler chickens. Poult Sci. 2016;95(11).(2016)2565–9.: 2565.
    10.3382/ps/pew171
    Article|CAS|PubMed|Google Scholar
  20. 20.Zou X, Ji J, Wang J, Qu H, Shu DM, Guo FY, et al. Dextran sulphate sodium (DSS) causes intestinal histopathology and inflammatory changes consistent with increased gut leakiness in chickens. Br Poult Sci. 2018;59(2).(2018)166–72.: 166.
    10.1080/00071668.2017.1418498
    Article|CAS|PubMed|Google Scholar
  21. 21.Chang Y, Zhang Z, Wu B, Zhao H, Liu G, Chen X, et al. Evaluating zinc glycine chelate in Cherry Valley ducks.(2021)Responses of growth performance, nutrient utilization, serum parameters, antioxidant status, meat quality and zinc accumulation.Anim Feed Sci Tech.: 114875.
    10.1016/j.anifeedsci.2021.114875
    Article|CAS|PubMed|Google Scholar
  22. 22.Wen M, Zhao H, Liu G, Chen X, Wu B, Tian G, et al. Effect of zinc supplementation on growth performance, intestinal development, and intestinal barrier-related gene expression in Pekin ducks. Biol Trace Elem Res. 2018;183(2).(2018)351–60.: 351.
    10.1007/s12011-017-1143-7
    Article|CAS|PubMed|Google Scholar
  23. 23.National Research Council. Nutrient requirements of poultry.(1994)9th revised edition.Washington, DC: The National Academies Press;.: 9.
    Article|CAS|PubMed|Google Scholar
  24. 24.Vicuna EA, Kuttappan VA, Tellez G, Hernandez-Velasco X, Seeber-Galarza R, Latorre JD, et al. Dose titration of FITC-D for optimal measurement of enteric inflammation in broiler chicks. Poult Sci. 2015;94(6).(2015)1353–9.: 1353.
    10.3382/ps/pev111
    Article|CAS|PubMed|Google Scholar
  25. 25.Chang Y, Mei J, Yang T, Zhang Z, Liu G, Zhao H, et al. Effect of dietary zinc methionine supplementation on growth performance, immune function and intestinal health of Cherry Valley ducks challenged with avian pathogenic Escherichia coli. Front Microbiol. 2022;13.(2022)849067.: 849067.
    10.3389/fmicb.2022.849067
    Article|CAS|PubMed|Google Scholar
  26. 26.Livak KJ, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods. 2001;25(4).(2001)402–8.: 402.
    10.1006/meth.2001.1262
    Article|CAS|PubMed|Google Scholar
  27. 27.Odenwald MA, Turner JR. The intestinal epithelial barrier.(2017)a therapeutic target? Nat Rev Gastroenterol Hepatol.: 9.
    10.1038/nrgastro.2016.169
    Article|CAS|PubMed|Google Scholar
  28. 28.Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, et al. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med. 2008;5(3).(2008)e54.
    10.1371/journal.pmed.0050054
    Article|CAS|PubMed|Google Scholar
  29. 29.Heuberger J, Kosel F, Qi J, Grossmann KS, Rajewsky K, Birchmeier W. Shp2/MAPK signaling controls goblet/paneth cell fate decisions in the intestine. P Natl Acad Sci USA. 2014;111(9).(2014)3472–7.: 3472.
    10.1073/pnas.1309342111
    Article|CAS|PubMed|Google Scholar
  30. 30.Stanger BZ, Datar R, Murtaugh LC, Melton DA. Direct regulation of intestinal fate by Notch. P Natl Acad Sci USA. 2005;102(35).(2005)12443–8.: 12443.
    10.1073/pnas.0505690102
    Article|CAS|PubMed|Google Scholar
  31. 31.Thummayot S, Tocharus C, Jumnongprakhon P, Suksamrarn A, Tocharus J. Cyanidin attenuates Aβ25-35-induced neuroinflammation by suppressing NF-κB activity downstream of TLR4/NOX4 in human neuroblastoma cells. Acta Pharmacol Sin. 2018;39(9).(2018)1439–52.: 1439.
    10.1038/aps.2017.203
    Article|CAS|PubMed|Google Scholar
  32. 32.Maares M, Keil C, Straubing S, Robbe-Masselot C, Haase H. Zinc deficiency disturbs mucin expression, O-Glycosylation and secretion by intestinal goblet cells. Int J Mol Sci. 2020;21(17).(2020)6149.: 6149.
    10.3390/ijms21176149
    Article|CAS|PubMed|Google Scholar
  33. 33.Wu X, Zhu Y, Zhang K, Ding X, Bai S, Wang J, et al. Growth performance, zinc tissue content, and intestinal health in meat ducks fed different specific surface area of micronized zinc oxide. Poult Sci. 2019;98(9).(2019)3894–901.: 3894.
    10.3382/ps/pez108
    Article|CAS|PubMed|Google Scholar
  34. 34.Yutao Y, Vasantha K, Guillaume D, Hang N, Hamed L, Sitaraman SV, et al. Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis. PloS One. 2009;4(6).(2009)e6073.
    Article|CAS|PubMed|Google Scholar
  35. 35.Wang L, Wang Z, Luo P, Bai S, Chen Y, Chen W. Dietary zinc glycine supplementation improves tibia quality of meat ducks by modulating the intestinal barrier and bone resorption. Biol Trace Elem Res. 2023;201(2).(2023)888–903.: 888.
    10.1007/s12011-022-03207-5
    Article|CAS|PubMed|Google Scholar
  36. 36.Wang Y, Tang J, Ma W, Feng J, Feng J. Dietary zinc glycine chelate on growth performance, tissue mineral concentrations, and serum enzyme activity in weanling piglets. Biol Trace Elem Res. 2010;133(3).(2010)325–34.: 325.
    10.1007/s12011-009-8437-3
    Article|CAS|PubMed|Google Scholar
  37. 37.Chand N, Ali P, Alhidary IA, Abdelrahman MA, Albadani H, Khan MA, et al. Protective effect of grape (Vitis vinifera) seed powder and zinc-glycine complex on growth traits and gut health of broilers following eimeria tenella challenge. Antibiotics-Basel. 2021;10(2).(2021)186.: 186.
    10.3390/antibiotics10020186
    Article|CAS|PubMed|Google Scholar
  38. 38.Jarosz L, Marek A, Gradzki Z, Laskowska E, Kwiecien M. Effect of zinc sulfate and zinc glycine chelate on concentrations of acute phase proteins in chicken serum and liver tissue. Biol Trace Elem Res. 2019;187(1).(2019)258–72.: 258.
    10.1007/s12011-018-1346-6
    Article|CAS|PubMed|Google Scholar
  39. 39.Zhang L, Wang J, Wang Q, Li K, Guo T, Xiao X, et al. Effects of maternal zinc glycine on mortality, zinc concentration, and antioxidant status in a developing embryo and 1-day-old chick. Biol Trace Elem Res. 2018;181(2).(2018)323–30.: 323.
    10.1007/s12011-017-1028-9
    Article|CAS|PubMed|Google Scholar
  40. 40.Kwiecien M, Winiarska-Mieczan A, Milczarek A, Tomaszewska E, Matras J. Effects of zinc glycine chelate on growth performance, carcass characteristics, bone quality, and mineral content in bone of broiler chicken. Livest Sci. 2016;191.(2016)43–50.: 43.
    10.1016/j.livsci.2016.07.005
    Article|CAS|PubMed|Google Scholar
  41. 41.Jarosz L, Marek A, Gradzki Z, Kwiecien M, Kalinowski M. The effect of feed supplementation with zinc chelate and zinc sulphate on selected humoral and cell-mediated immune parameters and cytokine concentration in broiler chickens. Res Vet Sci. 2017;112.(2017)59–65.: 59.
    10.1016/j.rvsc.2016.09.007
    Article|CAS|PubMed|Google Scholar
  42. 42.Koelink PJ, Bloemendaal FM, Li B, Westera L, Brink G. Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling. Gut. 2019;69(6).(2019)1053–63.: 1053.
    10.1136/gutjnl-2019-318264
    Article|CAS|PubMed|Google Scholar
  43. 43.Xie Y, Wen M, Zhao H, Liu G, Chen X, Tian G, et al. Effect of zinc supplementation on growth performance, intestinal development, and intestinal barrier function in Pekin ducks with lipopolysaccharide challenge. Poult Sci. 2021;100(12).(2021)101462.: 101462.
    10.1016/j.psj.2021.101462
    Article|CAS|PubMed|Google Scholar
  44. 44.Al-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front in Biosci. 2009;14.(2009)2765–78.: 2765.
    10.2741/3413
    Article|CAS|PubMed|Google Scholar
  45. 45.Kim YS, Ho SB. Intestinal goblet cells and mucins in health and disease.(2010)recent insights and progress.Current Gastro Rep.: 319.
    10.1007/s11894-010-0131-2
    Article|CAS|PubMed|Google Scholar
  46. 46.Van der Sluis M, De Koning BAE, De Bruijn ACJM, Velcich A, Meijerink JPP, Van Goudoever JB, et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology. 2006;131(1).(2006)117–29.: 117.
    10.1053/j.gastro.2006.04.020
    Article|CAS|PubMed|Google Scholar
  47. 47.Shirazi T, Longman RJ, Corfield AP, Probert CSJ. Mucins and inflammatory bowel disease. Postgrad Med J. 2000;76.(2000)473–8.: 473.
    10.1136/pmj.76.898.473
    Article|CAS|PubMed|Google Scholar
  48. 48.Zhao H, Cheng N, Zhou W, Chen S, Cao W. Honey polyphenols ameliorate DSS-induced ulcerative colitis via modulating gut microbiota in rats. Mol Nutr Food Res. 2019;63(23).(2019)e1900638.
    10.1002/mnfr.201900638
    Article|CAS|PubMed|Google Scholar
  49. 49.Peng Z, Wan P, Deng Y, Shen W, Liu R. Lipopolysaccharide exacerbates to the migration, invasion, and epithelial-mesenchymal transition of esophageal cancer cells by TLR4/NF-kappa B axis. Environ Toxicol. 2023;38(6).(2023)1090–9.: 1090.
    10.1002/tox.23750
    Article|CAS|PubMed|Google Scholar

Acknowledgements

The authors also wish to thank the laboratory staff for their ongoing assistance and International Science Editing ( http://www.internationalscienceediting.com ) for editing this manuscript.

Funding

This study was supported by the Natural Science Foundation of Sichuan Province (No. 2022NSFSC0060). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Ethics Declaration

Ethics approval and consent to participate

All experimental procedures followed the regulations of the Animal Care and Use Committee of Sichuan Agricultural University (No. 20180718), and were in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

All authors declare no other competing interests.

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