Dietary ethylenediamine dihydroiodide improves intestinal health in Cherry Valley ducks

This study investigated the effect of ethylenediamine dihydroiodide (EDDI) on the growth performance, thyroid function, immune function, intestinal development, intestinal permeability, intestinal barrier functions and microbial characteristics of Cherry Valley ducks. The results showed that the addition of EDDI significantly increased body weight, average daily gain, serum level of lymphocytes, basophils, triiodothyronine, thyroxine and thyrotropin, villus height, and villus height-to-crypt depth ratio, and significantly decreased crypt depth, diamine oxidase, serum D-Lactic acid of ducks (P < 0.05). EDDI also significantly up-regulated the mRNA expression of zonula occludens-1, zonula occludens-2, zonula occludens-3, mucin 2, secretory immunoglobulin A, interleukin-10 and avian β-defensin 2 in the jejunum and ileum (P < 0.05), and down-regulated the mRNA expression of occludin and interleukin-6 in the jejunum and ileum. Additionally, the addition of EDDI significantly increased cecal level of acetic acid, propionic acid, butyric acid (P < 0.05). Cecal microbiome analysis indicated that the addition of EDDI significantly increased the relative abundance of these microorganisms that can produce short-chain fatty acids, mainly including Actinobacteria, Verrucomicrobia, Clostridiales and Lactobacillales, and decreased the relative abundance of pathogenic bacteria Deferribactere. Interestingly, triiodothyronine and thyroxine levels were highly positively correlated with the relative abundance of Actinobacteria. These results revealed that the addition of EDDI could promote the growth and development of meat ducks by improving their thyroid function, immune function, intestinal development and intestinal barrier functions of ducks.


INTRODUCTION
Iodine is one of the essential trace elements in animals, which promotes the growth performance of animals by regulating metabolism, enhancing immunity, eliminating reactive oxygen species, and other physiological functions (Iannaccone et al., 2019).Sirakov and Plateroti (2011) found that iodine deficiency or excess caused thyroid dysfunction, led to decreased immune function, and then affect the growth and development of the body.At present, the dietary iodine proposed by the National Research Council may not meet the nutritional requirements of fast-growing poultry, and its genetic growth potential ca not be fully exerted (Inal et al., 2001).Therefore, in the current poultry breeding process, iodine is mainly added to trace mineral premix in inorganic form (Ammerman and Miller, 1972).However, a study reported that inorganic trace minerals have high loss rate and low utilization rate, but organic chelated trace minerals can solve these problems to some extent because of their higher bioavailability (Bao and Choct, 2009;Yu et al., 2022).
Additionally, the growth and development of poultry is also closely related to intestinal health.It has been reported that the intestinal barrier dysfunction is considered as one of the main reasons for the decrease of production performance and the increase of health problems in poultry (Lambert, 2009).However, in the current intensive farming process, factors such as highdensity farming, heat stress and toxins will cause intestinal barrier dysfunction of poultry, destroy intestinal health, lead to reduced production performance, and thus cause serious economic losses (Goo et al., 2019).Therefore, it is still one of the important challenges in the field of poultry nutrition research to increase the growth performance by improving the intestinal barrier function and maintaining the intestinal health of ducks through nutritional strategies.Fomichev et al. (2019) found that organic iodine can regulated the microbial composition of sheep to maintain intestinal health.Additionally, organic iodine also significantly improved the antibacterial activity and bioavailability of broilers (Ledesma et al., 2018).The result of the earlier studies indicated that organic iodine has a positive effect on the intestinal health of animals.Ethylenediamine dihydroiodide (EDDI) is an easily available organic chelated iodine (Ammerman et al., 1995).However, there was no scientific research on the positive effects of EDDI on the intestinal health of animals.In view of this fact, we hypothesized that EDDI could improve the intestinal health and growth performance of Cherry Valley ducks by positively influencing their thyroid function, immune function, intestinal development, intestinal barrier function and microbial characteristics.

Animals and Experimental Design
A total of 360 one-day-old Cherry Valley ducks (Sichuan Mianying Duck Industry Co., Ltd., Mianyang, China) were randomly divided into 6 groups with 6 replicates in each group and 10 ducks in each replicate.The ducks fed diets with 0, 1, 2, 4, 8, and 16 mg/kg iodine (EDDI form, Jilongda Co., Ltd, Guanghan, China) for 35 d.The nutritional components and requirements were listed in Table S1.The iodine concentration of the 6 diets were analyzed to be 0.82, 1. 90, 3.23, 4.35, 9.27, 17.16 mg/kg for the diets at the age of 1 to 14 d, and 0.97, 2.05, 3.54, 5.47, 9.86, 17.65 mg/kg for the diets at the age of 15 to 35 d.The weight and feed consumption of ducks were recorded at each stage during the experiment.

Sample Collection
Ducks were fasted for 12 h on 35 d, and an average weight duck (n = 6) was randomly selected from each repetition.After 10 mL of blood was collected by jugular vein and centrifuged at 4°C for 3,500 r/min for 15 min to separate serum, which was placed in a refrigerator at -20°C for analysis.The selected experimental ducks (36 ducks) were anesthetized and slaughtered.The jejunum and ileum segments with a length of 4 cm were washed with PBS and fixed in 4% paraformaldehyde for histomorphological analysis.Additionally, jejunum and ileum tissues were collected and frozen in liquid nitrogen, and then stored to -80°C for indicator analysis.The contents of caeca were removed and collected in sterile 1.5-mL tubes.Then caecum contents were flash-frozen in liquid N 2 and stored at −80°C until microbial community analysis.

Hematological Analyses
Hematological index including lymphocytes, basophils and eosinophils, were detected by automatic analyzer (Coulter Max M, Coulter Electronics Ltd, Luton, UK).Additionally, the levels of triiodothyronine (T3), thyroxine (T4) and thyrotropin (TSH) were examined by kit (Chengdu Weskong Bioscience and Technology Co., Ltd, Chengdu, China), and the specific steps were carried out according to the instructions.

Histomorphological Analysis
After dehydration, pruning, embedding, slicing and hematoxylin-eosin staining, the intestinal segment was observed by BA210Digital microscope camera (Motic, Xiamen, China), and the areas with better slices were randomly selected for photographing, and the villi height (VH) and crypt depth (CD) of each slice were measured, and the ratio of villi to crypt (V: C) was calculated on the basis of the method reported by Qin et al. (2019).

Determination of Diamine Oxidase and d-Lactate
The levels of diamine oxidase (DO) and D-lactic acid (D-LA) in serum were determined by ELISA kit.The kit was purchased from Chengdu Micro-Space Biotechnology Co., Ltd.(Chengdu, China), and the results were read by SpectraMax 190 full-wavelength microplate reader.

Quantitative Real-Time PCR
Total RNA was isolated from frozen jejunum and ileum tissue using trizol reagent (Takara, Beijing, China) according to the manufacturer's specifications.Real-time quantitative PCR was conducted using the primers listed in Table S2.All PCR contained 5.0 mL of SYBR Green qPCR Mix, 0.2 mL of cDNA, 0.3 mL of each primer, and 4.2 mL of double distilled water in a final volume of 20 mL.Amplification program was 95°C 15 min, followed by forty cycles of 95°C 5 s and 60°C 30 s, and a final melting curve analysis.The reference gene b-actin served as a control to normalize the mRNA expression level.

Determination of Short Chain Fatty Acids
Approximately 0.5g cecum contents were mixed in 2 mL ultrapure water and centrifuged at 3,000 g for 15 min.The supernatant was mixed with ice-cold metaphosphoric acid solution at 4°C for 30min.After centrifugation again, the concentrations of acetic acid (AA), propionic acid (PA), isobutyric acid (IBA), butyric acid (BA), valeric acid (VA), and isovaleric acid (IVA) were determined by GC system (Varian, GC CP3800).

Microbiome Analysis
16S rDNA gene sequencing analysis was performed by shanghai applied protein technology Co., Ltd.(China, Shanghai).The total genome DNA of the cecal contents was extracted using CTAB/SDS method.After detecting the integrity of DNA, the concentration of extracted DNA samples were measured using a NanoDrop Spectrophotometer, and only qualified samples were used for 16S rDNA sequencing analysis.The 16S rDNA V3-V4 region (Forward primer: CCTACGGGRBGCASCAG; Reverse primer: GGACTACNNGGGTATCTAAT) was selected for PCR amplification.After the PCR amplification products were purified, sequencing was performed using the Illumina Miseq sequencing platform (NovaSeq PE250).After the raw reads were spliced, filtered and de-chimeric, the last available data (highquality clean tags) was subjected to operational taxonomic units (OTUs) clustering analysis according to the principle of 97% similarity.The species classification information corresponding to each OTU could be obtained through annotation in the SILVA database through OTU sequence alignment (Liu et al., 2021).

Statistical Analyses
The experimental data were preliminarily sorted and calculated by Microsoft office 2016.SPSS 21.0 (SPSS Inc., Chicago, IL) software was used for 1-way analysis of variance among different levels of EDDI treatment groups, and Duncan method was used for multiple comparisons.Quadratic curve model fitting was used to determine the appropriate dosage of iodine added in the form of EDDI in duck diet.The experimental results were presented by the mean § standard error, and P < 0.05 was regarded as significant difference.Pearson correlation coefficient between cecal microbial abundance and phenotypic characteristics was analyzed by SPSS 21.0.

Growth Performance Response to Dietary EDDI Supplementation
As shown in Table 1, the addition of 2, 4, 8, and 16 mg/kg EDDI significantly increased (P < 0.05) the BW of ducks at the d 14 and d 35 compared with the control group.The addition of 4 and 8 mg/kg EDDI significantly increased (P < 0.05) the ADG of ducks at the d 1 to d 14.Additionally, the ADG of ducks at the d 1 to d 35 was also significantly increased (P < 0.05) in the 4 mg/kg EDDI group.However, no differences in ADG of the ADG of ducks at the specified d 15 to d 35, as well as the ADFI and F/G in all periods (P > 0.05).Interestingly, the ADG of ducks at the d 1 to d 35 and in the BW of ducks at the d 14 and d 35 showed linear and quadratic relationship with EDDI (P < 0.05).Based on the quadratic regression analysis for BW of ducks at the d 14, d 35 and the ADG of ducks at the d 1 to d 35, the optimal dietary EDDI of ducks was estimated to be 7.40, 9.60, and 9.40 mg/kg (as iodine), respectively.

Immune Function and Thyroid Function Response to Dietary EDDI Supplementation
The impact of EDDI on immune and thyroid function of ducks was presented in Table 2. Compared with the control group, the levels of lymphocytes, T3 and T4 in the blood of ducks in the addition of EDDI group were significantly increased (P < 0.05).And the addition of 16 mg/kg EDDI also significantly increased (P < 0.05) the level of basophils compared with the control group.However, the levels of eosinophils and TSH was not found to be significantly different among the experimental groups (P > 0.05).

Integrity of Intestinal Mucosal Morphology and Intestinal Permeability in Response to Dietary EDDI Supplementation
Table 3 and Figure 1 showed the integrity of intestinal mucosal morphology.Compared with the control group, the VH of ileum was significantly improved in all EDDI addition groups (P < 0.05).Additionally, the 16 mg/kg EDDI group significantly increased the V/C of jejunum, decreased the CD of ileum (P < 0.05) compared with the control group.Table 4 showed the effect of EDDI on intestinal permeability of ducks, compared with the control group, the addition of 4, 8 and 16 mg/kg EDDI significantly decreased the content of DAO and D-LA of ducks (P < 0.05).

Intestinal Barrier Function Response to Dietary EDDI Supplementation
The influence of EDDI on intestinal physical barrier function of ducks was presented in Figure 2. Compared with the control group, the relative expression levels of ZO-1, ZO-2, and ZO-3 in intestine were markedly increased by the addition of 4, 8, and 16 mg/kg EDDI (P < 0.05).And the addition of EDDI observably also decreased the relative expression level of OCLN in intestine (P < 0.05).Compared with the control group, the relative expression of MUC2 in intestine was significantly increased by the addition of EDDI (P < 0.05) (Figure 2).However, no differences in the relative expression level of TFF2 were observed among the treatment groups (P > 0.05).The influence of EDDI on intestinal immune barrier of ducks was presented in Figure 3. Compared with the control group, the addition of EDDI observably increased the relative expression of sIgA and IL-10 in intestine (P < 0.05).And 16 mg/kg EDDI markably increased the relative expression of AvBD-2 in intestine compared with the control group (P < 0.05).The addition of 2, 4, 8 and 16 mg/kg EDDI also markedly decreased the relative expression of IL-6 in intestine (P < 0.05).

The Concentration of Short-Chain Fatty Acids in Cecum Response to Dietary EDDI Supplementation
Figure 4 showed the effect of EDDI on the concentration of short-chain fatty acids (SCFAs) in cecum of ducks, compared with the control group, the addition of 1, 4, and 16 mg/kg EDDI observably increased (P < 0.05) the level of AA, PA, and BA in cecum of ducks.However, no differences in the levels of IBA, VA and IVA were observed among the treatment groups (P > 0.05).

Sequencing, Microbial Abundance, and Diversity
After 2,214,394 raw reads were spliced, quality controlled and de-chimerized, 2,182,697 joined tags were obtained, with an average of 60,630 joined tags per sample (Table S3).OTU cluster analysis showed that there were 115,480 unique OTU candidates with 97% sequence similarity, among which 4,546 candidates were shared by 6 treatment groups (Figure 5A).Based on the results of OTU annotation, Figure 5B and Figure 5C showed the top 10 class and genus of relative richness, respectively.And the most abundant class and genus of ducks were Clostridia and Bacteroidetes, respectively.

Intestinal Microbial Barrier Function Response to Dietary EDDI Supplementation
Table 5 and Figure S1A showed the addition of EDDI observably up-regulated the richness of Firmicutes, Actinobacteria, and Verrucomicrobia, and the relative richness of Deferribactere down-regulated (P < 0.05) compared with the control group at the phylum level.Additionally, the order-taxa level of Clostridiales, Selenomonadales, and Lactobacillales were up-regulated (P < 0.05), and the relative richness of Bacteroidales, Betaproteobacteriales, Mollicutes RF39, and Gastranaerophilales were down-regulated in the EDDI treatment groups compared with the control group (P < 0.05) (Figure S1B and Table S4).

DISCUSSION
Our results demonstrated that EDDI diets promoted growth performance in ducks.Behroozlak et al. (2020) found that the diet supplemented with 5 mg/kg potassium iodide had no effects on growth performance of broiler.Similar results were reported that dietary 2.96 mg/kg calcium iodate had no significant effects on growth performance of broiler chickens (Eila et al., 2012).Above results seems to be inconsistent with our  research findings.The possible reason was that EDDI was an organic chelating iodine, which has the characteristics of small molecular weight and high absorption, thus playing a positive role in the growth performance of ducks.
As an important part of thyroid hormone synthesis, iodine plays an important role in the growth and metabolism of animals.Usually, iodine exists in blood in the form of T3 and T4, with the proportion of T3 being low and the proportion of T4 being above 95% (Lin et al., 2014).The current experimental results showed that dietary EDDI supplementation significantly increased blood T3 and T4 concentrations.It is suggested that adding EDDI to the diet can ensure the iodine intake of meat ducks, promote the full synthesis of thyroid hormones in meat ducks and improve thyroid function, which on the one hand can promote the metabolism of sugar, fat and protein, thus promoting the digestion and absorption of nutrients (Eila et al., 2012).On the other hand, it can promote tissue differentiation, growth and maturity, and play an allowable role in growth hormone, so as to act on growth hormone secondarily and improve the growth and development of meat ducks (Zimmermann, 2011).Additionally, the number of lymphocytes and basophils were significantly increased, suggested that EDDI had a beneficial effect on the immune function of ducks.In consistency with the present study, Ali et al. (2017) reported that adding 2 mg/kg potassium iodide supplementation significantly increased the number of lymphocytes and heterophiles in ducks, thereby enhancing immune function.
As we all know, the main place for digestion of nutrients is intestine, so the health of intestinal environment is very important for the metabolism of nutrients, immune response and the regulation of body homeostasis (Kim and Ho, 2010).However, intestinal health mainly depends on intestinal development, intestinal permeability and barrier function (Liu et al., 2020).Intestinal integrity is mainly reflected by indicators such as VH, CD, and V/C.Generally speaking, lower VH and V/C values and higher CD value indicate worse intestinal development integrity and worse intestinal digestion and nutrition absorption capacity (Chang et al., 2022).In this study, dietary supplementation of EDDI exerted beneficial effects on intestinal morphology in VH and V/C in intestine, which suggested that EDDI might promote the digestion of nutrients by improving the integrity of intestine.Serum D-LA and DAO levels reflect intestinal permeability (Odenwald and Turner, 2017).In our study, the addition of EDDI was found to improve the intestinal permeability of ducks by decreasing DO and D-LA level, suggesting that EDDI may change the intestinal structure by improving the intestinal permeability.
Intestinal barrier is also very important to maintain intestinal health, including mechanical, chemical, immunological and microbial barriers (Ghosh et al., 2021;Ma et al., 2022).Among them, intestinal mechanical barrier depend on the expression of tight junction protein (Camara-Lemarroy et al., 2018).The chemical barrier mainly works through enzymes and mucus in the intestine (Birchenough et al., 2015).Immune barrier is mainly played by immune active molecules such as immunoglobulin, complement and cytokines (Uematsu and Fujimoto, 2010).Microbial barrier mainly means that the normal flora living in the intestinal tract secretes some bactericidal and bacteriostatic substances, which act as a barrier to pathogenic bacteria (Wang et al., 2018).In this study, EDDI enhanced mechanical barrier (ZO-1, ZO-2, ZO-3, and OCLN), chemical barrier (MUC2) and immune barrier (sIgA, AvBD-2, IL-10, and IL-6).Additionally, EDDI also improved microbial barrier function by changing the alpha and beta diversity, up-regulating the abundance of Firmicutes, Actinobacteria, Epsilonbacteraeot, and Verrucomicrobia, down-regulating the abundance of Deferribactere in phylum level.And EDDI also changed the abundance of Clostridiales, Selenomonadales, Lactobacillales, Bacteroidales, Betaproteobacteriales, Mollicutes RF39, and Gastranaerophilales in order level.Interestingly, the addition of EDDI was also found to increase the content of AA, PA, and BA in cecum of ducks, which may be attributed to the increase of the abundance of bacteria producing AA, PA, and BA.Actinobacteria is absolute players in maintaining gut barrier homeostasis because they have an ability to produce SCFAs, especially Aa and Ba (Ashida et al., 2012;Barczynska et al., 2015).Verrucomicrobia is mainly the Akkermansia muciniphila of the Verrucomicrobiaceae that produces AA, PA and BA.Importantly, A. muciniphila regulated metabolism and immune function, as well as protected intestinal health (Zhai, 2019).Deferribactere is an opportunistic pathogen, which can promote inflammation (Long et al., 2010).Clostridiales is also a bacterium that mainly produces butyrate, and it has been reported that the decrease of butyrate will destroy the intestinal integrity, resulting in the increase of colon permeability (Pichler et al., 2020).Lactobacillus produces lactic acid, prevents the invasion and colonization of pathogenic bacteria to the intestinal tract, and maintains the micro-ecological balance of the intestines (Castro-Bravo et al., 2018).It has been reported that intestinal microorganisms not only influenced the barrier function of the host intestine, but also regulated intestinal immune response, and then affected other physiological characteristics (Maloy and Powrie, 2011).This study discovered that the addition of EDDI had a significant impact on the abundance of microbiota, and these microbiota were positively or negatively correlated with the related characteristics of duck's thyroid function, immune function, intestinal development, intestinal permeability and barrier function.This indicated that there was a complex and beneficial interaction between intestinal microorganisms and animal physiological networks to enhance the intestinal health and promote the growth and development of ducks.

CONCLUSIONS
This study revealed that the addition of EDDI to the diet had a strong positive effect on the intestinal health of cherry valley ducks.That is, the addition of 7.40 to 9.60 mg/kg iodine (EDDI form) improved the growth and development of the meat ducks by enhancing the thyroid function and immune function, improving the integrity and permeability of the intestinal structure, promoting the digestion and absorption of nutrients, changing the expression levels of genes related to the intestinal physical, chemical and immune barriers, and increasing the abundance of SCFA-producing microorganisms and reducing the abundance of pathogenic bacteria.

Figure 1 .
Figure 1.Effect of dietary EDDI on the histological structure of the jejunum and ileum in Cherry Valley ducks (magnification 10£, scale bar = 100 mm).(A) The histological structure of the jejunum in each group.(B) The histological structure of the ileum in each group.Capital letters A−F refer to 0, 1, 2, 4, 8, and 16 mg/kg EDDI groups, respectively.

Figure 5 .
Figure 5. Common and specific OUT distribution of the fecal microbiota among 7 groups.(A) Venn Graph representation of the shared and exclusive OTU at the 97% similarity level of cecal microbiota; (B) The class level in each group; (C) The genus level in each group.Capital letters A−F refer to 0, 1, 2, 4, 8, and 16 mg/kg EDDI groups, respectively.

Table 1 .
Effect of different levels of EDDI on growth performance in Cherry Valley ducks.

Table 2 .
Effect of different levels of EDDI on routine hematological and thyroid function parameters in Cherry Valley ducks.

Table 3 .
Effect of different levels of EDDI on intestinal morphology in Cherry Valley ducks.
Abbreviations: CD, crypt depth; V:C, villus height-to-crypt depth ratio; VH, villus height.a-c Mean values (n = 6) in a row without a common superscript are significantly different in variance analysis (P ˂ 0.05).

Table 4 .
Effect of different levels of EDDI on the intestinal permeability in Cherry Valley ducks.
Abbreviations: D-LA, D-Lactic acid; DO, diamine oxidase.a-c Mean values (n = 6) in a row without a common superscript are significantly different in variance analysis (P ˂ 0.05).

Table 5 .
The aligned percentages that annotated at phylum level (%).