Integrated omics analysis reveals the differentiation of intestinal microbiota and metabolites between Pekin ducks and Shaoxing ducks (2024)

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Poult Sci
v.103(9); 2024 Sep
PMC11315098

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Poult Sci. 2024 Sep; 103(9): 103976.

Published online 2024 Jun 23. doi:10.1016/j.psj.2024.103976

PMCID: PMC11315098

PMID: 39024692

Li Chen,^*^† Ying Bao,^* Dandan Wang,^* Yong Tian,^* Tao Zeng,^* Tiantian Gu,^* Wenwu Xu,^* and Lizhi Lu^*,¹

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Supplementary Materials

Abstract

Pekin ducks and Shaoxing ducks are 2 Chinese local duck breeds, both domesticated from mallard, but after domestication and long-term artificial selection, the body weight of Pekin ducks is significantly higher than that of Shaoxing ducks. It is no debate that genetic factors are the main factors responsible for this difference, but whether intestinal microbiota contribute to this difference is yet unknown. Thus, we performed comparative intestinal metagenomics and metabolomics analysis between Pekin ducks and Shaoxing ducks. We found obvious differentiation of intestinal metagenome and metabolome between the 2 breeds. Four cecal microbial genera, including Fusobacterium, Methanobrevibacter, Butyricicoccus, and Anaerotignum showed higher abundance in Pekin ducks. Among them, Methanobrevibacter and Butyricicoccus may positively correlate with fat deposition and body weight. A total of 310 metabolites showed difference between the 2 breeds. Functions of these differential metabolites were mainly enriched in amino acid metabolism, including energy metabolism-related histidine metabolism. Integrated omics analysis showed that microbial changes were closely related to altered metabolites. Especially, Butyricicoccus showing higher abundance in Pekin ducks was significantly negatively correlated with D-glucosamine-6-phosphate, which has been reported to prevent body weight gains. These findings may contribute to further understand the difference in body weight between Pekin ducks and Shaoxing ducks.

Key words: body weight, duck, metabolite, microbiota

INTRODUCTION

The Pekin duck and Shaoxing duck are 2 Chinese local duck breeds, both of which were domesticated from mallard (Zhang et al., 2018; Zhou et al., 2018). However, after domestication and long-term artificial selection, the body weight and growth rate of the 2 breeds were significantly different. The body weight of Pekin ducks at 42 d of age can reach about 2,781 g (Ding et al., 2021), while Shaoxing ducks at 85 d of age is only about 1167 g (Sun et al., 2022). In addition, the fat deposition rate of Pekin ducks is relatively high (Ding et al., 2021).

Many factors can influence the body weight of animals, including genetics, diet, and management. Intestinal microbiota produce diverse metabolites and play important roles in affecting the host's physiology and metabolism. In humans and animals, cumulative studies have demonstrated that intestinal microbiota is an important factor affecting host's body weight. For instance, germ-free mice are lean compared to conventionally raised mice, and colonization of germ-free mice leads to rapid weight gain (Bäckhed et al., 2004). In low birth weight neonates, increased diversity of intestinal microbiota was demonstrated to be associated with weight gain (Jacquot et al., 2011). Germ-free mice colonized with donor microbiota from healthy infants gained significantly more body weight than those colonized with microbiota from undernourished donors (Blanton et al., 2016). It is generally considered that intestinal microbiota influence body weight primarily by affecting energy intake from the diet and intestinal absorption of nutrients (Dao and Clément, 2018; Martinez-Guryn et al., 2018). With accumulated evidences showing that intestinal microbiota is close correlated with the body weight, intestinal microbiota has become an important target for weight modification in clinical or livestock production (Sergeev et al., 2020).

In the modern poultry industry, body weight is an economically important trait. To obtain rapid body weight gain, researchers have made effort from the perspective of intestinal microbiota in addition to genetic improvement. As 2 excellent Chinese local duck breeds, it is no doubt that genetic factors are the main contributors to the differences in body weight between Pekin ducks and Shaoxing ducks, and it has been found that IGF2BP1 is the major gene affecting the body weight of Pekin ducks (Zhou et al., 2018). However, whether the intestinal microbiota contribute to the body weight difference between the 2 breeds has never been noticed. In addition, microbiome was heritable and it was influenced by host genetics (Grieneisen et al., 2021), thus understanding the difference in intestinal microbiota between Pekin ducks and Shaoxing ducks may provides a new perspective for understanding the differentiation of the 2 breeds. Therefore, in this study, we compared intestinal microbiota and metabolites between Pekin ducks and Shaoxing ducks, hoping to provide clues for regulating body weight through changing the intestinal microbial composition as well as hoping to provide a new perspective for understanding the difference between these 2 breeds.

MATERIALS AND METHODS

Ethics Statement

Animals used in this study were slaughtered according to the national standard of Laboratory animal-Guideline for ethical review of animal welfare. All experiment procedures were approved by Zhejiang Academy of Agricultural Sciences with the authorization number 2022ZAASLA32 issued on 12 March 2022.

Animals Rearing

A total of 160 male Pekin ducks and 122 male Shaoxing ducks were fed with a starter diet (from 1 d to 3 wk of age) containing 18.7% crude protein and 12.81 MJ/kg dietary metabolizable energy (ME), and a grower diet (from 4 wk of age to 6 wk of age) containing 17.1% crude protein and 11.67 MJ/kg ME. All the ducks were fed according to the same feeding and management conditions: brooding temperature of 32℃ from 1 d to 3 wk of age, and natural lighting during all the experiment. During the experiment, all the ducks were raised on the ground, fed ad libitum and had free access to water.

Body Weight Measurement

The birth weights of Pekin ducklings and Shaoxing ducklings were measured within 24 h of their hatching. After feeding to 42 d of age, all the ducks were weighed after fasting for 12 h. The average daily gain (ADG) was calculated according to the following formula: Average daily gain (ADG) = Weight gain /42 d.

Sample Collection

When fed to 42 d of age, 10 Pekin ducks and 10 Shaoxing ducks were randomly selected for slaughter by neck bloodletting after 12 h of fasting. Ileum and cecum tissues of 3 individuals per breed were collected and fixed in 4% paraformaldehyde for histological analysis. Ileum and cecum contents of ten individuals per breed were snap-frozen in liquid nitrogen for metagenomic sequencing and metabolomics analysis.

Histological Analysis

Paraformaldehyde-fixed tissues were made into paraffin blocks and cut into 5 μm sections, then stained with hematoxylin and eosin following the standard protocols (Thompson and Richter, 1960). Samples were evaluated using light microscopy, and the intestinal villus height and crypt depth were measured using Image-Pro Plus 6.0 software (Media Cybernetics, MD)

Metagenomic Sequencing

The ileum and cecum digesta of 10 Pekin ducks and 10 Shaoxing ducks were selected for metagenomic sequencing. Genomic DNA was extracted from these samples using DNeasy PowerSoil Pro Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's protocol. DNA libraries were constructed after DNA quality test and then sequenced on the Illumina platform (Illumina, CA) to generate 150-bp paired-ends reads.

Metagenomic Data Analysis

Raw sequencing data were processed to obtain high-quality reads by using fastp (v0.20.0) (Chen et al., 2018) and Cutadapt (v1.2.1) (Martin, 2011). Host contamination were removed by aligning reads to the host genome using BMTagger (Rotmistrovsky and Agarwala, 2017). Then the high-quality reads were assembled for each sample using Megahit (v1.1.2) (Li et al., 2015). Gene prediction of metagenomic contigs longer than 300 bp were predicted by MetaGeneMark (Zhu et al., 2010). To assess the abundance of the gene, high-quality reads from each sample were mapped onto the predicted gene using salmon (Patro, 2015), and the CPM (copy per kilobase per million mapped reads) was used to normalize the abundance value of the metagenome. Alpha diversity and PLS-DA were constructed using R (v3.6.1). Based on the taxonomic of non-redundant genes, linear discriminant analysis effect size (LEfSe) was used to detect differentially abundant taxa. The function of the non-redundant genes were annotated by using mmseqs2 (Steinegger and Söding, 2017) against databases of KEGG.

Metabolomics

Untargeted metabolomics from the ileum and cecum digesta of 10 Pekin ducks and 10 Shaoxing ducks were performed using the liquid chromatographymass spectrometry (LC-MS) analysis. Chromatographic separations were performed using Vanquish ultra-high performance liquid chromatography (UHPLC) system (Thermo, MA). After chromatographic separations, Q Exactive HF mass spectrometer (Thermo, MA) was operated in both PIM and NIM to detect metabolites. The raw LC–MS data were processed for peak detection and annotation using package XCMS and CAMERA package, respectively. Compound identification was identified by combining the retention time and m/z value.

Statistical Analysis

Spearman's correlation analysis was conducted to identify the correlation between altered intestinal microbiota and metabolites. Significant differences were detected using Student's t-test. The P value was adjusted for multiple tests using a false discovery rate (Benjamini–Hochberg) and the adjusted P-value of < 0.05 was recognized as significant.

RESULTS

Differences in the Body Weight and Intestinal Morphology Between Pekin Ducks and Shaoxing Ducks

Pekin ducks and Shaoxing ducks were fed to 42 d in the same batch according to the same feeding conditions. At 42 d of age, the body weight of Pekin ducks was significantly higher than that of Shaoxing ducks (Figure 1A, B), and the average daily gain (ADG) was also significantly higher than that of Shaoxing ducks (Figure 1B). Hematoxylin-eosin (HE) staining of the ileum and cecum tissues of Pekin ducks and Shaoxing ducks showed that the villus height of the ileum of Pekin ducks was significantly higher than that of Shaoxing ducks, while the crypt depth was significantly lower than that of Shaoxing ducks (Figure 1C, Table 1). In the cecum, the intestinal gland depth and the thickness of mucosal layer of Pekin ducks were significantly higher than those of Shaoxing ducks (Figure 1D, Table 1).

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Figure 1

Differences in the body weight and intestinal morphology between Pekin ducks and Shaoxing ducks. (A) Photographs of 42-day-old Pekin ducks and Shaoxing ducks. (B) Body weight difference between Pekin ducks and Shaoxing ducks. Hematoxylin-eosin staining of the ileum (C) and cecum (D) tissues of Pekin ducks and Shaoxing ducks. ** indicates P < 0.01.

Table 1

Differences in intestinal morphology between Pekin ducks and Shaoxing ducks.

Breeds	Ileum			Cecum
Breeds	Villus height (mm)	Crypt depth (mm)	Villus height/crypt depth ratio	Intestinal gland depth (mm)	Thickness of mucosal layer (mm)
Pekin ducks	0.94 ± 0.12^A	0.13 ± 0.02^A	7.36 ± 0.8^A	0.34 ± 0.01^A	0.59 ± 0.11^A
Shaoxing ducks	0.36 ± 0.07^B	0.18 ± 0.01^B	1.97 ± 0.45^B	0.24 ± 0.01^B	0.30 ± 0.02^B

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Different superscripts in the same column indicate significant differences (p < 0.01).

Differences in Intestinal Microbiota Between Pekin Ducks and Shaoxing Ducks

To identify the difference in intestinal microbiota between Pekin ducks and Shaoxing ducks, we performed metagenomic sequencing on ileum and cecum contents of the 2 breeds. After quality filtering, an average of 68,885,317 high quality reads in each sample were obtained (^{Table S1}). There was no significant difference in the alpha diversity of the microbiota in the same intestinal segment between Pekin ducks and Shaoxing ducks (Figure 2A). The plots of partial least squares discriminant analysis (PLS-DA) was then applied to reveal the differences in taxa composition between the 2 breeds. The PLS-DA plot showed that an obvious separation were existed between Pekin ducks and Shaoxing ducks (Figure 2B).

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Figure 2

Analysis of intestinal microbial diversity in Pekin ducks and Shaoxing ducks. (A) Alpha diversity analysis based on indices of Chao and Shannon. (B) Differences in taxa composition between Pekin ducks and Shaoxing ducks were revealed using partial least squares discriminant analysis (PLS-DA). SX_H and PK_H represent ileum contents of Shaoxing ducks and Pekin ducks, respectively; SX_M and PK_M represent cecum contents of Shaoxing ducks and Pekin ducks, respectively, and the same below.

Microbial community structure showed that Proteobacteria (53.8% and 51.6%) was the most abundant phylum in the ileum, followed by Firmicutes (19.1% and 25.5%), Bacteroidota (5.8% and 10.1%) and Actinobacteria (10.3% and 3.3%), whereas Bacteroidota (43.4% and 43.9%) and Firmicutes (42.8% and 44.4%) were 2 dominant phyla in the cecum, with a total combined abundance of 87% (Figure 3A). At the species level, Acinetobacter baumannii (38.1% and 35.9%), which belongs to the phylum of Proteobacteria, was the most abundant species in the ileum, whereas Bacteroides caecigallinarum (5.3% and 6.9%) and Phocaeicola plebeius (5.1% and 4.3%) were predominant in the cecum (Figure 3B). Functional analysis of the intestinal microbiota showed that carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins were the 3 most enriched functions in both ileum and cecum of Pekin ducks and Shaoxing ducks (Figure 3C). Then, PLS-DA plot was applied to reveal the differences in microbial functions between the 2 breeds. The result showed a distinct separation of microbial functions between Pekin ducks and Shaoxing ducks (Figure 3D).

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Figure 3

Composition and function of intestinal microbiota of Pekin ducks and Shaoxing ducks. Microbial taxonomic composition in the phylum level (A) and species level (B) in Pekin ducks and Shaoxing ducks. (C) Microbial functions predicted based on the associated KEGG orthologous group markers. (D) Separation in microbial functions between Pekin ducks and Shaoxing ducks were revealed using PLS-DA.

In order to screen the differential microbiota between the 2 duck breeds, LEfSe analysis with P < 0.05 and LDA (linear discriminant analysis) > 2 was conducted. In the ileum, only 3 taxa including Oceanitalea stevensii, Ruoffia and Chlamydia abortus showed differential abundance between Pekin ducks and Shaoxing ducks, and all of them exhibited higher abundance in Shaoxing ducks (Figure 4A). However, the changes of microbial abundance between Pekin ducks and Shaoxing ducks were complex in the cecum. Four genera, including Fusobacterium, Anaerotignum, Methanobrevibacter, and Butyricicoccus showed higher abundance in Pekin ducks, whereas a few taxa including Prevotella, Brachyspira, Helicobacter and so on exhibited higher abundance in Shaoxing ducks (Figure 4B).

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Figure 4

The main altered microbiota between Pekin ducks and Shaoxing ducks analyzed using LEfSe analysis in the ileum (A) and cecum (B). The prefixes ‘p’, ‘c’, ‘o’, ‘f’, ‘g’, and ‘s’ represent the annotated level of phylum, class, order, family, genus, and species.

Intestinal Metabolomic Changes Between Pekin Ducks and Shaoxing Ducks

To investigate intestinal metabolomic changes between Pekin ducks and Shaoxing ducks, untargeted metabolomic analysis was performed in the contents of ileum and cecum. A total of 1,597 metabolites in the positive ion mode (PIM) and 1,052 metabolites in the negative ion mode (NIM) were detected. Further comparative metabolomic analysis identified that 83 and 227 metabolites in the ileum and cecum respectively showed significantly altered abundances between Pekin ducks and Shaoxing ducks (Figure 5A). The cluster analysis based on these altered metabolites showed that despite the mixing of several samples, an obvious division between Pekin ducks and Shaoxing ducks could also be achieved (Figure 5B). The KEGG pathway analysis of the significantly altered metabolites found that amino acid metabolism was significantly enriched (Figure 5C), such as histidine metabolism, alanine, aspartate, and glutamate metabolism.

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Figure 5

Alternations in intestinal metabolites between Pekin ducks and Shaoxing ducks. (A) Venn diagram of differential metabolites in the ileum and cecum between Pekin ducks and Shaoxing ducks. PIM and NIM represent metabolites identified in the positive ion mode and negative ion mode respectively. (B) Heatmaps showing top 40 differential metabolites in the ileum and cecum respectively between Pekin ducks and Shaoxing ducks. The top 40 differential metabolites consist of top 20 differential metabolites in PIM as well as top 20 differential metabolites in NIM. (C) Top 20 pathways enriched in differential metabolites in the content of ileum and cecum respectively.

Association Analysis of the Metagenome and Metabolome

Association analyses were performed on the metagenome and metabolome of the ileum and cecum, respectively. In the ileum samples, Ruoffia and Oceanitalea stevensii were very prominent, as they almost correlated (P < 0.05) with all differential metabolites (Figures 6A and 66B).B). In cecum samples, the result of correlation analysis was more complicated (Figures 6C and 66D).D). Altered microbiota showed closely correlated with multiple altered metabolites. For example, Butyricicoccus highly abundant in Pekin ducks showed significantly negatively correlated with D-glucosamine-6-phosphate, 3-methyl-2-oxopentanoate, alpha-ketoisovaleric acid and trans-2,3-dimethylacrylic acid, but positively correlated with methylphosphonic acid, 2,3-dimercaptosuccinic acid and 3-ethylphenol. Methanobrevibacter reported to be significantly correlated with fat deposition were also found correlated with multiple metabolites. These results suggested that changes in intestinal microbiota were closely correlated with alternations of metabolites.

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Figure 6

Heatmaps of correlations between altered intestinal microbiota and metabolites. Heatmaps of correlations in NIM (A) and PIM (B) in the ileum. Heatmaps of correlations in NIM (C) and PIM (D) in cecum. The circle size is positively correlated with the correlation value; Significant correlations are marked by * P < 0.05, ** P < 0.01.

DISCUSSION

Pekin ducks and Shaoxing ducks are 2 Chinese local duck breeds both derived from mallards, but after domestication and long term artificial selection they exhibit evident physiological, behavioral and phenotypic differences. The differentiation between the 2 breeds have been well demonstrated from the molecular perspective (Zhang et al., 2018; Zhou et al., 2018), but it have never been demonstrated from the perspective of intestinal microbiota and metabolites. In this study, metagenomic sequencing and metabolomics analysis were performed on the ileum and cecum contents of Pekin ducks and Shaoxing ducks. Both metagenomic analysis and metabolomic analysis showed that an obvious division were found between Pekin ducks and Shaoxing ducks (Figures 2B, ,3D,3D, and and5B),5B), indicating that the intestinal microbiota and metabolites have differentiated between the 2 breeds, which provides a new perspective for understanding the divergence between Pekin ducks and Shaoxing ducks.

The fat deposition and body weight of Pekin ducks are significantly higher than that of Shaoxing ducks. Body weight is affected by many factors, including genetics, feeding and management. Intestinal morphology and intestinal microbiota which affect the host's nutrient digestion and absorption are also critical factors affecting the host's body weight (Bäckhed et al., 2004; Jacquot et al., 2011; Blanton et al., 2016). In this study, we found that the villus height and V/C ratio of the ileum of Pekin ducks were higher than those of Shaoxing ducks, but the crypt depth was lower than that of Shaoxing ducks, indicating that Pekin ducks had a greater mucosal surface area, which implied stronger digestive and absorptive capacity (Zeitz et al., 2015; Wang et al., 2017). In addition, the intestinal gland depth and thickness of mucosal layer of the cecum were higher in Pekin ducks, which indicating a stronger intestinal mucosal barrier function and was beneficial to intestinal health. These factors may partly account for the higher body weight of Pekin ducks than Shaoxing ducks.

Intestinal microbiota is an important factor affecting the host's body weight. In this study, we found that the abundance of some microorganisms was different between Pekin ducks and Shaoxing ducks. In the ileum, 2 taxa, Ruoffia and Oceanitalea stevensii, were very prominent. They showed higher abundance in Shaoxing ducks and exhibited significantly correlated with almost all differential metabolites. The 2 taxa are very puzzling, as their functions have never been reported. Perhaps the related metabolites are a breakthrough to study the functions of the 2 taxa. Compared with Shaoxing ducks, 4 genera, including Fusobacterium, Methanobrevibacter, Butyricicoccus and Anaerotignum showed higher abundance in the cecum of Pekin ducks. It has been reported that Methanobrevibacter was significantly correlated with fat deposition in the chicken (Wen et al., 2019). Chickens with lower Methanobrevibacter abundance had significantly lower fat deposition than those with higher abundance of Methanobrevibacter. Body weight is highly positively correlated with fat content (Leclercq et al., 1980), and it has been reported that the higher body weight of Pekin ducks than that of Shaoxing ducks was accompanied by higher fat content (Ding et al., 2021). Thus, we speculate that the higher Methanobrevibacter abundance in Pekin ducks may be positively correlated with the higher fat deposition and body weight in Pekin ducks. Intestinal microbiota regulates host physiology through producing a myriad of metabolites (Krautkramer et al., 2021). Our association analysis of the differential metabolites and microbiota revealed that Butyricicoccus was significantly negatively correlated with D-glucosamine-6-phosphate. D-glucosamine-6-phosphate is the natural form of glucosamine which has been reported to inhibit the adipocyte differentiation leading to the preventions of body weight gains in obese rats (Huang et al., 2015). The higher abundance of Butyricicoccus in Pekin ducks may contribute to the heavier body weight of Pekin ducks by negatively regulating D-glucosamine-6-phosphate abundance.

Changes in intestinal microbiota can contribute to metabolic alternation. In this study, a total of 310 differential metabolites were identified between Pekin ducks and Shaoxing ducks. Integrated analysis of the metagenome and metabolome showed that these differential metabolites were closely related to the altered microbiota. These differential metabolites were mainly enriched in amino acid metabolism, implying the possibility that microbial changes between Pekin ducks and Shaoxing ducks may ultimately result in the changes in amino acid metabolism. Among these enriched amino acid metabolism, histidine metabolism was found. Histidine metabolism was also found to be altered in obese adults (Bellissimo et al., 2019; Li et al., 2021), and dietary histidine was found inversely associated with body mass index (BMI) and energy intake in overweight/obese individuals. Histidine is obtained mainly from the daily intake of protein. It can be utilized for metabolic energy production, accounting for 15% of the metabolic energy production (Zapata et al., 2019). Therefore, the changes in histidine metabolism might lead to the alteration of energy homeostasis between Pekin ducks and Shaoxing ducks.

In summary, this study demonstrated for the first time the differentiation between Pekin ducks and Shaoxing ducks from the perspective of intestinal microbiota and metabolites. Four genera showed higher abundance in the cecum of Pekin ducks, among which Methanobrevibacter may positively correlate with the fat accumulation, and Butyricicoccus may contribute to the body weight by negatively correlated with D-glucosamine-6-phosphate. Microbial changes were closely related to altered metabolites which mainly enriched in amino acid metabolism and may finally altered energy metabolism. These findings may contribute to understand the body weight difference between Pekin ducks and Shaoxing duks.

DISCLOSURES

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by Major Science and Technology Projects of Zhejiang Province (grant no. 2021C02068-10), China Agriculture Research System of MOF and MARA (grant no. CARS-42-6).

SUPPLEMENTARY DATA

Metagenomic sequencing data have been submitted to the SRA database in NCBI with the BioProject accession number PRJNA1031405. And metabolomic data have been submitted to MetaboLights with the accession number MTBLS9857.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2024.103976.

Appendix. Supplementary materials

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