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Accepted Manuscript

Title: Evaluating the association between body weight and the
intestinal microbiota of weaned piglets via 16S rRNA
sequencing

Author: Geon Goo Han Jun-Yeong Lee Gwi-Deuk Jin Jongbin
Park Yo Han Choi Byung Jo Chae Eun Bae Kim Yun-Jaie Choi

PII: S0378-1135(16)30504-1
DOI: http://dx.doi.org/doi:10.1016/j.vetmic.2016.10.020
Reference: VETMIC 7420

To appear in: VETMIC

Received date: 19-8-2016
Accepted date: 14-10-2016

Please cite this article as: Han, Geon Goo, Lee, Jun-Yeong, Jin, Gwi-
Deuk, Park, Jongbin, Choi, Yo Han, Chae, Byung Jo, Kim, Eun Bae, Choi,
Yun-Jaie, Evaluating the association between body weight and the intestinal
microbiota of weaned piglets via 16S rRNA sequencing.Veterinary Microbiology
http://dx.doi.org/10.1016/j.vetmic.2016.10.020

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Evaluating the association between body weight and the intestinal microbiota of weaned

piglets via 16S rRNA sequencing

Geon Goo Hana, Jun-Yeong Leea, Gwi-Deuk Jinb, Jongbin Parkc, Yo Han Choib, Byung Jo

Chaeb, Eun Bae Kimb,d,* and Yun-Jaie Choia,e,*

a
Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of

Korea

b
Department of Animal Life Science, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

c
Department of Animal Life System, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

d
Division of Applied Animal Science, Kangwon National University, Chuncheon, Gangwon-

do, Republic of Korea

e
Research Institute for Agriculture and Life Science, Seoul National University, Seoul,

Republic of Korea

*
Corresponding authors with equal contribution

Yun-Jaie Choi: E-mail: cyjcow@snu.ac.kr

Eun Bae Kim: E-mail: itanimal@kangwon.ac.kr

1

Highlights

 Microbial richness was higher in the heavier piglets than in the lighter piglets.

 FB ratio was higher in the heavier piglets than in the lighter piglets.

 Several genera were significantly different in the lighter and heavier piglets.

 Several metabolic pathways were different in the lighter and heavier piglets.

ABSTRACT

Due to the ban on the use of antimicrobial growth promoters in livestock feeds,

understanding the relationship between intestinal microbiota and the physiology of the host

has become very important for improving livestock performance. In this study, we

investigated the relationship between intestinal microbiota and body weights of weaned

piglets. Lighter (n=9) and heavier (n=9) 9-week-old weaned piglets were selected from

approximately one-hundred individuals based on their body weights. Their fecal microbial

communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The

microbial richness estimators of the heavier piglets, were significantly higher than those of

the lighter piglets. At the phylum level, the microbiota of the heavier group had significantly

higher levels of Firmicutes and a higher Firmicutes-to-Bacteroidetes ratio than that of the

lighter group. At the genus level, the levels of several genera, such as Anaerococcus and

Lactococcus, were significantly different in the two groups. In particular, the lighter group

had significantly higher levels of opportunistic pathogenic bacteria, such as Anaerotruncus

2

Keywords: Fecal microbiota. such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).. Metagenome. modulating the intestinal microbiota of livestock through administering prebiotics and probiotics is considered an effective strategy by many researchers. The development of alternatives to antibiotics to solve these problems has been an important issue in the livestock industry. compared with those of the heavier group. such as a reduction in growth performance and the increasing therapeutic use of antibiotics (Heo et al. Body weight. Weaned piglet. The microbiota of the heavier group had a significantly higher involvement in three KEGG pathways concerned with xenobiotic degradation than that of the lighter group. 3 . however. 2013. effective strategies have not yet been developed.and Bacteroides. 16S rRNA gene. These results may provide insights into host-microbe interactions occurring in the piglet intestine and will be useful in establishing a strategy for improving growth performance in the swine industry. including the swine industry. Moreover. Given this situation. the levels of bacteria expressing the components of several metabolic pathways were significantly different in the two groups. Wierup. and since July 2011 in the Republic of Korea due to the emergence of bacteria that are resistant to a broad range of antibiotics (known as super bacteria). To develop an effective strategy for modulating the intestinal microbiota. various problems were observed in the livestock industry. Introduction Using antibiotics as antimicrobial growth promoters (AGPs) in livestock feeds has been banned since 1986 in Sweden. After the ban on the use of AGPs. 2001). since 2006 in the EU. High-throughput sequencing 1.

studies of the gut microbiota of livestock in their youth are needed because it is during this crucial period that the intestinal microbiota is stabilized. there is insufficient knowledge of this topic at present. such as 454 pyrosequencing and Illumina sequencing. To investigate the relationship. discovered obesity-associated bacteria in three distinct gut locations in pigs (Yang et al. Other researchers have found body weight-related bacterial groups in mice and humans (Everard et al. For example. 2011. 2016).. and many researchers have reported host-microbe interactions. 4 . has enabled culture-independent analysis of the composition of microbial communities. 2009). particularly a relationship between the composition of the intestinal microbiota and body weight. particularly regarding growth performance. Yang et al. Turnbaugh et al. In particular. we analyzed the microbial communities and inferred the functions of the fecal microbiota of lighter and heavier piglets.. The development of high-throughput sequencing technologies. The aim of this study was to investigate the relationship between the intestinal microbiota and body weights of weaned piglets under a commercial environment.. However. is very important.understanding the relationship between the intestinal microbial composition and the physiology of the host.

.60 ± 4.2. according to the manufacturer’s protocol. Fecal samples were collected from each piglet and were stored at -70 °C until DNA extraction was performed. 2013). Sample preparation Nine-week-old weaned LYD piglets raised on a local commercial farm (Gangneung.33 kg). The mean body weights of the lighter and heavier groups were significantly different (P < 0. 2011.09 to 11. and the body weights of the members of the heavier group ranged from 16.20 ± 1. and was stored at -20 °C until further analysis. Germany). with the total body weights ranging from 8. Republic of Korea) were used in this study. which provides sufficient information regarding the phylogenetic diversity of microbial communities (Caporaso et al. The weaned piglets were individually weighed.. and a total 18 piglets were selected from approximately one-hundred individuals based on their body weights. 2.2.75 kg (mean values ± SD = 14. The body weights of the members of the lighter group ranged from 8.001). Zhao et al. with 9 piglets being placed in the lighter group and 9 piglets placed in the heavier group (Table 2).89 kg (mean values ± SD = 10.38 kg).09 to 22. The piglets were fed a commercial diet designed for weaned piglets (Table 1) and had access to feed and water ad libitum. DNA extraction and sequencing DNA was extracted from 250 mg of each fecal sample using a NucleoSpin®Soil Kit (Macherey-Nagel.75 kg (mean values ± SD = 19. Materials and methods 2.00 ± 2.1. Düren.70 to 22. The V4 region of the bacterial 16S rRNA gene.90 kg) (Table 3). and the experimental protocols followed in this study were approved by the Institutional Animal Care and Use Committee of Kangwon National University (IACUC #: KW-140509-1). was amplified from the total extracted 5 .

Microbial community analysis The microbial communities were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) version 1. MA. The construction of the DNA libraries was confirmed by agarose gel electrophoresis. Republic of Korea). 2. SNU. The amplicons were separated by agarose gel electrophoresis and were purified using a QIAquick Gel Extraction Kit (Qiagen. Ipswich. The raw sequence reads were quality trimmed and demultiplexed. Valencia. Valencia. CA. 1 cycle of 72 °C for 10 min.9.0 (http://qiime. The size selection steps for the adaptor-ligated DNAs and the cleanup steps were replaced by PCR product purification using a QIAquick PCR Purification Kit (Qiagen.3. USA). MA. 55 °C for 1 min. The 16S rRNA gene sequences determined in this study were deposited in the NCBI Sequence Read Archive (SRA) database with the accession number SRP080895. and 72 °C for 1. USA). Shiga. The DNA libraries were constructed using a NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs. The adaptor and index primers were added to the amplicons using the NEBNext Multiplex Oligos for Illumina Kit (New England Biolabs. CA. The amplification program consisted of 1 cycle of 94 °C for 3 min. USA). The sequence reads were then clustered into operational 6 . Ipswich. and the libraries were purified using a QIAquick Gel Extraction Kit. with some modifications of the manufacturer’s instructions. The components of the libraries were then sequenced using an Illumina MiSeq platform (NICEM.5 min and finally. Seoul. followed by 40 cycles of 94 °C for 45 sec. Japan) and universal primers (forward: 5’-GGACTACHVGGGTWTCTAAT-3’ and reverse: 5’-GTGCCAGCMGCCGCGGTAA-3’). USA).DNA.org) software. PCR amplification was performed using Takara Ex-taq polymerase (Takara Bio.

2.4. A two-sided Welch’s t-test was used to identify significant differences in the microbial taxa represented and the alpha diversity of the microbiota of the two groups. The taxonomic assignments for each representative sequence were obtained using the uclust consensus taxonomic classifier using the GreenGenes 13_8 database. and the representative sequences were aligned using the PyNAST program. phylogenetic diversity (PD). The resulting biom files were normalized according to known/predicted 16S rRNA gene copy numbers. The abundance of the microbial taxa was expressed as a percentage of the total 16S rRNA gene sequences. Shannon.1. and the data were normalized for 2.0 (http://picrust. Closed reference OTU picking against the GreenGenes 13_5 database was conducted at 97% sequence similarity using the QIIME and OTU tables. and Simpson methods.. observed OTUs.github. and these indices were calculated from 5.05 considered significant. and the results were visualized using the STAMP version 2.0. and the differences between the groups were compared. 2014). with 10 iterations. with P < 0. and the 7 .3 program (Parks et al.000 sequence reads through rarefaction. Chao1. The statistical analyses were performed using Microsoft Excel 2013 and STAMP software. Principal component analysis (PCA) was performed at the phylum and the genus level.627 reads per sample by single rarefaction. Prediction of the functions of the microbial communities The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) version 1. The microbial diversity indices of the samples (alpha diversity) were determined using the abundance-based coverage estimator (ACE).io/picrust/) program was used to predict the functional profile of the microbial communities based on the 16S rRNA gene sequences obtained.taxonomic units (OTUs) by de novo OTU picking at a 97% level of sequence similarity.

The predicted metagenomes were collapsed into a specified level in a hierarchy using the KEGG pathway metadata and were analyzed using the STAMP program. A two-sided Welch’s t-test was used to identify significant differences in the microbial taxa represented and the alpha diversity of the microbiota of the two groups. with P < 0.05 considered significant. 8 .metagenomes were predicted using precalculated Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs. Unclassified functional categories were eliminated from the analysis.

82 (± 10.720. The diversity indices (PD.485.99 (± 0.437 (± 8. Shannon index.41) for the lighter group and 9. observed OTUs.605. The richness estimators (ACE.642. Differences in the alpha diversity of the intestinal microbiota of the lighter and heavier piglets A total of 337. PD. The Simpson index value was 0.243.3.2. Results 3.480) reads per piglet in the heavier group.96) for the lighter group and 18. The number of observed OTUs was 2. 3.675) 16S rRNA gene sequence reads were generated. although the differences were not significant.261) reads per piglet in the lighter group and 14.92 (± 1.95) for the heavier group.067 (± 13. The Chao1 metric was 15. Chao1.70) for the lighter group and 181.534 (mean value = 18.452.05). with an average of 23. Differences in the microbial taxa represented in the intestinal microbiota of the lighter and heavier piglets At the 97% similarity level.41 (± 0. Chao1.18) for the heavier group.887.60 (± 1.00) for the heavier group. The Shannon index value was 9.99 (± 0.1.56 (± 10.257.44) for the lighter group and 17. The PD value was 174.29 (± 2. and Simpson index values were used as parameters of the alpha diversity of intestinal microbiota in this study (Table 3). all of the OTUs observed were classified into 26 phyla and 9 .628.29) for the heavier group.19) for the lighter group and 2.309. Shannon.01) for the lighter group and 0. The ACE metric was 16.08 (± 100.69) for the heavier group. and Simpson) were also higher for the heavier group than for the lighter group.86) for the heavier group. ACE.752 ± 11.56 (± 1. and observed OTUs) were significantly higher for the heavier group than for the lighter group (P < 0.78 (± 180.63 (± 0.

whereas the three dominant genera in the intestinal microbiota of the heavier group were Prevotella (18. respectively.08%).86%). the level of Bacteroidetes members was significantly higher in the lighter group than in the heavier group (Fig.01%). although they were not separated at the genus level (Fig.34%) uniquely identified in the lighter group and the heavier group. At the phylum level. Next. and Proteobacteria (5. 2B).25% in the lighter group and 4. the intestinal microbiota of the two groups shared 180 genera (69. Bacteroidetes (39. In the resulting PCA plot.59% in the heavier group). Lactobacillus (6.66%). At the genus level. 10 . and Faecalibacterium (3. the microbiota of the lighter and heavier group shared 24 phyla (92. with 41 (15. Three dominant genera.77%).42% in the lighter group and 53. and Bacteroides (4.54%). The Firmicutes-to-Bacteroidetes ratio (FB ratio) was significantly higher in the heavier group than in the lighter group (Table 4).31% in the lighter group and 34.258 genera (Fig.89%) and 37 genera (14.31%). 1). 3A). Lactobacillus (7. the microbial communities were clustered into two groups by body weight at the phylum level (Fig. with members of Acidobacteria and Armatimonadetes uniquely identified in that of the lighter group and the heavier group. we compared the relative abundance of the members of microbial taxa in the lighter and heavier groups and found that some phyla and genera were represented at significantly different levels in the groups (Table 4). Prevotella (20.15% in the heavier group). were found in the intestinal microbiota of the lighter group.23% in the heavier group). 2A). The three dominant phyla detected in both groups were Firmicutes (48. At the phylum level. the levels of Firmicutes and Planctomycetes members were significantly higher in the heavier group than in the lighter group (Fig. We then compared the overall composition of the microbiota of the two groups at the phylum and genus levels using PCA. respectively. 2B). In contrast.36%).

and 0. 11 . respectively) than in that of the lighter group (0. In contrast. respectively).16%. 1.06%. although the difference was not significant (P = 0. and ‘benzoate degradation’ pathways that are involved in ‘xenobiotics biodegradation and metabolism’ at the higher KEGG pathway hierarchical level. ‘xylene degradation’.24%. Bacteroides. 4). and 0.28%. Sediminibacterium. ‘alanine.05%. [Prevotella]. ‘dioxin degradation’. respectively) than in that of the heavier group (0.056). (0.3. ‘Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway’. Bilophila. In contrast. aspartate and glutamate metabolism’.17%. Streptococcus was more abundant in the heavier group than in the lighter group.05%. [Eubacterium]. respectively). 3B). and ‘novobiocin biosynthesis’ pathways were predicted at significantly higher levels in the microbiota of the lighter group. Anaerotruncus. and Butyrivibrio were significantly higher in the intestinal microbiota of the heavier group than in that of the lighter group (Fig. 0. were predicted at significantly higher levels in the microbiota of the heavier group (0.06%. At the genus level. 1.05%. and Corynebacterium were significantly higher in the lighter group than the heavier group (Fig. 3B).23%. the levels of Anaerococcus.05%. the levels of Lactococcus.26%. Comparison of the KEGG pathways of the fecal microbiota of the lighter and heavier piglets We compared the KEGG pathways predicted for the fecal microbiota of the lighter and heavier piglets and found that various KEGG pathways were significantly differentially identified in the microbiota of the two groups (Fig. and 0. and 0. 3. 0.

2008.. 2014. reported that the level of intestinal microbial diversity was reduced in obese mice and humans compared with those of lean individuals (Turnbaugh et al. many studies have shown that intestinal microbial diversity and richness are negatively related to body weight. 2016). Turnbaugh et al. the levels of the microbial richness estimators (ACE. some researchers have reported contrary results (Clarke et al. Louis et al.. Chao1. whereas a significantly higher Bacteroidetes level was observed in the microbiota of the lighter group. consistent with the results observed in many previous studies (Delzenne and Cani. 2014. 2013). However.. However..4. 2009.. we observed differences in the levels of several different phyla in the microbiota of the lighter and heavier groups. Our previous study revealed that the number of OTUs observed in the cecum of broiler chickens is negatively related to their body weights (Han et al.. Discussion In this study. and observed OTUs) were significantly higher for the heavier group than for the lighter group. reported that individuals with a low body mass index (BMI) showed a higher level of gut bacterial richness than did high BMI individuals (Le Chatelier et al. 2015.. Mejia-Leon et al. Ley et al.. 2016)... Hildebrandt et al. In this study. Le Chatelier et al.. Walters et al. Kasai et al. 2014). 2014). However. The microbiota of the heavier group had a significantly higher Firmicutes level and FB ratio compared with that of the lighter group. several studies have suggested that the level of intestinal microbial diversity is not related to obesity or to the existence of obesity-related disorders (de Goffau et al. 2015.. More studies of the host-microbe interactions in various animals in various 12 . 2006). 2011. and other researchers have reported that there was no relationship between the FB ratio and BMI (Hu et al. 2009).. Turnbaugh et al. The relationship between intestinal microbial diversity and body weight or obesity remains controversial issues.

environments are required to explain these results. In this study... This pathway is associated with the host innate immune response.. inflammatory bowel disease. 2016. we can hypothesize that the presence of certain pathogenic bacteria in the intestine causes the relatively reduced body weight of the host animal. reported that Bacteroides species are more abundant in the microbiota of low BMI men than in that of high BMI men. the fecal metagenomes were predicted using PICRUSt and several different KEGG pathways were observed in the lighter and heavier piglets. Ngo et al. 2006). Rhee et al. the relative abundance of members of the genera Bacteroides and Anaerotruncus were significantly lower in the microbiota of the heavier group than they were in that of the lighter group. 1995. bloating.. For example. Zhu et al. and heart disease (Choi et al. More studies of the relationship between infections with opportunistic pathogens and body weight are required to clarify our hypothesis.. Haro et al. In this study. is associated with bacteremia. inflammatory diarrhea. although many Bacteroides species are normally mutualistic in the mammal intestine. 2011. Infection with Anaerotruncus colihominis. although the abundance of Bacteroides species in the microbiota of women was not affected by their BMI values (Haro et al. and infection with this species causes malignancy. Hu et al. Robert et al. Some species of Bacteroides are opportunistic pathogens. 2015)... Redondo et al. 2016). Bacteroides fragilis is associated with anaerobic bacteremia and sepsis... the only known species of the genus Anaerotruncus. Based on these data. 2013. 2008. Lau et al. 2013). and cachexia (Bindels et al. 2016.. and its 13 . reported that Bacteroides species are more abundant in the microbiota of normal adolescents than they are in that of obese adolescents (Hu et al. 2009.. ‘NLR signaling pathway’- related genes were more abundant in the fecal microbiota of the lighter group than in that of the heavier group. Jalanka-Tuovinen et al.

consistent with the results of other studies. as part of a pathogen-associated molecular pattern.. reported that the administration of the atypical antipsychotic risperidone was related to weight gain and the level of ‘xenobiotics biodegradation and metabolism’ pathway-related genes and the FB ratio in the gut microbiome were higher in the risperidone-treated group than in the controls (Bahr et al.activation affects the activity of alternative signaling pathways. such as the caspase activation and cell death pathways (Wells et al. the body weight gain of the piglets with a microbiota that had a more activated NLR signaling pathway might be less than that of the piglets in whose microbiota that pathway was less activated. and antibody formation (Carroll and Forsberg. 2015). 2016).. ‘xylene degradation’. such as meso- diaminopimelic acid and muramyl dipeptide. NLR is a member of the pattern recognition receptor family. 2015). Yang et al.(‘dioxin degradation’. The immune response is very energy intensive. and this term is commonly used in the context of harmful substances. 14 . as are the febrile processes of an animal and the various subsequent responses. which recognizes bacterial cell wall components. such as the production of cytokines.. and ‘benzoate degradation’) related genes belonging to the ‘xenobiotics biodegradation and metabolism’ pathway were significantly more abundant in the microbiota of the heavier group than in that of the lighter group. We hypothesize that the activation of NLR signaling induces a host immune response and would lead to the expenditure of more energy to maintain immune homeostasis. 2011). We observed that three KEGG pathway. Xenobiotics are foreign chemical substances. Therefore. the reduction of amylase activity. found that the ‘benzoate degradation’ pathway-related genes were significantly enriched in the cecal microbiome of high fatness pigs compared with that of lean fatness pigs (Yang et al. 2007. Liu et al.. Bahr et al.

dioxin is a well-known carcinogen. These toxic substances are harmful to a host and may inhibit its growth. and long-term exposure to xylene is toxic to the central nervous system and liver.For example. In the microbiota of the heavier group. microbial degradation pathways for these toxic substances were more activated than they were in that of the lighter group. which may have led to the body weight gain of the former group. 15 .

Bacteroidetes. Anaerotruncus. we compared the fecal microbiota of lighter and heavier piglets and their predicted metagenomes. Based on these results. Firmicutes. The level of microbial richness was higher in the microbiota of the heavier group than in that of the lighter group..g. Additional. the levels of genes related to several metabolic pathways were significantly different in the microbiota of the two groups. and several different bacterial phyla and genera that were differentially represented in the two groups were identified (e.5. These results may provide insights into understanding the host-microbe interactions occurring in the piglet intestine and will be useful in establishing a strategy for improving growth performance in the swine industry. such as the immune response and xenobiotic degradation. such as ‘NLR signaling pathway’ and ‘xenobiotics biodegradation and metabolism’ pathway- related genes. 16 . we can infer that the intestinal microbiota affects the growth performance of piglets through host-microbe interactive processes. Conclusion In this study. and Bacteroides).

Ministry of Agriculture. Republic of Korea (grant number 914005-04). Geon Goo Han was supported by the BK21 program. 17 . Food and Rural Affairs.Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements This study was supported by the Strategic Initiative for Microbiomes in Agriculture and Food.

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24 . Compositions of the fecal microbiota of the lighter and heavier piglets.Figure 1. The number of phyla (A) and genera (B) shared by the two groups are shown in Venn diagrams. The overall compositions of the fecal microbiota of the lighter and heavier groups were represented as bar plots at the phylum level (C) and the genus level (D).

and P < 0. Comparison of the compositions of the fecal microbiota of the lighter and heavier piglets at the phylum level.05 was considered significant. The differences in the phylum level compositions were tested using a two-sided Welch’s t-test.Figure 2. (B) The phyla represented at significantly different levels in the microbiota of two groups are shown in an extended error bar plot. 25 . (A) Principal component analysis (PCA) plot.

Figure 3. and P < 0. 26 . (B) The genera represented at significantly different levels in the microbiota of the two groups are shown in an extended error bar plot.05 was considered significant. Comparison of the compositions of the fecal microbiota of the lighter and heavier piglets at the genus level. The differences in the genus level compositions were tested using a two-sided Welch’s t-test. (A) Principal component analysis (PCA) plot.

and P < 0. The different functions of the fecal microbiota of the lighter and heavier piglets. The differences between the levels of the predicted functions were tested using a two-sided Welch’s t-test. The microbial functions were predicted using PICRUSt at the third level of the KEGG pathway and were expressed as relative abundances.05 was considered significant. 27 .Figure 4.

06 Vitamin 0.04 Oat 5.10 Enzyme complex 0.00 Whey powder 10.26 Methionine 0.09 Threonine 0.00 Lactose 4.00 Wheat bran 2.05 Emulsifier 0. Composition of the commercial diet of the weaned piglets Item Contents.00 Dehulled soybean meal 21.00 Fish meal 4.10 Zinc oxide 0.20 Phytase 0.65 Limestone 1.70 Soy oil 3.00 Glucose 3.25 Lysine 0.20 Choline chloride 0.10 Organic acids 0.60 Salt 0. % Corn 39.30 Mineral 0.30 28 .00 Dried sweets by-products 4.00 Monocalcium phosphate 0.Tables Table 1.

23 Lighter 9 L6 11.22 Lighter 9 L3 9.23 Lighter 9 L9 11.98 Heavier 9 H6 18.50 Heavier 9 H9 22.75 Heavier 9 The piglets were sorted in ascending order by their body weights 29 .05 Lighter 9 L7 11.01 Heavier 9 H3 17. Characteristics of the weaned piglets used in this study Piglet Body weight (kg) Group Age (week) L1 8.Table 2.09 Lighter 9 L2 8.18 Lighter 9 L5 10.70 Heavier 9 H8 22.70 Lighter 9 L4 10.78 Heavier 9 H7 20.70 Heavier 9 H2 17.89 Lighter 9 H1 16.13 Heavier 9 H4 17.17 Lighter 9 L8 11.45 Heavier 9 H5 17.

935 The data were expressed as the mean values ± standard deviation (SD) a The P values were determined using Welch’s t-test (* P < 0. ** P < 0.243.01.20 ± 1.000 sequence reads with 10 iterations 30 .86 0.19 2.05.720.08 ± 100.Table 3.014 * PD 174.92 ± 1.56 ± 10.309.887.41 9.18 0.001) b The alpha diversity indices were calculated from 5.78 ± 180.82 ± 10.485.56 ± 1. *** P < 0.29 ± 2.037 * Chao1 15.642.63 ± 0.29 0.41 ± 0.257.60 ± 1.00 ± 2.99 ± 0.208 Simpson 0.001 *** Alpha diversity b ACE 16.00 0.029 * Observed OTUs 2.69 0. Differences in the fecal microbial diversity of the two groups Item Lighter Heavier P value a Body Weight (kg) 10.96 18.205 Shannon 9.99 ± 0.44 17.628.33 19.95 0.605.452.70 181.38 < 0.01 0.

05.01 ± 0.14 ± 0.59 ± 4.54 ± 1.01 0.01± 0.040 * Bacteroides 4.84 0.020 * FB ratio 1.24 ± 0.52 ± 0.36 ± 0.14 ± 0.00 0.02 ± 0.05 0.01 ± 0.38 53.25 ± 0.01) 31 .00 0.045 * [Prevotella] 3.02 ± 0.19 0.21 ± 1. ** P < 0.05 ± 0.02 0.31 ± 3.056 The data were expressed as the mean values ± standard deviation (SD) a The P values were determined using Welch’s t-test (* P < 0.05 ± 0.01 0.Table 4.035 * Sediminibacterium 0.02 0.03 ± 0.07 ± 0.15 ± 4.02 0.03 ± 0.03 0.01 0.01 0.03 0.88 0.010 ** Genus (%) Anaerococcus 0.025 * Anaerotruncus 0.39 34.61 ± 0.13 ± 0.01 ± 0.00 ± 0.024 * Lactococcus 0.034 * [Eubacterium] 0.040 * Butyrivibrio 0.13 0.03 0.42 0.009 ** Planctomycetes 0.00 ± 0.01 0.019 * Bacteroidetes 39.038 * Bilophila 0.65 ± 1.12 0.13 0. Differences in the fecal microbiota of the two groups Taxon Lighter Heavier P value a Phylum (%) Firmicutes 48.00 ± 0.42 ± 2.67 2.049 * Corynebacterium 0.049 * Streptococcus 0.75 2.09 0.32 0.01 ± 0.01 0.16 1.00 0.