Effect of Dietary Amylose/amylopectin Ratio on Diarrhea and Gut Microorganism of Weaned Pigs Challenged With Escherichia Coli Lipopolysaccharide

can yang Hunan Normal University https://orcid.org/0000-0001-8973-6994 min wang Hunan Normal University xiaowu tang Hunan vocational technical college of environment and biology huansheng yang (  yhs@hunnu.edu.cn ) Hunan Normal University https://orcid.org/0000-0003-1164-5771 fengna li Institute of Subtropical Agriculture Chinese Academy of Sciences yancan wang Hunan Normal University yulong yin Hunan Normal University

Diarrhea of piglets was recorded every day during the experimental period. On day 29, 12 h post-challenge, pigs were euthanized. Immediately after removal of the gastrointestinal tract from the carcass, digesta samples from stomach, proximal duodenum, distal jejunum, end of ileum, cecal and colon were taken.
Mucosa from jejunum and ileum were sampled using glass slide scraping intestinal wall. A 5 cm section of cecal and colon was xed by immersion in 10% buffer neutral formalin. In addition, the remaining digesta samples underwent determination of pH value (Testo 206, pH meter, Testo AG, Lenzkirch, Germany).
Mucosa and chyme samples were stored at -80℃ until required for analysis.

Diarrhea incidence
Fecal consistency was scored as follows: 0 = normal, 5 = liquid. Diarrhea degree was calculated as the sum of the fecal scores for every piglet each week.
Piglets diarrhea rates were recorded daily and calculated as follows: Diarrhea rate = total number of pigs with diarrhea/(total number of pigs × experimental days) × 100, where total number of pigs with diarrhea was de ned as the number of pigs with diarrhea observed on each day.

Intestinal morphology
Fixed intestine was dehydrated, embedded, sectioned and stained with hematoxylin and eosin. The mean crypt depth was quanti ed using a 40 × combined magni cation, and an image processing and analysis system (Version 1, Leica Imaging Systems Ltd., Cambridge, UK). A minimum of 20 crypts were randomly chosen and measured per subject. Crypt depth was measured using a light microscope tted with an image analysis system (AxioScope A1, Carl Zeiss, Germany).

Gene expression analysis by RT-qPCR
Total RNA was isolated from mucosa using RNAiso Plus (TaKaPa) and then reverse transcription reactions were performed using RT reagent kit (TaKaPa). All the procedures were carried out as described by the manufacturer's protocol. The quantity and quality of RNA were determined using NanoDrop ND-2000 spectrophotometer system (Thermo Fisher Scienti c). Real-time RT-PCR primers were designed to assay genes related to tight connection and in ammation (Table 2). 18S were used as reference gene. Real-time RT-PCR for target genes were performed on MyIQ instrument (Bio-Rad, Hercules, California) using SYBR Green quantitative PCR mix (TaKaRa).

16S ribosomal RNA sequencing
Total genome DNA from cecal digesta of saline stressed piglets was extracted using CTAB/SDS method. The hypervariable regions V3-V4 of the 16S rRNA genes were chosen for pyrosequencing to investigated the taxonomic compositions of the microbial community. The PCR amplification was conducted in triplicate utilizing the barcoded universal bacterial primers. Triplicates were pooled, and the PCR amplicons were sequenced using IlluminaHiSeq2500 platform and 250 bp paired-end reads were generated according to previous study [9,10]. The raw sequence data were processed and analyzed with a QIIME software package. Then, sequences with a threshold of 97% similarity were assigned to the same operational taxonomic units (OTUs) on the basis of representative sequences using Uparse v7.0.1001 and GreenGene Database. R software (version 2.15.3) with the 'vegan' package was used to perform bacterial analysis of Bray-Curtis dissimilarities based on the levels of changed gut microbiota.

Statistical analysis
Gene expression data from replicate measurement within the same RNA extraction were averaged and analyzed using Livak's (2001) [11] method to measure the difference between the ve DAR diets. Data of diarrhea occurrence, microorganism were examined by single factor design using GLM procedure. Other data were analyzed by double factor with the DAR and LPS stress as main effects, using general linear model (GLM) procedure of SAS 8.0. Duncan differences were determined to compare differences among the groups. Results were presented as means ± sem. Differences with P < 0.05 were considered to be signi cant.

Diarrhea occurrence
Diarrhea rate and diarrhea degree were higher in group 0.40 than in group 0.20 and 0.80 during third week (P<0.05). No statistical difference of diarrhea rate and degree could be observed between different experimental treatments during rst, second, fourth and total four weeks (P 0.05) ( Table 3).
pH value and VFA of digesta No signi cant difference of pH could be observed between ve groups in digesta of stomach, jejunum and ileum (P 0.05) ( Table 4). Cecal pH was affected by DAR and LPS stress, pH value was higher in group 0.20 than in groups 0.80 and 0.00 (P<0.05), but with no signi cant difference when compared with groups 0.40 and 0.60 (P>0.05). Cecal and colonic pH value increased after LPS stress (P<0.05). No effect of interaction between DAR and LPS stress was found on pH value.
The DAR had no signi cant effect on SCFA such as acetic acid, propionic acid, iso-butyrate acid, iso-Valeric acid and total amount of SCFA concentration in jejunum (P>0.05) ( Table 5). Jejunal butyrate acid was affected by DAR, it was higher in group 0.80 compared with groups 0.00, 0.40, 0.60 (P<0.05). Jejunal butyrate acid increased in group 0.00 and 0.20 but decreased in groups 0.60 and 0.80 after LPS stress (P<0.05). Valeric acid concentration in jejunum increased after LPS stress in groups 0.00, 0.20, 0.80 but decreased in groups 0.40 and 0.60 (P<0.05). Ileum acetic acid, propionic acid, butyrate acid, isobutyrate acid, valeric acid and total amount of SCFA were not affected by DAR or LPS stress except that iso-valeric acid of ileum decreased after LPS stress (P<0.05). Cecal SCFA except butyrate acid was affected by DAR (P<0.05). Acetic acid, propionic acid and total SCFA concentrations were higher in group 0.40 compared with groups 0.00, 0.60, 0.80 (P<0.05). Iso-butyrate acid and iso-valeric acid concentrations increased after LPS stress (P<0.05), they were both higher in group 0.20 than in other groups (P<0.05). Valeric acid concentration was higher in group 0.20 compared with groups 0.00, 0.60, 0.80 (P<0.05). Total amount of SCFA was higher in group 0.40 when compared with groups 0.00, 0.60, 0.80 (P<0.05), but with no signi cant difference compared with group 0.20 (P>0.05). Colonic SCFA except valeric acid was affected by LPS stress but not DAR, all of SCFA such as acetic acid, propionic acid, iso-butyrate acid, butyrate acid, iso-valeric acid decreased after LPS stress (P<0.05). Crypt depth of large intestine Crypt depth of cecal was lower in group 0.80, lower than other groups except group 0.40 (P<0.05). Crypt depth of colon was not affected by DAR and LPS stress(P>0.05) ( Table 6).

Expression of genes related to gut health
Expression of genes such as claudin, zo-1 in mucosa were showed in Table 7. DAR did not alter the mRNA expression of ZO-1,IL-1β, TNFα in mucosa of jejunal, the Occludin,ZO-1,IL-1β in mucosa of ileum (P 0.05). LPS stress caused lower mRNA expression of claudin in jejunal (P<0.05). Claudin mRNA expression was higher in group 0.60 when compared with other groups (P=0.045) in jejunal, and it was higher in group 0.20 than in other groups in mucosa of ileum (P<0.01). Ingestion of diet with DAR 0.00 resulted in lowest TNFα mRNA levels in ileum mucosa, lower than groups 0.40, 0.60 and 0.80 (P<0.05).

The bacterial community composition in the cecum
The reads for each sample is in the range of 70049 to 96176. After quality trimming and chimera checking, each sample has 77296±7459 tags with a minimum length of 410 nucleotides, a maximum length of 426. 791 OUTs were shared by the ve groups, and 169, 211, 194, 368, 247 OTUs were only found in ceca of group 0.00, 0.20, 0.40, 0.60, 0.80 respectively (Fig. 1). No signi cant differences were found on Shannon, Simpson, ACE, PD_whole tree indexes and beta diversity of bacteria between different DAR groups ( Fig.2 A and Fig.2 B). Alpha diversity representative by chao1 of group 0.40 tended to be lower than group 0.60 (P=0.053), with no signi cant difference compared with other groups (P>0.05). Alpha diversity representative by goods coverage of groups 0.20 and 0.40 was lower than group 0.60 (P<0.05), with no signi cant difference compared with other groups (P>0.05).
At the phylum level, Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes were predominantly found in the cecal samples from different DAR groups. No signi cant difference was found in phylum between different DAR groups (P>0.05) (Fig.3A). At phyla level, no signi cant difference was found between different DAR groups (P>0.05). At the genus level, the abundance of Ruminococcaceae_NK4A214_group and Anaerotruncus in cecal chyme of Group 0.20 was signi cantly higher than that in Group 0.60 (P<0.05), with no signi cant difference compared with other groups (P>0.05) (Fig.3B). The abundance of Cetobacterium was signi cant lower in cecal chyme from Group 0.20 compared with pigs from Group 0.60 (P<0.01), with no signi cant difference compared with other groups (P>0.05).

Discussion
An inappropriate balance between pro-in ammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α), and the antiin ammatory cytokines such as IL-10 would lead to in ammation in local tissues such as bowel [12]. Elevation in expression of the pro-in ammatory cytokines such as TNFα and IL-6 are the hallmark of acute bowel in ammation [13]. Therefore, our cytokine pro ling results suggest that acute bowel in ammation had occurred in Group 0.40 but not in Group 0.00 and 0.20 under 100 µg/kg of lipopolysaccharide (LPS) stress in weaning pigs. This is inconsistent with other reports. High level of amylose is associated with high resistant starch (RS) level. Chronic kidney disease rats consuming diets supplemented with amylopectin exhibited in ammation, activation of NF k B, upregulation of pro-in ammatory, pro-oxidant molecules, impaired nuclear transcription factor (Nrf2) activity, down-regulation of antioxidant enzymes, and disruption of colonic epithelial tight junction, but the high resistant starch diet signi cantly attenuated these abnormalities [14].RS supplementation has proven to be effective in reducing markers of in ammation in the state of the disease [15]. Studiers found a reduction of TNF-α concentrations in prediabetes patients supplemented with 45 g/d high-amylose maize for 12 weeks [16,17]). Supplementation of HAM-RS2 20 g/d in the rst month and 25 g/d during the second month led to a decrease in serum urea, IL-6, TNFα in end-stage renal disease patients [17]. Moreover, consumption of retrograded high-amylose corn resistant starch at 15% may protect the colon from developing in ammation by enhancing IL-10 abundance in pigs, but not affected TNFα and IL-6 abundance in colon [18].Though gut injury was observed in piglets that received 60 [19] or 100 [20] µg/kg of E.coli LPS and injection of LPS stimulated the production of cytokines such as IL-1, TNF-α and IFN-γ [21]. But 11 h post-challenge showed no effect of LPS on TNFα mRNA expression in jejunal and ileal mucosa of weaning pigs in our result. The acute bowel in ammation in Group 0.40 was due to severe diarrhea happened during the last week before LPS stress. Diarrhea resulted adverse effect on average daily gain (ADG) and feed e ciency (F:G) during the next week (data not shown).
Prebiotic approaches also typically fail to consider the potential to cause detrimental changes in individuals in whom the gut microbiota is already substantially disrupted, such as the selective promotion of potentially pathogenic taxa. Phylum Proteobacteria abundance was higher in Group 0.40. Previous studies indicated that the abnormal increase of Prevotellaceae abudndance exacerbated the occurrence of in ammation [22]. Genus Sutterella belongs to Prevotellaceae phylum, and it has been found to elevated in feces from dogs with acute hemorrhagic diarrhea [23]. The genus Rikenellaceae RC9 gut group was signi cantly increased in the high-fat diet with high-dose genistein mice group [24] and in isoproterenol-induced acute myocardial ischemia group [25]. In the present study, we observed the signi cant negative correlation between abundance of Rikenellaceae RC9 gut group and diarrhea rate during third week. Thus, the increase of Rikenellaceae RC9 gut group might associate with the gut in ammation. Although piglets from Group 0.40 suffered severe diarrhea, they got the same ADG and feed intake as other groups during the whole four experimental weeks. This result, in part, be due to SCFAs' modulating in ammation response. In this study, 11 h post-LPS-challenge pigs showed a signi cant reduction in acetate, propionic acid, butyric acid and total short-chain fatty acid (SCFA) in colonic digesta. Supplementation of diet with DAR 0.40 in weaning piglets resulted in a signi cant increase in these SCFA in cecum digesta compared with Group 0.00. SCFAs have a wide range of anti-in ammatory properties, including the ability to increase colonic regulatory T cells [26], alter dendritic cell and macrophage function [27], and production of pro-in ammatory cytokines [28]. Gut microbiotal conversion of inulin-type fructose into propionate could inhibit malignant cell proliferation, lessens systemic in ammation in liver [29]. The reduction in intestinal pH due to increased SCFA production decreases the formation of pro-in ammatory and pro-oxidant uremic toxins from colonic bacteria [14]. 0.20-fed piglets exhibited less microbial diversity than 0.6-fed piglets. More microbial diversity is generally associated with a healthier phenotype [30]. Both Ruminococcaceae_ NK4A214_group and Anaerotruncus abundance in cecal chyme were higher in Group 0.20 than that in Group 0.60. It is well known that intestinal microbiota is a crucial factor in maintaining intestinal barrier and harmful metabolites' transfer. They can affect the intestinal permeability, enhance the transfer of harmful substances in blood, and stimulated in ammatory response [31].The regulation of gut microbiota such as Ruminococcaceae_NK4A214_group and Anaerotruncus could be developed to an increase in cecal SCFA such as iso-butyrate acid, iso-valeric acid and valeric acid concentration. The higher levels of iso-BCFAs may be associated with alteration in the metabolism of BCAAs, namely valine, leucine, and isoleucine, which can serve as precursors of BCFAs [32]. Thus, the increased production of iso-butyric acid and iso-valeric acid should indicate increased protein degradation during LPS stress in Group 0.20. LPS challenge increases the plasma-urea nitrogen level in piglets due to muscle proteolysis as result of increased in ammation [33]. Study found inverse correlations between circulating iso-branched-chain fatty acids and C-reactive protein (CRP, an in ammatory marker) in patients with morbid obesity [34]. Released amino acids due to in ammation may be channeled to the liver to synthesize acute phase proteins and/or to serve as an energy source [35]. Further examination of the these and other related markers of in ammation and oxidative stress are warranted in studies of longer duration. Claudin-1 and occluding are key components of the epithelial tight junction. LPS stress caused lower claudin mRNA expression in jejunal mucosa. As a result, intestinal barrier function was affected as claudin mRNA expression decreased in Group 0.20 compared with Group 0.60.

Conclusions
In conclusion, diarrhea was affected by dietary amylose/amylopectin ratio (DAR). Diarrhea rate and diarrhea degree were higher when DAR was 0.40 and lower when it was 0.20 or 0.80 during third weed after weaning. Consumption of diet with DAR 0.20 leads to increase in cecal pH, iso-and valeric acid concentration and in claudin mRNA expression in mucosa of ileum. After diarrhea, microbial composition changed to restore intestinal health in piglets supplemented diet with DAR 0.40. This was con rmed by higher alpha diversity and higher cecal acetic acid and propionic acid concentration in this group (0.40).

Declarations
Ethics approval and consent to participate Experimental procedure in this study was reviewed and approved by the Animal Care and Use Committee of the Hunan international joint laboratory of animal intestinal ecology and health, Hunan Normal University.

Not applicable
Availability of data and material The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Yin Yulong and Yang Huansheng organized the experiment and gave some advice on experiment idea. Yang Can conducted the experiment and was a major contributor in writing the manuscript. Wang Min, Tang Xiaowu and Wang Yancan conducted the experimental analysis. Li Fengna reviewed the manuscript and gave some advice on experiment idea. All authors read and approved the nal manuscript 21. Kluger MJ. Fever: role of pyrogens and cryogens. Physiol Rev, 1991