Abstract

   Feeding HC diets has been found to induce metabolic dysregulation in
   the colon. However, the mechanisms by which changes in colonic flora
   and metabolites damage the colonic epithelium are poorly studied.
   Therefore, the present experiment used a multi-omics technique to
   investigate the mechanism of colonic injury induced by high-concentrate
   diets in lambs. Twelve male Dumont lambs were randomly split into two
   groups: a low-concentrate diet (LC = concentrate/forage = 30:70) group
   and a high-concentrate diet (HC = concentrate/forage = 70:30) group.
   The results showed that the HC group presented significantly increased
   lipopolysaccharide (LPS) concentrations in the colonic epithelium and
   significantly decreased serum total cholesterol (TC), superoxide
   dismutase (SOD), and glutathione peroxidase (GSH-Px) levels (p < 0.05),
   which led to cavities and inflammatory cell infiltration in the colonic
   epithelium. The HC group had significantly lower pH and less VFAs in
   colon contents, as well as a significantly increased abundance of
   bacteria of the genera [Eubacterium]_coprostanoligenes_group,
   Rikenellaceae_RC9_gut_group, Treponema, Clostridia_UCG-014, Alistipes,
   Ruminococcus, Christensenellaceae_R-7_group, UCG-002,
   Bacteroidales_RF16_group and Lachnospiraceae_AC2044_group compared to
   the LC diet group. These microorganisms significantly increased the
   level of metabolites of cholic acid, chenodeoxycholic acid, LysoPA
   (P-16:0/0:0), methapyrilene, and fusaric acid. A transcriptome analysis
   showed that cytokine–cytokine receptor interaction, glutathione
   metabolism, and the peroxisome signaling pathway were downregulated in
   the colon epithelium of the lambs fed the HC diet. Therefore, the HC
   diet caused epithelial inflammation and oxidative damage by affecting
   the interaction between the microbial flora of the colon and
   metabolites and the host epithelium, which eventually disrupted colon
   homeostasis and had a negative impact on sheep health.

1. Introduction

   Feeding HC diets to satisfy the increased nutritional demands of
   high-production ruminants is a common practice in livestock management.
   However, this dietary composition alteration often increases the
   incidence of nutritional metabolic disorders [[40]1]. Digestion in the
   hindgut is essential for ruminants; approximately 17% of digestible
   cellulose is metabolized in the cecum and 13% is metabolized in the
   colon [[41]2], resulting in the production of around 12% of VFAs
   [[42]3] and contributing about 8% to the metabolic energy requirements
   of sheep [[43]4]. When ruminants consume a high proportion of grain
   feed and a small amount of forage, the VFA and lactic acid
   concentrations in the rumen increase and the pH decreases, inducing
   subacute ruminal acidosis (SARA), which causes metabolic disorders of
   rumen microorganisms and reduces the absorption and barrier ability of
   rumen epithelial cells [[44]1]. At the same time, the amount of rumen
   bypass starch entering the hindgut increases [[45]5,[46]6,[47]7]. Due
   to the absence of saliva and protozoan buffering in the hindgut,
   coupled with the presence of a mucus layer in the intestinal lumen,
   which leads to a decrease in pH, VFA absorption is not promoted,
   causing hindgut acidosis [[48]8,[49]9]. Furthermore, the hindgut
   epithelium is a monolayer columnar epithelium, which renders the
   permeability and integrity of the hindgut mucosa more susceptible to
   compromise [[50]1].

   The cecum and colon are colonized by a rich and complex microbiome,
   dominated by bacteria (more than 95%), including archaea [[51]10].
   During digestion and metabolism, intestinal microorganisms communicate
   with host cells via the production of metabolites and signaling
   molecules, which affect the metabolism and immunity of the host
   [[52]11]. Intestinal microorganisms are influenced by many factors,
   such as diet [[53]12,[54]13,[55]14], feeding method [[56]15], season
   [[57]16] and genetics [[58]17]. HC diets have been shown to
   significantly reduce microbial diversity and richness in the ruminant
   hindgut [[59]12], accompanied by a marked decline in the relative
   abundance of fibrinolytic bacteria [[60]18] and an increase in
   amylolytic and pathogenic bacteria, which ultimately affects the
   intestinal health of the host [[61]19,[62]20]. However, most current
   studies examining the effects of HC diets on ruminants have primarily
   focused on the rumen, with less information on metabolic dysregulation
   in the colon and even less still on the interactions of changes in
   colon flora and metabolites induced by HC diets with the host
   epithelium. Therefore, this experiment investigated the interactions of
   colonic microorganisms and their metabolites with the host on an HC
   diet.

2. Materials and Methods

2.1. Animal Feeding and Experimental Design

   An experiment was conducted at Hailutu Farming Base (40°40′30″ N,
   111°21′34″ E) with 12 male Dumont lambs (who had an average weight of
   26.37 ± 2.29 kg and were 3 months old). The lambs, sourced from Inner
   Mongolia Sino Breeding Sheep Technology Co., Ltd (Ulanqab, Inner
   Mongolia, China). were randomly assigned to two groups. The lambs were
   housed in individual metabolic cages (1.5 m × 1 m × 1 m) with ad
   libitum access to water and fed twice daily at 08:00 and 18:00. The
   control group received a low-concentrate diet (LC = concentrate/forage
   = 30:70), while the experimental group received an HC diet (HC =
   concentrate/forage = 70:30). The pre-feeding period lasted 15 days,
   followed by a 60-day experimental period. Diet composition and
   nutritional details are provided in [63]Table A1.

2.2. Sample Collection

   After the experiment, blood was collected from the jugular vein of the
   lambs immediately post-mortem (the LC group’s average weight was 36.70
   ± 1.20 kg, and the HC group’s average weight was 41.79 ± 3.89 kg).
   Lambs were exsanguinated according to the Interim Regulations on the
   Management of Livestock and Poultry Slaughtering in Hohhot issued by
   the People’s Government of Hohhot, China. Colonic contents were
   collected for pH measurement. Furthermore, the colon tissue was washed
   with PBS post-mortem. Approximately 1 cm of the intestinal segment of
   the colon was collected and fixed using a 4% paraformaldehyde solution
   for a histological analysis. Colon contents were collected with a
   sterile medicine spoon and placed into a freezing tube for quick
   freezing with liquid nitrogen. Colonic epithelial tissues were rinsed
   with pre-cooled phosphate-buffered saline (PBS) and then epithelium was
   separated from underlying layers by scraping using a coverslip, and the
   cells were frozen in a tube containing 0.5 mL RNase inhibitor
   (BeyotimeBiotechnology, Shanghai, China) and subsequently stored at −80
   °C.

2.3. Histological Analysis of Colon Tissue

   The fixed colon tissues were subjected to paraffin sectioning and
   hematoxylin–eosin (HE) staining according to conventional methods.
   Colon morphology was observed under a microscope (Nikon, Minato-ku,
   Tokyo Metropolis, Japan).

2.4. Measurement of Colonic Epithelial LPS Content and Serum Parameters

   Serum total protein (TP), albumin (ALB), globulin (GLB), glucose (GLU),
   total cholesterol (TC), and triglyceride (TG) levels were measured
   using a biochemical analyzer, with kits supplied by Lepu Diagnostic
   Technology Co., Ltd., (Beijing, China). The kits for serum
   immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG),
   serum smyloid A (SAA), interleukin-1β (IL-1β), tumor necrosis factor–α
   (TNF-α), interleukin–6 (IL-6), superoxide dismutase (SOD), glutathione
   peroxidase (GSH-Px), and colon LPS concentrations were provided by
   Wuhan Gene Biotechnology Co., Ltd., (Wuhan, Hubei, China).

2.5. Quantitative Real-Time PCR Analysis

   Total colonic RNA was extracted using Trizol (TaKaRa Bio, Kyoto, Kyoto
   Prefecture, Japan) and reverse-transcribed into cDNA after confirming
   the concentration and purity using a Nanodrop 2000 (Thermo Scientific,
   Waltham, MA, USA). The primer sequences are listed in [64]Table A2.
   qRT-PCR was performed, with β-actin and glyceraldehyde-3-phosphate
   dehydrogenase (GAPDH) as the internal references. The amplification