Galacto-Oligosaccharides Alleviate LPS-Induced Immune Imbalance in Small Intestine through Regulating Gut Microbe Composition and Bile Acid Pool.

Recent evidence suggests that the protective effect of gut microbiota on intestinal inflammation can be achieved through a microbe-bile acids (BAs) mechanism. Galacto-oligosaccharides (GOS) are a kind of prebiotic that alter gut microbiota composition. To verify whether GOS has a protective effect on intestinal inflammation through a microbe-BAs mechanism, this research was performed in a lipopolysaccharide (LPS) porcine model with the presence or absence of GOS. GOS prevented LPS-induced production of pro-inflammatory cytokines, the decrease of bacterial bile salt hydrolase-containing bacteria abundance, and the decrease of chendoxycholic acid (CDCA) level in piglets. Additionally, CDCA decreased LPS-induced production of pro-inflammatory cytokines, induced the expression of the takeda G-protein receptor 5 (TGR5), and its downstream cyclic adenosine monophosphate (cAMP) production in lamina propria-derived CD11b+ cells. The cAMP inhibitor eliminated the protective effect of CDCA on lamina propria-derived CD11b+ cells. These results suggested that GOS reduced the production of pro-inflammatory cytokines and inhibited NF-κB activation via microbe-BA-dependent TGR5-cAMP signaling in LPS-challenged piglets.

Dynamic analysis of metabolomics reveals the potential associations between colonic peptides and serum appetite-related hormones.

Gut signals, including hormones and metabolites are crucial zeitgebers that regulate the circadian rhythm of host metabolism, but the potential links have been explored more in rodents. Herein, we performed an hour-scale metabolomics analysis of serum and colonic digesta to characterize the circadian rhythmic metabolic patterns using a pig model under ad libitum feeding conditions. Importantly, our findings identified potential associations between colonic and body metabolism, revealing the potential relationships between colonic peptides and host appetite regulation. Concretely, amino acids accounted for the highest proportion in rhythmic serum metabolites, whereas lipids accounted for the highest proportion in rhythmic colonic metabolites. The diurnal difference analysis revealed that the levels of most amino acids and peptides were higher in the light phase, while the levels of most lipids were higher in the dark phase. And more correlations were be checked between serum amino acids, lipids, peptides and colonic metabolites in the light and more correlations were be checked between serum carbohydrates, cofactors and vitamins, energy, nucleotides, xenobiotics and colonic metabolites in the dark. Interestingly, peptides oscillated to a similar extent in serum and colonic digesta. Of note, colonic peptides composed of valine, proline and leucine were checked in positive associations to glucagon-like peptide-1 (GLP-1) in serum. And these peptides were positive with the genera Butyricicoccus, Streptococcus, Clostridioides, Bariatricus and Coriobacteriia_norank, and negative with Prevotella, and showed the potential relationships with colonic microbial biosynthesis of amino acids. Collectively, we mapped the rhythmic profiling on pig serum and colonic metabolites and revealed the relationships between host and gut metabolism. However, the underlying regulatory mechanisms remains to be further investigated.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of α-linolenic acid and 12,13-epoxyoctadecenoic acid.

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium (DSS)-induced colitis in mice. To explore the protective effects of L. plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC), 28 weaned piglets were assigned into four groups, namely, CON group-orally given 10 mL/d phosphate buffer saline (PBS), LI47 group-orally given a mixture of 10 mL/d L. plantarum L47 and inulin, ECON group-orally given 10 mL/d PBS and challenged by ETEC, and ELI47 group-orally given 10 mL/d L. plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L. plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased anti-inflammatory factors. Transcriptome analysis revealed that L. plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA (PLA2G2A) (P < 0.05) as well as affected alpha-linolenic acid (ALA) metabolism and linoleic acid metabolism. Moreover, L. plantarum L47 and inulin increased the levels of ALA (P < 0.05), lipoteichoic acid (LTA) (P < 0.05), and 12,13-epoxyoctadecenoic acid (12,13-EpOME) (P < 0.05) and the protein expression of Toll-like receptor 2 (TLR2) (P = 0.05) in the ileal mucosa. In conclusion, L. plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.

Duodenal infusion of soy protein hydrolysate activates cAMP signaling and hypothalamic neurotransmitter synthesis in pigs.

Neurotransmitters in the brain are important for cognition and memory. As bioactive substrates, whether increased soy protein levels in pigs can promote hypothalamic neurotransmitter synthesis remains unclear. The effect of increased soy protein hydrolysate (SPH) levels in the small intestine of pigs on neurotransmitter precursor supply, hypothalamic neurotransmitter synthesis and underlying molecular processes was investigated by using sixteen pigs (35.2 ± 0.3 kg) infused either with SPH (70 g day-1) or sterile saline (control) twice daily for 15 days via a duodenal fistula. It demonstrated that SPH infusion increased the expression of the neutral amino acid transporter B0AT1 in the jejunal mucosa, the serum tyrosine/large neutral amino acid ratio, the concentrations of serum tyrosine, hypothalamic tyrosine, dopamine and brain-derived neurotrophic factor (BDNF) (P < 0.05). It also increased the jejunal and serum choline, hypothalamic choline and acetylcholine levels (P < 0.05). Hypothalamic transcriptome revealed that differential genes were significantly enriched in the cholinergic synapse, dopaminergic synapse and cyclic adenosine monophosphate (cAMP) signalling pathways, and that the expression of key enzyme genes in the synthesis of acetylcholine and dopamine and dopamine receptors 1 (DRD1) was upregulated by SPH (P < 0.05). Western blotting showed that SPH infusion activated the hypothalamic cAMP signalling pathways. Overall, SPH infusion promoted the synthesis of hypothalamic dopamine and acetylcholine, and the synthesised dopamine promoted BDNF production most likely through the activation of the cAMP signalling pathways by the DRD1.

Effects of lactic acid bacteria-fermented formula milk supplementation on ileal microbiota, transcriptomic profile, and mucosal immunity in weaned piglets.

BACKGROUND: Lactic acid bacteria (LAB) participating in milk fermentation naturally release and enrich the fermented dairy product with a broad range of bioactive metabolites, which has numerous roles in the intestinal health-promoting of the consumer. However, information is lacking regarding the application prospect of LAB fermented milk in the animal industry. This study investigated the effects of lactic acid bacteria-fermented formula milk (LFM) on the growth performance, intestinal immunity, microbiota composition, and transcriptomic responses in weaned piglets. A total of 24 male weaned piglets were randomly divided into the control (CON) and LFM groups. Each group consisted of 6 replicates (cages) with 2 piglets per cage. Each piglet in the LFM group were supplemented with 80 mL LFM three times a day, while the CON group was treated with the same amount of drinking water. RESULTS: LFM significantly increased the average daily gain of piglets over the entire 14 d (P < 0.01) and the average daily feed intake from 7 to 14 d (P < 0.05). Compared to the CON group, ileal goblet cell count, villus-crypt ratio, sIgA, and lactate concentrations in the LFM group were significantly increased (P < 0.05). Transcriptomic analysis of ileal mucosa identified 487 differentially expressed genes (DEGs) between two groups. Especially, DEGs involved in the intestinal immune network for IgA production pathways, such as polymeric immunoglobulin receptor (PIGR), were significantly up-regulated (P < 0.01) by LFM supplementation. Moreover, trefoil factor 2 (TFF2) in the LFM group, one of the DEGs involved in the secretory function of goblet cells, was also significantly up-regulated (P < 0.01). Sequencing of the 16S rRNA gene of microbiota demonstrated that LFM led to selective enrichment of lactate-producing and short-chain fatty acid (SCFA)-producing bacteria in the ileum, such as an increase in the relative abundance of Enterococcus (P = 0.09) and Acetitomaculum (P < 0.05). CONCLUSIONS: LFM can improve intestinal health and immune tolerance, thus enhancing the growth performance of weaned piglets. The changes in microbiota and metabolites induced by LFM might mediate the regulation of the secretory function of goblet cells.

Effects of the increased protein level in small intestine on the colonic microbiota, inflammation and barrier function in growing pigs.

BACKGROUND: An increased level of the dietary protein alters the colonic microbial community and metabolic profile of pigs, but it remains unclear whether this leads to colonic inflammation and impairs barrier function in growing pigs. RESULTS: Sixteen pigs (35.2 ± 0.3 kg) were infused with sterile saline (control) or soy protein hydrolysate (SPH) (70 g/day) through a duodenal fistula twice daily during a 15-day experimental period. The SPH treatment did not affect their average daily feed intake and daily weight gain (P > 0.05), but reduced colon index and length (P < 0.05). Illumina MiSeq sequencing revealed that species richness was increased following SPH intervention (P < 0.05). Furthermore, SPH reduced the abundance of butyrate- and propionate-producing bacteria-such as Lachnospiraceae NK4A136 group, Lachnospiraceae_uncultured, Coprococcus 3, Lachnospiraceae UCG-002, and Anaerovibrio-and increased the abundance of potentially pathogenic bacteria and protein-fermenting bacteria, such as Escherichia-Shigella, Dialister, Veillonella, Prevotella, Candidatus Saccharimonas, Erysipelotrichaceae UCG-006, Prevotellaceae_uncultured, and Prevotellaceae UCG-003 (P < 0.05). In addition, a lower content of total short-chain fatty acids, propionate, and butyrate and a higher concentration of cadaverine, putrescine, total biogenic amines, ammonia, and isovalerate were observed following SPH infusion (P < 0.05). Further analysis revealed that SPH increased the concentration of tumour necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-8 in the colonic mucosa (P < 0.05). Interestingly, SPH intervention increased the expression of occludin, zonula occludens (ZO)-1, and claudin-1 in colonic mucosa (P < 0.05). Correlation analysis showed that different genera were significantly related to the production of metabolites and the concentrations of pro-inflammatory cytokines. CONCLUSION: An increased soy protein level in the small intestine altered the colonic microbial composition and metabolic profile, which resulted in the secretion of colonic proinflammatory cytokines and the increased expression of tight junction proteins.

Proteomic identification of ruminal epithelial protein expression profiles in response to starter feed supplementation in pre-weaned lambs.

The present study aimed to comparatively characterize the ruminal epithelial protein expression profiles in lambs fed ewe milk or milk plus starter diet using proteome analysis. Twenty new-born lambs were randomly divided into a group receiving ewe milk (M, n = 10) and a group receiving milk plus starter diet (M + S, n = 10). From 10 d old, M group lambs remained with the ewe and suckled ewe milk without receiving the starter diet. The lambs in the M + S group were separated from the ewe and received starter feed. All lambs were slaughtered at 56 d old. Eight rumen epithelia samples (4 per group) were collected to characterize their protein expression profiles using proteomic technology. Proteome analysis showed that 31 upregulated proteins and 40 downregulated proteins were identified in the rumen epithelium of lambs in response to starter diet supplementation. The results showed that starter feeding regulates a variety of biological processes in the epithelium, especially blood vessel development and extracellular matrix protein expression. Meanwhile, the expression of proteins associated with synthesis and degradation of ketone bodies, butanoate metabolism, and citrate cycle signaling transduction pathway were upregulated in the group with starter diet supplementation, including 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGCS2, fold change [FC] = 1.93), 3-hydroxybutyrate dehydrogenase 1 (BDH1, FC = 1.91), and isocitrate dehydrogenase 1 (IDH1, FC = 8.12). The metabolic processes associated with ammonia detoxification and antioxidant stress were also affected by starter diet supplementation, with proteins, microsomal glutathione S-transferase 3 (MGST3, FC = 2.37) and IDH1, linked to the biosynthesis of glutamate and glutathione metabolism pathway being upregulated in the group with starter diet supplementation. In addition, starter feeding decreased the expression of Ras-related protein rap-1A (RAP1A, FC = 0.48) enriched in Rap1 signaling pathway, Ras signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. In summary, starter feed supplementation changed the expression of proteins related to energy production, ammonia detoxification, antioxidant stress, and signaling pathways related to proliferation and apoptosis, which facilitates the rumen epithelia development in lambs. The results provide new insights into the molecular adaptation of rumen epithelia in response to starter diet supplementation at the protein level in lambs.

Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs.

Although soybean protein is the major component in livestock feeds, its effect on pigs' appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorectic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannulated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be attenuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study's result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/TRPM5 pathway.

Low crude protein diets supplemented with casein hydrolysate enhance the intestinal barrier function and decrease the pro-inflammatory cytokine expression in the small intestine of pigs.

To reduce nitrogen excretion and lower feeding costs, low crude protein (CP) diets are sometimes proposed, however, a great reduction of dietary CP concentration (>4% reduction vs. recommended concentration), even supplemented with essential and nonessential amino acids (AA) can detrimentally affect small intestinal barrier function and immunity, possibly due to the excessive lack of peptides. Here we hypothesize that with an extremely low CP concentration diet, protein-derived peptides, rather than AA supplementation, can improve intestinal barrier development and health. To test this hypothesis, 21 growing pigs (19.90 ± 1.00 kg body weight) were randomly assigned to 3 treatments with control diet (16% CP), or low CP diets (13% CP) supplemented with AA (LCPA) or casein hydrolysate (LCPC) for 28 days. In comparison with the control diet, the LCPA diet decreased the protein expression level of jejunal barrier factor zonula occludens-1 (ZO-1) and stem cell proliferation factor leucine-rich repeat-containing G-protein-coupled receptor-5, whereas the LCPC diet enhanced intestinal barrier function by increasing the protein expression level of jejunal occludin and ZO-1 and ileal mucin-2. The LCPA diet reduced Lactobacillus counts, whereas the LCPC diet increased Lactobacillus counts and reduced Escherichia coli counts in the ileum. The LCPA diet also increased protein expression levels of pro-inflammatory cytokine interleukin-6 (IL-6) and IL-22, whereas the LCPC diet decreased protein expression levels of pro-inflammatory IL-1ß, IL-17A and tumor necrosis factor-α in the ileum. Collectively, the casein hydrolysate supplementation of low CP diets showed beneficial effects on the small intestinal barrier, bacterial community, and immunity in pigs, pointing to the important role of protein-derived peptides in small intestinal health in cases of low crude protein diets.

Galacto-oligosaccharides improve barrier function and relieve colonic inflammation via modulating mucosa-associated microbiota composition in lipopolysaccharides-challenged piglets.

BACKGROUND: Galacto-oligosaccharides (GOS) have been shown to modulate the intestinal microbiota of suckling piglets to exert beneficial effects on intestinal function. However, the modulation of intestinal microbiota and intestinal function by GOS in intestinal inflammation injury models has rarely been reported. In this study, we investigated the effects of GOS on the colonic mucosal microbiota composition, barrier function and inflammatory response of lipopolysaccharides (LPS)-challenged suckling piglets. METHODS: A total of 18 newborn suckling piglets were divided into three groups, the CON group, the LPS-CON group and the LPS-GOS group. Piglets in the LPS-GOS group were orally fed with 1 g/kg body weight of GOS solution every day. On the d 14, piglets in the LPS-CON and LPS-GOS group were challenged intraperitoneally with LPS solution. All piglets were slaughtered 2 h after intraperitoneal injection and sampled. RESULTS: We found that the colonic mucosa of LPS-challenged piglets was significantly injured and shedding, while the colonic mucosa of the LPS-GOS group piglets maintained its structure. Moreover, GOS significantly reduced the concentration of malondialdehyde (MDA) and the activity of reactive oxygen species (ROS) in the LPS-challenged suckling piglets, and significantly increased the activity of total antioxidant capacity (T-AOC). GOS significantly increased the relative abundance of norank_f__Muribaculaceae and Romboutsia, and significantly decreased the relative abundance of Alloprevotella, Campylobacter and Helicobacter in the colonic mucosa of LPS-challenged suckling piglets. In addition, GOS increased the concentrations of acetate, butyrate and total short chain fatty acids (SCFAs) in the colonic digesta of LPS-challenged suckling piglets. GOS significantly reduced the concentrations of interleukin 1ß (IL-1ß), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and cluster of differentiation 14 (CD14), and the relative mRNA expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) in the LPS-challenged suckling piglets. In addition, GOS significantly reduced the relative mRNA expression of mucin2 (MUC2), and significantly increased the protein expression of Claudin-1 and zonula occluden-1 (ZO-1) in LPS-challenged suckling piglets. CONCLUSIONS: These results suggested that GOS can modulate the colonic mucosa-associated microbiota composition and improve the intestinal function of LPS-challenged suckling piglets.

Nano chitosan-zinc complex improves the growth performance and antioxidant capacity of the small intestine in weaned piglets.

The present study was conducted to test the hypothesis that dietary supplementation with a nano chitosan-zinc complex (CP-Zn, 100 mg/kg Zn) could alleviate weaning stress in piglets challenged with enterotoxigenic Escherichia coli K88 by improving growth performance and intestinal antioxidant capacity. The in vivo effects of CP-Zn on growth performance variables (including gastrointestinal digestion and absorption functions and the levels of key proteins related to muscle growth) and the antioxidant capacity of the small intestine (SI) were evaluated in seventy-two weaned piglets. The porcine jejunal epithelial cell line IPEC-J2 was used to further investigate the antioxidant mechanism of CP-Zn in vitro. The results showed that CP-Zn supplementation increased the jejunal villus height and decreased the diarrhoea rate in weaned piglets. CP-Zn supplementation also improved growth performance (average daily gain and average daily feed intake), increased the activity of carbohydrate digestion-related enzymes (amylase, maltase, sucrase and lactase) and the mRNA expression levels of nutrient transporters (Na+-dependent glucose transporter 1, glucose transporter type 2, peptide transporter 1 and excitatory amino acid carrier 1) in the jejunum and up-regulated the expression levels of mammalian target of rapamycin (mTOR) pathway-related proteins (insulin receptor substrate 1, phospho-mTOR and phospho-p70S6K) in muscle. In addition, CP-Zn supplementation increased glutathione content, enhanced total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-px) activity, and reduced malondialdehyde (MDA) content in the jejunum. Furthermore, CP-Zn decreased the content of MDA and reactive oxygen species, enhanced the activity of T-SOD and GSH-px and up-regulated the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway-related proteins (Nrf2, NAD(P)H:quinone oxidoreductase 1 and haeme oxygenase 1) in lipopolysaccharide-stimulated IPEC-J2 cells. Collectively, these findings indicate that CP-Zn supplementation can improve growth performance and the antioxidant capacity of the SI in piglets, thus alleviating weaning stress.

Swine gut microbiota and its interaction with host nutrient metabolism.

Gut microbiota is generally recognized to play a crucial role in maintaining host health and metabolism. The correlation among gut microbiota, glycolipid metabolism, and metabolic diseases has been well reviewed in humans. However, the interplay between gut microbiota and host metabolism in swine remains incompletely understood. Given the limitation in conducting human experiments and the high similarity between swine and humans in terms of anatomy, physiology, polyphagy, habits, and metabolism and in terms of the composition of gut microbiota, there is a pressing need to summarize the knowledge gained regarding swine gut microbiota, its interplay with host metabolism, and the underlying mechanisms. This review aimed to outline the bidirectional regulation between gut microbiota and nutrient metabolism in swine and to emphasize the action mechanisms underlying the complex microbiome-host crosstalk via the gut microbiota-gut-brain axis. Moreover, it highlights the new advances in knowledge of the diurnal rhythmicity of gut microbiota. A better understanding of these aspects can not only shed light on healthy and efficient pork production but also promote our knowledge on the associations between gut microbiota and the microbiome-host crosstalk mechanism. More importantly, knowledge on microbiota, host health and metabolism facilitates the development of a precise intervention therapy targeting the gut microbiota.

Stimulation of Gastric Transit Function Driven by Hydrolyzed Casein Increases Small Intestinal Carbohydrate Availability and Its Microbial Metabolism.

Gastrointestinal (GI) functions affect gut nutrient flow and microbial metabolism. Dietary peptides modulate GI functions and improve small intestinal health, but the mechanism remains elusive. This study aims to investigate whether dietary peptides affect small intestinal microbial metabolism, and the underlying mechanisms. An ileal-cannulated pig model is adopted to explore the relationship between gut nutrient flow and microbial metabolism after treatment with hydrolyzed casein (peptides) or intact casein (Control)-based diet. The results demonstrate that hydrolyzed casein enhances microbial carbohydrate metabolism with higher Streptococcus abundance and higher lactate level in the ileum. Meanwhile, hydrolyzed casein increases ileal flows of nutrients, especially carbohydrate, leading to a higher carbohydrate availability in ileal digesta. To unveil the mechanisms, it is found that the hydrolyzed casein enhances the ghrelin signal and improves development of interstitial cells of Cajal and muscular layer in gastric corpus, indicating the enhanced upper GI transit function. In addition, hydrolyzed casein improves small intestinal health, as indicated by higher villus heights and luminal lactate concentrations in the jejunum and ileum. In conclusion, hydrolyzed casein stimulates upper GI transit function, enhances gut nutrient flow, and increases small intestinal carbohydrate availability and its microbial metabolism, which favor the small intestinal health.

Early galactooligosaccharide intervention alters the metabolic profile, improves the antioxidant capacity of mitochondria and activates the AMPK/Nrf2 signaling pathway in suckling piglet liver.

The suckling stage is a critical window time for the development of human and animal neonates. Our previous study found that an early-life galactooligosaccharide (GOS) intervention could improve the growth status of suckling piglets; however, the difference in the metabolic status between different treatment groups still remains unclear. In the present study, sixty neonatal piglets were used to investigate the effects of early-life GOS intervention on the hepatic metabolic profiles of piglets. In the first week after birth, the piglets in the GOS and control (CON) groups were fed with GOS solution (10 mL for 1 g per kg body weight per day) and physiological saline, respectively. On days 8 and 21, six piglets from each group were euthanized for sampling. Compared with the CON piglets, the GOS piglets showed a lower total serum cholesterol concentration on day 8, but a higher concentration of serum HDL-C and albumin on day 21. Principal component analysis and partial least-squares discriminant analysis showed a dramatic difference in the composition of hepatic metabolic profiles between the two groups. The GOS intervention mainly affected the lipid and amino acid metabolism of the piglets. Meanwhile, the mRNA levels of CPT-1 and PCG1α increased in the GOS piglets' liver. Additionally, with the accumulation of antioxidant-related metabolites in the GOS piglet liver, the hepatic mitochondrial antioxidant capacity increased, and the phosphorylation of AMP-activated protein kinase and the protein expression levels of nuclear factor-erythroid-2-related factor 2 and heme oxygenase-1 were also upregulated in the liver. In conclusion, our findings provide new insights into the effects of galactooligosaccharides on the growth of suckling piglets.

Metagenomic analysis reveals significant differences in microbiome and metabolic profiles in the rumen of sheep fed low N diet with increased urea supplementation.

Urea is a cost-effective replacement for feed proteins in ruminant diets. However, its metabolism by the rumen microbiome is not fully understood. Here, rumen contents were collected from 18 male sheep fed one of the following three treatments: a low N basal diet with no urea (UC, 0 g/kg dry matter (DM)), low urea (LU, 10 g/kg DM) and high urea (HU, 30 g/kg DM). Principal coordinate analysis showed that the microbial composition and functional profiles of the LU treatment significantly differed from the UC and HU treatments. The genera Prevotella, Succinivibrio, Succinatimonas and Megasphaera were higher in the LU rumen, while the genera Clostridium, Ruminococcus and Butyrivibrio were enriched in the UC and HU rumen. The aspartate-glutamate and arginine-proline metabolic pathways and valine, leucine and isoleucine biosynthesis were higher in the LU rumen. The cysteine and methionine metabolism, lysine degradation and fructose and pentose phosphate metabolism pathways were higher in the UC and HU rumen. The protozoa population in the HU treatment was higher than in the UC and LU treatments. These findings suggest that the rumen microbiome of sheep fed low N diet with different urea supplementation are significantly different.

Co-cultured methanogen improved the metabolism in the hydrogenosome of anaerobic fungus as revealed by gas chromatography-mass spectrometry analysis.

OBJECTIVE: The purpose of this study was to reveal the metabolic shift in the fungus cocultured with the methanogen (Methanobrevibacter thaueri). METHODS: Gas chromatography-mass spectrometry was used to investigate the metabolites in anaerobic fungal (Pecoramyces sp. F1) cells and the supernatant. RESULTS: A total of 104 and 102 metabolites were detected in the fungal cells and the supernatant, respectively. The partial least squares-discriminant analysis showed that the metabolite profiles in both the fungal cell and the supernatant were distinctly shifted when co-cultured with methanogen. Statistically, 16 and 30 metabolites were significantly (p<0.05) affected in the fungal cell and the supernatant, respectively by the co-cultured methanogen. Metabolic pathway analysis showed that co-culturing with methanogen reduced the production of lactate from pyruvate in the cytosol and increased metabolism in the hydrogenosomes of the anaerobic fungus. Citrate was accumulated in the cytosol of the fungus co-cultured with the methanogen. CONCLUSION: The co-culture of the anaerobic fungus and the methanogen is a good model for studying the microbial interaction between H2-producing and H2-utilizing microorganisms. However, metabolism in hydrogenosome needs to be further studied to gain better insight in the hydrogen transfer among microorganisms.

Low-protein diets supplemented with casein hydrolysate favor the microbiota and enhance the mucosal humoral immunity in the colon of pigs.

BACKGROUND: High-protein diets can increase the colonic health risks. A moderate reduction of dietary crude-protein (CP) level can improve the colonic bacterial community and mucosal immunity of pigs. However, greatly reducing the dietary CP level, even supplemented with all amino acids (AAs), detrimentally affects the colonic health, which may be due to the lack of protein-derived peptides. Therefore, this study evaluated the effects of supplementation of casein hydrolysate (peptide source) in low-protein (LP) diets, in comparison with AAs supplementation, on the colonic microbiota, microbial metabolites and mucosal immunity in pigs, aiming to determine whether a supplementation of casein hydrolysate can improve colonic health under very LP level. Twenty-one pigs (initial BW 19.90 ± 1.00 kg, 63 ± 1 days of age) were assigned to three groups and fed with control diet (16% CP), LP diets (13% CP) supplemented with free AAs (LPA) or casein hydrolysate (LPC) for 4 weeks. RESULTS: Compared with control diet, LPA and LPC diet decreased the relative abundance of Streptococcus and Escherichia coli, and LPC diet further decreased the relative abundance of Proteobacteria. LPC diet also increased the relative abundance of Lactobacillus reuteri. Both LP diets decreased concentrations of ammonia and cadaverine, and LPC diet also reduced concentrations of putrescine, phenol and indole. Moreover, LPC diet increased total short-chain fatty acid concentration. In comparison with control diet, both LP diets decreased protein expressions of Toll-like receptor-4, nuclear factor-κB, interleukin-1ß and tumor necrosis factor-α, and LPC diet further decreased protein expressions of nucleotide-binding oligomerization domain protein-1 and interferon-γ. LPC diet also increased protein expressions of G-protein coupled receptor-43, interleukin-4, transforming growth factor-ß, immunoglobulin A and mucin-4, which are indicators for mucosal defense activity. CONCLUSIONS: The results showed that supplementing casein hydrolysate showed beneficial effects on the colonic microbiota and mucosal immunity and barrier function in comparison with supplementing free AAs in LP diets. These findings may provide new framework for future nutritional interventions for colon health in pigs.

Effects of early-life lactoferrin intervention on growth performance, small intestinal function and gut microbiota in suckling piglets.

The early postnatal stage is a critical period for suckling animals in developing intestinal function and stabilizing gut microbiota. Lactoferrin (LF) plays a critical role in promoting gut development and regulating gut microbiota. This study investigates the impact of early-life lactoferrin (LF) intervention on the growth performance, small intestinal function and gut microbiota in suckling piglets. Sixty suckling piglets (1.51 ± 0.05 kg) obtained from six sows (10 piglets per litter) were assigned to a control (CON) group and an LF group in each litter, which were sow-fed. Piglets in the LF group were orally administered 8-12 mL LF solution (0.5 g per kg body weight per day) for a week, and piglets in the CON group were orally administered the same dose of physiological saline. Six piglets (n = 6) from each group were euthanized on days 8 and 21. The early-life LF intervention increased growth performance, with higher villi height of the jejunum and greater disaccharidase activity of the jejunum and ileum (P < 0.05). Diarrhoea incidence decreased in the LF group from day 1 to day 7 (P < 0.05). Urinary lactulose-mannitol ratios decreased in the LF group, whereas the gene and protein expressions of jejunal occludin increased in the LF group on day 8 and day 21, and higher gene and protein levels of ileal occludin were observed on day 8 (P < 0.05). Additionally, the LF piglets had lower concentrations of IL-1ß and TNF-α, and higher concentration of IL-10 in the jejunum (P < 0.05). For the ileum, higher concentration of IL-10 and lower concentration of TNF-α were observed in the LF group (P < 0.05). LF piglets had a greater abundance of Lactobacillus and lower abundance of Veillonella and Escherichia-Shigella in the jejunum on day 8 (P < 0.05). In the ileum, the abundance of Actinobacillus was decreased in the LF piglets on day 8 and day 21 (P < 0.05). The early-life LF intervention enhanced the growth performance and decreased diarrhoea incidence in the suckling piglets by promoting the development of intestinal function and changing the microbiota in the small intestine.

The Effects of the Combination of Oral Lactoferrin and Iron Injection on Iron Homestasis, Antioxidative Abilities and Cytokines Activities of Suckling Piglets.

Iron deficiency is considered a common nutritional problem for suckling piglets. The aim of this study was to evaluate the effects of the combination of oral lactoferrin and iron injection on iron levels, antioxidant ability and cytokine activity in suckling piglets. A total of sixty suckling piglets taken from six sows (10 piglets per litter) with a similar parity were chosen. The lactoferrin (LF) group was orally administrated with lactoferrin solution (0.5 g/kg body weight per day) for a week, the CON group was orally administrated with the same dose of physiological saline. Each piglet (all groups) was given 100 mg of iron dextran (FeDex) by intramuscular injection at the third day of age. Six piglets (n = 6) from each group were euthanized on days 8 and 21. The oral lactoferrin improved the iron level of suckling piglets by increasing the concentrations of serum hemoglobin and hepatic iron on day 8. Gene expression of lactoferrin receptor (LFR) was significantly increased in the LF group piglets on day 8, while duodenal protein expression of the divalent metal transporter 1 (DMT1) was significantly reduced in the LF group on day 8. In addition, oral lactoferrin enhanced serum T-AOC activities and duodenal SOD activities on day 21. The LF piglets had a significantly increased serum concentration of IL-10 on day 8. These results indicated that a combination of oral lactoferrin and iron injection is a more effective method of improving the iron level by up-regulating the expression of the LFR gene, enhancing the antioxidant ability and modulating the cytokine activity in the suckling piglets.

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon-like peptide-2 in pre-weaned lambs.

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon-like peptide -2 (GLP-2) in pre-weaned lambs using animal and cell culture experiments. In vivo, twelve 14-day-old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP-2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin-dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin-like growth factor 1 (IGF-1) in the jejunum and ileum and mRNA expression of GLP-2 receptor (GLP-2R) in the jejunum. In vitro, when jejunal cells were treated with GLP-2 for 2 hr, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) OD, IGF-1 concentration, and the mRNA expression of IGF-1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF-1 receptor (IGF-1R) inhibitor decreased (p < .05) the mRNA expression of IGF-1, cyclin A2, cyclin D1 and CDK6 in GLP-2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP-2 in pre-weaning lambs. Furthermore, GLP-2 can indirectly promote the proliferation of jejunal cells mainly through the IGF-1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.