Variations in colostrum metabolite profiles in association with sow parity.

Information about the full spectrum of metabolites present in porcine colostrum and factors that influence metabolite abundances is still incomplete. Parity number appears to modulate the concentration of single metabolites in colostrum. This study aimed to 1) characterize the metabolome composition and 2) assess the effect of parity on metabolite profiles in porcine colostrum. Sows (n = 20) were divided into three parity groups: A) sows in parity 1 and 2 (n = 8), B) sows in parity 3 and 4 (n = 6), and C) sows in parity 5 and 6 (n = 6). Colostrum was collected within 12 h after parturition. A total of 125 metabolites were identified using targeted reversed-phase high-performance liquid chromatography-tandem mass spectrometry and anion-exchange chromatography-high resolution mass spectrometry. Gas chromatography additionally identified 19 fatty acids (FAs). Across parities, colostrum was rich in creatine and creatinine, 1,3-dioleyl-2-palmitatoylglycerol, 1,3-dipalmitoyl-2-oleoylglycerol, and sialyllactose. Alterations in colostrum concentrations were found for eight metabolites among parity groups (P < 0.05) but the effects were not linear. For instance, colostrum from parity group C comprised 75.4% more valine but 15.7%, 34.1%, and 47.9% less citric, pyruvic, and pyroglutamic acid, respectively, compared to group A (P < 0.05). By contrast, colostrum from parity group B contained 39.5% more spermidine than from group A (P < 0.05). Of the FAs, C18:1, C16:0, and C18:2 n6 were the main FAs across parities. Parity affected four FAs (C18:3n3, C14:1, C17:0ai, and C17:1), including 43.1% less α-linolenic acid (C18:3n3) in colostrum from parity group C compared to groups A and B (P < 0.05). Signature feature ranking identified 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine and the secondary bile acid hyodeoxycholic acid as the most discriminative metabolites, showing a higher variable importance in the projection score in colostrum from parity group A than from groups B and C. Overall, results provided a comprehensive overview about the metabolome composition of sow colostrum. The consequences of the changes in colostrum metabolites with increasing parity for the nutrient supply of the piglets should be investigated in the future. The knowledge gained in this study could be used to optimize feeding strategies for sows.

An insight into the temporal dynamics in the gut microbiome, metabolite signaling, immune response, and barrier function in suckling and weaned piglets under production conditions.

Little information is available on age- and creep-feeding-related microbial and immune development in neonatal piglets. Therefore, we explored age- and gut-site-specific alterations in the microbiome, metabolites, histo-morphology, and expression of genes for microbial signaling, as well as immune and barrier function in suckling and newly weaned piglets that were receiving sow milk only or were additionally offered creep feed from day of life (DoL) 10. The experiment was conducted in two replicate batches. Creep feed intake was estimated at the litter level. Piglets were weaned on day 28 of life. Gastric and cecal digesta and jejunal and cecal tissue were collected on DoL 7, 14, 21, 28, 31, and 35 for microbial and metabolite composition, histomorphology, and gene expression. In total, results for 10 piglets (n = 5/sex) per dietary group (sow milk only versus additional creep feed) were obtained for each DoL. The creep feed intake was low at the beginning and only increased in the fourth week of life. Piglets that were fed creep feed had less lactate and acetate in gastric digesta on DoL 28 compared to piglets fed sow milk only (p < 0.05). Age mainly influenced the gastric and cecal bacteriome and cecal mycobiome composition during the suckling phase, whereas the effect of creep feeding was small. Weaning largely altered the microbial communities. For instance, it reduced gastric Lactobacillaceae and cecal Bacteroidaceae abundances and lowered lactate and short-chain fatty acid concentrations on DoL 31 (p < 0.05). Jejunal and cecal expression of genes related to microbial and metabolite signaling, and innate immunity showed age-related patterns that were highest on DoL 7 and declined until DoL 35 (p < 0.05). Weaning impaired barrier function and enhanced antimicrobial secretion by lowering the expression of tight junction proteins and stimulating goblet cell recruitment in the jejunum and cecum (p < 0.05). Results indicated that age-dependent alterations, programmed genetically and by the continuously changing gut microbiome, had a strong impact on the expression of genes for gut barrier function, integrity, innate immunity, and SCFA signaling, whereas creep feeding had little influence on the microbial and host response dynamics at the investigated gut sites.

Plant-oriented microbiome inoculum modulates age-related maturation of gut-mucosal expression of innate immune and barrier function genes in suckling and weaned piglets.

In the immediate time after weaning, piglets often show symptoms of gut inflammation. The change to a plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite profile in digesta may be causative factors for the observed inflammation. We used the intestinal loop perfusion assay (ILPA) to investigate jejunal and colonic expression of genes for antimicrobial secretion, oxidative stress, barrier function, and inflammatory signaling in suckling and weaned piglets when exposed to "plant-oriented" microbiome (POM) representing postweaning digesta with gut-site specific microbial and metabolite composition. Two serial ILPA were performed in two replicate batches, with 16 piglets preweaning (days 24 to 27) and 16 piglets postweaning (days 38 to 41). Two jejunal and colonic loops were perfused with Krebs-Henseleit buffer (control) or with the respective POM for 2 h. Afterward, RNA was isolated from the loop tissue to determine the relative gene expression. Age-related effects in jejunum included higher expression of genes for antimicrobial secretions and barrier function as well as reduced expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Age-related effects in the colon comprised downregulation of the expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Likewise, age reduced the colonic expression of genes encoding for cytokines, antimicrobial secretions, antioxidant enzymes, and tight-junction proteins post- compared to preweaning. Effect of POM in the jejunum comprised an increased the expression of toll-like receptors compared to the control (P < 0.05), demonstrating a specific response to microbial antigens. Similarly, POM administration upregulated the jejunal expression of antioxidant enzymes (P < 0.05). The POM perfusion strongly upregulated the colonic expression of cytokines and altered the expression of barrier function genes, fatty acid receptors and transporters, and antimicrobial secretions (P < 0.05). In conclusion, results indicated that POM signaled via altering the expression of pattern-recognition receptors in the jejunum, which in turn activated the secretory defense and decreased mucosal permeability. In the colon, POM may have acted pro-inflammatory via upregulated cytokine expression. Results are valuable for the formulation of transition feeds for the immediate time after weaning to maintain mucosal immune tolerance towards the novel digesta composition.

After weaning, piglets often show symptoms of gut inflammation and reduced performance. The plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite composition in digesta may be causative. However, the acute response of the gut mucosa when exposed to the novel digesta composition has not been fully elucidated. Here, we used the intestinal loop perfusion assay to characterize the immediate effect of a plant-oriented microbiome inoculum (POM) representing postweaning digesta composition on gene expression related to innate immune pathways and barrier function at the jejunal and colonic mucosa in suckling and weaned piglets. Results showed that the recognition of microbial components and barrier function changed in the jejunal and colonic mucosa from pre- to postweaning, indicating age-related maturation and priming by digesta compounds prior to the intestinal loop perfusion assay. In the jejunum, exposure to POM increased expression of receptors recognizing microbial components. In the colon, POM exposure upregulated the expression of genes for pro-inflammatory cytokines and other components of the first line of defense. Results have implications for the formulation of transition feeds for the immediate time after weaning. Inclusion of bioactive porcine milk components may help maintain mucosal immune tolerance towards the novel digesta composition.

Moringa oleifera and Propolis in Cattle Nutrition: Characterization of Metabolic Activities in the Rumen In Vitro.

Moringa oleifera by-products such as seed cake and leaves are protein-rich ingredients, while raw propolis has the potential to influence ruminal protein metabolism. These substances are also known to be sources of functional compounds. With these properties, they could modulate ruminal fermentation activities. Using the rumen simulation technique, we investigated ruminal fermentation and the antioxidant properties of four dietary treatments. These included a control diet (CON) without supplementation; the CON diet top-dressed on a dry matter (DM) basis, either with moringa seed cake (MSC, containing 49% crude protein (CP)), moringa leaf powder (ML, containing 28% CP), or raw propolis (PRO, 3% CP). MSC, ML, and PRO accounted for 3.8, 7.4, and 0.1% of the total diet DM, respectively. Both ML and MSC resulted in 14 and 27% more ammonia concentration, respectively than CON and PRO (p < 0.05). MSC increased the propionate percentage at the expense of acetate (p < 0.05). Both ML and MSC decreased methane percentages by 7 and 10%, respectively, compared to CON (p < 0.05). The antioxidant capacity of the moringa seed cake, moringa leaf powder, and raw propolis were 1.14, 0.56, and 8.56 mg Trolox/g DM, respectively. However, such differences were not evident in the fermentation fluid. In conclusion, the supplementation of moringa seed cake desirably modulates rumen microbial activities related to protein and carbohydrate metabolism.

Exposure to plant-oriented microbiome altered jejunal and colonic innate immune response and barrier function more strongly in suckling than in weaned piglets.

Weaning often leaves the piglet vulnerable to gut dysfunction. Little is known about the acute response of a gut mucosa primed by a milk-oriented microbiome before weaning to a plant-oriented microbiome (POM) after weaning. We evaluated the epithelial structure, secretory response and permeability in the small and large intestines of piglets receiving a milk-based (i.e., preweaning) or plant-based diet (i.e., postweaning) to POM inocula using intestinal loop perfusion assays (ILPA). The POM were prepared from jejunal and colonic digesta of four 7 week-old weaned (day 28 of life) piglets, having gut-site specific microbial and metabolite composition. Two consecutive ILPA were performed in 16 piglets pre- (days 24 to 27) and 16 piglets postweaning (days 38 to 41) in two replicate batches. Two jejunal and colonic loops per piglet were perfused with Krebs-Henseleit buffer (control) or the respective POM. The outflow fluid was analyzed for antimicrobial secretions. Jejunal and colonic loop tissue were collected after each ILPA for histomorphology and electrophysiology using Ussing chambers. ANOVA was performed using the MIXED procedure in SAS. The POM stimulated the secretory response by increasing mucin in the jejunal and colonic outflow by 99.7% and 54.1%, respectively, and jejunal IgA by 19.2%, whereas colonic lysozyme decreased 25.6% compared to the control (P < 0.05). Fittingly, the POM raised the number of goblet cells by 96.7% in jejunal and 56.9% in colonic loops compared to control loops (P < 0.05). The POM further flattened jejunal villi by 18.3% and reduced crypt depth in jejunal and colonic loops by 53.8% and 9.0% compared to the control (P < 0.05); observations typically made postweaning and indicative for mucosal recognition of 'foreign' compounds. The POM altered the jejunal and colonic net ion flux as indicated by 22.7% and 59.2% greater short-circuit current compared to control loops, respectively; the effect being stronger postweaning (P < 0.05). Colonic barrier function improved with age (P < 0.05), whereas POM perfusion compromised the mucosal barrier as suggested by 17.7% and 54.1% greater GT and mucosal-to-serosal flux of fluorescein-isothiocyanate dextran, respectively, compared to the control (P < 0.05). In conclusion, results demonstrated that the preweaning gut epithelium acutely responds to novel compounds in postweaning digesta by upregulating the first line of defense (i.e., mucin and lysozyme secretion) and impairment of the structural integrity.

Creep feed is offered during the suckling period to prepare the piglet's gut for the dietary transition from a milk- to a plant-based diet at weaning. Nevertheless, the discontinuation of sow milk consumption after weaning can lead to disturbed interactions between the host mucosa and the gut microbiota. Little information is available on the immediate mucosal response towards the altered microbial and metabolite composition in digesta. Therefore, the main objective of this study was to evaluate the immediate effect of the exposure of the jejunal and colonic mucosa to a plant-oriented microbiome (POM), prepared from intestinal digesta of weaned pigs, on the mucosal structure, secretory response, and permeability in piglets before and after weaning using the intestinal loop perfusion assay. The perfusion with POM stimulated the host's secretory response, altered the gut structure and decreased the epithelial integrity before and after weaning. Effects were less strong postweaning, indicating that adaptation processes at the gut epithelium occurred from pre- to postweaning which increased the tolerance towards the POM inoculum.

Short-Chain Fatty Acids Modulate Permeability, Motility and Gene Expression in the Porcine Fetal Jejunum Ex Vivo.

Postnatally, short-chain fatty acids (SCFA) are important energetic and signaling agents, being involved in host nutrition, gut imprinting and immune and barrier function. Whether SCFA exert similar effects during the late fetal phase has been insufficiently elucidated. This study aimed to evaluate whether the fetal jejunum senses SCFA and whether SCFA modify the muscle tension and epithelial permeability and related signaling in jejunal tissue from the porcine fetus in late gestation. Exposure of fetal jejunal tissue to a mix of SCFA (70 µmol/mL) in an organ bath for 20 min lowered the muscle tension. Moreover, SCFA decreased the transepithelial conductance while increasing the short-circuit current in the Ussing chamber, indicating reduced permeability and increased SCFA absorption. Gene expression in the tissues harvested from the Ussing chamber after 30 min indicated downregulation of the expression of receptors (i.e., FFAR2 and TLR2), MCT1 and tight-junction and adherens proteins, which may be a negative feedback response to the applied high SCFA concentration compared with the micromolar concentration detected in fetal gastric fluid. Taken together, our data demonstrate that the fetal jejunum senses SCFA, which trigger electrophysiological, muscle contraction and related gene transcription responses. Hence, SCFA may play a role in prenatal gut nutrition and imprinting.

Supplementation With Phytogenic Compounds Modulates Salivation and Salivary Physico-Chemical Composition in Cattle Fed a High-Concentrate Diet.

Saliva facilitates feed ingestion, nutrient circulation, and represents an important pH buffer for ruminants, especially for cattle fed high-concentrate diets that promote rumen acidification. This experiment evaluated the short-term effects of nine phytogenic compounds on salivation, saliva physico-chemical composition as well as ingested feed boli characteristics in cattle. A total of nine ruminally cannulated Holstein cows were used. Each compound was tested in four of these cows as part of a high-concentrate meal (2.5 kg of total mixed ration in dry matter basis for 4 h) in low or high dose, and was compared to a control meal without compound. Saliva was sampled orally (unstimulated saliva) for physico-chemical composition analysis. Composition of the ingested saliva (stimulated saliva), salivation and feed boli characteristics were assessed from ingesta collected at the cardia during the first 30 min of the meal. Analysis of unstimulated saliva showed that supplementation with capsaicin and thyme oil increased buffer capacity, while supplementation with thymol, L-menthol and gentian root decreased saliva pH. In addition, supplementing angelica root decreased saliva osmolality. Regression analysis on unstimulated saliva showed negative associations between mucins and bicarbonate as well as with phosphate when garlic oil, thyme oil or angelica root was supplemented. Analysis of stimulated saliva demonstrated that supplementation with garlic oil increased phosphate concentration, thyme oil tended to increase osmolality, capsaicin and thymol increased buffer capacity, and ginger increased phosphate content. Furthermore, salivation rate increased with ginger and thymol, and tended to increase with garlic oil, capsaicin, L-menthol and mint oil. Feed ensalivation increased with capsaicin. A positive association was found between feed bolus size and salivation rate when any of the phytogenic compounds was supplemented. Overall, our results demonstrate positive short-term effects of several phytogenic compounds on unstimulated and stimulated saliva physico-chemical properties, salivation or feed boli characteristics. Thus, the phytogenic compounds enhancing salivary physico-chemical composition have the potential to contribute to maintain or improve ruminal health in cattle fed concentrate-rich rations.

Short-, medium-, and long-chain fatty acid profiles and signaling is responsive to dietary phytase and lactic acid treatment of cereals along the gastrointestinal tract of growing pigs.

Dietary and microbially derived fatty acids (FA) play important roles in gut mucosal inflammatory signaling, barrier function, and oxidative stress response. Nevertheless, little information is available about gastrointestinal FA profiles and receptor distribution in pigs, especially for long-chain FA (LCFA). Therefore, the present pilot study aimed to (1) investigate the gastrointestinal FA profiles; (2) link the luminal FA profiles to the mucosal expression of genes related to FA sensing and signaling; and (3) assess potential dietary effects on gut and systemic lipid metabolism in pigs. Gut, liver, and serum samples were obtained from barrows (13.1 ± 2.3 kg) fed diets containing either phytase (500 phytase units/kg diet) or cereals treated with 2.5% lactic acid (LA; n = 8/diet) for 18 d. Results showed gut regional and diet-related differences in luminal FA profiles and mucosal receptor expression, whereas diet little affected hepatic expression levels and serum lipids. Short-chain fatty acids (SCFA) increased from stomach, jejunum, and ileum to the cecum (P < 0.05), whereas LCFA were higher in stomach, cecum, and colon than in jejunum and ileum (P < 0.05). LA-treated cereals enhanced cecal acetate and butyrate, whereas phytase and LA treated cereals decreased the LCFA by 35.9% and 14.4%, respectively (P < 0.05). Gut regional differences suggested stronger signaling via FFAR1 expression in the ileum, and via FFAR2, FFAR4, and HCAR1 expression in cecum and colon (P < 0.05). Expression of AMPK, FASN, PPARG, SREBP1, and SREBP2 was higher in the cecum and colon compared with the small intestine (P < 0.05), with stronger sensing via FASN and SREBP2. Phytase decreased expression of FFAR2 and FFAR4, whereas it increased that of FFAR3 and MCT1 in the cecum (P < 0.05). LA-treated cereals raised cecal expression of FFAR3 and HCAR1 (P < 0.05). Pearson's correlations (|r| > 0.35; P < 0.05) supported that FA receptor- and nuclear transcription factor-dependent pathways were involved in the mucosal regulation of gut incretin expression but differed across gut regions. In conclusion, results support regional differences in SCFA, lactate and LCFA sensing and absorption capacities in the small and large intestines of pigs. Effects of phytase and the LA-treated cereals on intestinal FA levels and signaling can be explained by differences in nutrient flows (e.g., phosphorus and carbohydrate fractions). This overview provides a solid basis for future intestinal FA sensing in pigs.

Dynamic changes in salivation, salivary composition, and rumen fermentation associated with duration of high-grain feeding in cows.

Salivary secretions are essential for the regulation of digestive processes, as well as rumen and cow health. This research evaluated the effects of the duration of high-grain feeding, and of the time relative to a meal, on salivation, saliva properties, feed bolus characteristics, chewing activity, ruminal and reticular volatile fatty acids, as well as salivary and ruminal pH. Nine nonlactating cannulated Holstein cows were sampled at 1 and 23 d after transition to a 65% grain diet (short term and long term, respectively). Both before and after a controlled meal (2.5 kg of dry matter, offered over 4 h), unstimulated saliva was taken orally for composition analysis. Stimulated salivation and feed boli characteristics were evaluated by collection of ingesta from cardia during 30 min. Chewing and ruminal pH were measured during the controlled meal and for a total of 6 h thereafter. Results from unstimulated saliva showed no effect of the duration of high-grain feeding on bicarbonate, phosphate, total proteins, mucins, lysozyme, and buffer capacity, but increased osmolality at the long term. Lysozyme activity did not differ with high-grain feeding duration, but tended to be lower after the meal. In contrast to short-term-fed cows, the long-term-fed cows increased both meal consumption and feed bolus size, but decreased chewing and feed ensalivation (5.2 vs. 4.6 ± 0.50 g of saliva/g of dry matter), and had lower pH of the stimulated saliva (7.00 vs. 6.67 ± 0.076). These cows also had decreased chewing index (66.5 vs. 45.4 min/kg of neutral detergent fiber), and despite the increase in stimulated saliva buffer capacity (0.027 vs. 0.039 ± 0.006), mean ruminal pH decreased (6.31 vs. 6.11 ± 0.065) during ad libitum feeding. Both in the rumen and reticulum, the concentration of total volatile fatty acids was lower and propionate proportion was higher at the long term. Linear regression analyses revealed a positive influence of the flow rates of salivary bicarbonate and phosphate on ruminal pH during the short term. For every 1-mol increment in the flow of bicarbonate or phosphate, ruminal pH increased by 0.062 or 0.439 units, respectively. Overall, salivary buffers are key determinants of ruminal pH regulation, especially during short-term grain feeding. However, in the long term, ruminal pH drop during ad libitum feeding was stronger, and this effect seems to be exacerbated by increased feed bolus size, accompanied by reductions in feed ensalivation, stimulated saliva pH, and chewing index.

Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?

AIM: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study. MATERIALS AND METHODS: The effects of extract on glucose uptake, insulin receptor-ß (IR-ß), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days. RESULTS: The effect of extract promoted glucose uptake, IR-ß and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC(50) 17.4 µg/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment). CONCLUSION: The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B.

Cinnamic acid, from the bark of Cinnamomum cassia, regulates glucose transport via activation of GLUT4 on L6 myotubes in a phosphatidylinositol 3-kinase-independent manner.

BACKGROUND: Cinnamomum cassia (Family: Lauraceae) is an Ayurvedic medicinal plant used traditionally for the treatment of a number of diseases, including diabetes. The hypoglycemic effect of this plant has been established in vivo. However, the effects of cinnamic acid, isolated from C. cassia, on the insulin signaling cascade in an in vitro model have not been elucidated. Hence, the aim of the present study was to evaluate the anti-diabetic effect of cinnamic acid on glucose transport by L6 myotubes. METHODS: The mechanism of action of cinnamic acid was determined using specific targets in the insulin signaling pathway, including protein tyrosine phosphatase (PTP) 1B, phosphatidylinositol 3-kinase (PI3-K) and the glucose transporter GLUT4. After differentiation of myoblast to myotubes, the cells were serum deprived for 5 h and then treated with 1 ng/mL cinnamic acid and 50 µmol/L rosiglitazone for 18 h and 100 nmol/L insulin for 20 min for gene expression studies. RESULTS: Expression of GLUT4 mRNA was increased following treatment of L6 myotubes with 1 ng/mL cinnamic acid. Furthermore, cinnamic acid inhibited PTP1B activity (by 96.5%), but had no significant effect on PI3-K activity. CONCLUSION: On the basis of the results of the present study, we postulate that cinnamic acid isolated from the hydro-alcoholic extract of Cinnamomum cassia activates glucose transport by a PI3-K-independent pathway. However, the detailed mechanism of action requires further analysis.

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors.

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P(2) region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure-activity relationship, and selectivity against related proteases are delineated.