Metagenomic Insight: Dietary Thiamine Supplementation Promoted the Growth of Carbohydrate-Associated Microorganisms and Enzymes in the Rumen of Saanen Goats Fed High-Concentrate Diets.

Subacute ruminal acidosis (SARA) is often caused by feeding a high-concentrate diet in intensive ruminant production. Although previous studies have shown that dietary thiamine supplementation can effectively increase rumen pH and modify rumen fermentation, the effect of thiamine supplementation on rumen carbohydrate-related microorganisms and enzymes in goats under SARA conditions remain unclear. Therefore, the objective of the present study was to investigate the effects of dietary thiamine supplementation on carbohydrate-associated microorganisms and enzymes in the rumen of Saanen goats fed high-concentrate diets. Nine healthy mid-lactating Saanen goats in parity 1 or 2 were randomly assigned into three treatments: A control diet (CON; concentrate:forage (30:70)), a high-concentrate diet (HC; concentrate:forage (70:30)), and a high-concentrate diet with 200 mg of thiamine/kg of DMI (HCT; concentrate:forage (70:30)). Compared with the HC group, dietary thiamine supplementation improved ruminal microbes associated with fiber, including Prevotella, Fibrobacter, Neocallimastix, and Piromyces (p < 0.05). In addition, an increase in the relative abundance of enzymes involved in both fiber degradation and starch degradation, such as CBM16, GH3, and GH97, was observed in the HCT treatment. (p < 0.05). Thus, thiamine supplementation can improve carbohydrate metabolism by increasing the abundance of the microorganisms and enzymes involved in carbohydrate degradation. In conclusion, this study revealed the relationship between ruminal microbiota and enzymes, and these findings contributed to solving the problems arising from the high-concentrate feeding in ruminant production and to providing a new perspective on ruminant health.

l-Arginine Alleviates Hydrogen Peroxide-Induced Oxidative Damage in Ovine Intestinal Epithelial Cells by Regulating Apoptosis, Mitochondrial Function, and Autophagy.

BACKGROUND: Previous studies demonstrated that dietary l-arginine (Arg) alters the equilibrium between reactive oxygen species (ROS) generation and biological defenses to resist oxidant-induced toxicity. Whether supplying Arg can protect ovine intestinal epithelial cells (OIECs) from hydrogen peroxide (H2O2)-induced oxidative damage is unclear. OBJECTIVES: The current study aimed to examine the effect of Arg on mitophagy, mitochondrial dysfunction, and apoptosis induced by H2O2 in OIECs. METHODS: The OIECs were incubated in Arg-free DMEM supplemented with 100 µM Arg (CON) or 350 µM Arg (ARG) alone or with 150 µM H2O2 (CON + H2O2, ARG + H2O2) for 24 h. Cellular apoptosis, mitochondrial function, autophagy, and the related categories of genes and proteins were determined. All data were analyzed by ANOVA using the general linear model procedures of SAS (SAS Institute) for a 2 × 2 factorial design. RESULTS: Relative to the CON and ARG groups, H2O2 administration resulted in 44.9% and 26.5% lower (P < 0.05) cell viability but 34.7% and 61.8% greater (P < 0.05) ROS concentration in OIECs, respectively. Compared with the CON and CON + H2O2 groups, Arg supplementation led to 40.7% and 28.8% lower (P < 0.05) ROS concentration but 14.9%-49.0% and 29.3%-64.1% greater (P < 0.05) mitochondrial membrane potential, relative mitochondrial DNA content, and complex (I-IV) activity in OIECs, respectively. Compared with the CON and CON + H2O2 groups, Arg supplementation led to 33.9%-53.1% and 22.4%-49.1% lower (P < 0.05) mRNA abundance of proapoptotic genes, respectively. Relative to the CON and CON + H2O2 groups, Arg supplementation resulted in 33.0%-59.2% and 14.6%-37.7% lower (P < 0.05) abundance of proapoptotic, mitophagy, and cytoplasmic cytochrome c protein, respectively. CONCLUSIONS: Supply of Arg protects OIECs against H2O2-induced damage partly by improving mitochondrial function and alleviating cellular apoptosis and autophagy.

l-Arginine Inhibits Apoptosis of Ovine Intestinal Epithelial Cells through the l-Arginine-Nitric Oxide Pathway.

BACKGROUND: In nonruminants, many of the biological roles of l-arginine (Arg) at the intestinal level are mediated through the Arg-nitric oxide (Arg-NO) pathway. Whether the Arg-NO pathway is involved in controlling the immune response and viability in ovine intestinal epithelial cells (IOECs) is unclear. OBJECTIVES: The current study aimed to examine the role of the Arg-NO pathway in apoptosis, antioxidant capacity, and mitochondrial function of IOECs. METHODS: The IOECs were incubated in Arg-free DMEM supplemented with 150 µM Arg (CON) or 300 µM Arg (ARG) alone or with 350 µM Nw-nitro-l-arginine methyl ester hydrochloride (l-NAME) (CON + NAME, ARG + NAME) for 24 h. The reactive oxygen species (ROS) concentration, antioxidant capacity, and cell apoptotic percentage were determined. RESULTS: Arg supplementation decreased (P < 0.05) the ROS concentration (38.9% and 22.7%) and apoptotic cell percentage (57.2% and 54.8%) relative to the CON and CON + NAME groups, respectively. Relative to the CON and ARG treatments, the l-NAME administration decreased (P < 0.05) the mRNA abundance of superoxide dismutase 2 (32% and 21.3%, respectively) and epithelial NO synthase (36% and 29.1%, respectively). Arg supplementation decreased (P < 0.05) the protein abundance of apoptosis antigen 1 (FAS) (52.0% and 43.9%) but increased (P < 0.05) those of nuclear respiratory factor 1 (31.3% and 22.9%) and inducible NO synthase (35.2% and 41.8%) relative to the CON and CON + NAME groups, respectively. CONCLUSIONS: The inhibition of apoptosis in IOECs due to the increased supply of Arg is associated with the mitochondria- and FAS-dependent pathways through the activity of the Arg-NO pathway. The findings help elucidate the role of the Arg-NO pathway in IOEC growth and apoptosis.

N-carbamylglutamate and l-arginine promote intestinal function in suckling lambs with intrauterine growth restriction by regulating antioxidant capacity via a nitric oxide-dependent pathway.

Data indicate that intrauterine growth restriction (IUGR) in newborns can be partly alleviated through the supply of l-arginine (Arg) and N-carbamylglutamate (NCG). The current work aimed to explore whether Arg and NCG promote intestinal function by regulating antioxidant capacity in suckling lambs with IUGR via a nitric oxide (NO)-dependent pathway. Forty eight newly born Hu lambs with normal weights at birth (CON) or suffering from IUGR were randomly divided into 4 groups (n = 12 per group), namely, the CON, IUGR, IUGR + 1% Arg, and IUGR + 0.1% NCG groups. The animals were used for experiments from the age of day 7 to 28. Compared with the lambs in the IUGR group, the lambs in the Arg or NCG group had higher (P < 0.05) final body weights. The plasma insulin, NO, and NO synthase (NOS) concentrations in the IUGR group were higher (P < 0.05) compared with those in IUGR + 1% Arg or IUGR + 0.1% NCG. The jejunal level of the tumor necrosis factor α (TNF-α) in the IUGR lambs was greater (P < 0.05) compared with that in IUGR + 1% Arg or IUGR + 0.1% NCG. The plasma and jejunal total antioxidant capacity (T-AOC) values for the IUGR + 1% Arg or IUGR + 0.1% NCG group were greater (P < 0.05) compared with those for the IUGR group. Compared with the IUGR + 1% Arg or IUGR + 0.1% NCG lambs, the IUGR lambs had lower (P < 0.05) abundance of mRNA and protein abundance of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), zonula occludens-1 (ZO-1), occludin, inducible NOS (iNOS), and epithelial NOS (eNOS). Overall, the data suggest that the Arg or NCG supplementation to suckling lambs with IUGR enhances the intestinal function by regulating the oxidant status via the NO-dependent pathway.

N-Carbamylglutamate and l-Arginine Promote Intestinal Absorption of Amino Acids by Regulating the mTOR Signaling Pathway and Amino Acid and Peptide Transporters in Suckling Lambs with Intrauterine Growth Restriction.

BACKGROUND: Previous studies have revealed that dietary N-carbamylglutamate (NCG) and l-arginine (Arg) improve intestinal integrity, oxidative state, and immune function in Hu suckling lambs with intrauterine growth restriction (IUGR). Whether these treatments alter intestinal nutrient absorption is unknown. OBJECTIVE: The aim of this study was to determine the influence of dietary NCG and Arg treatment during the suckling period on intestinal amino acid (AA) absorption, alterations in the mechanistic target of rapamycin (mTOR) signaling pathway, and the abundance of AA and peptide transporters in IUGR lambs. METHODS: On day 7 after birth, 48 newborn Hu lambs were selected from a cohort of 424 twin lambs. Normal-birth-weight and IUGR Hu lambs were allocated randomly (n = 12/group) to a control (4.09 ± 0.12 kg), IUGR (3.52 ± 0.09 kg), IUGR + 0.1% NCG (3.49 ± 0.11 kg), or IUGR + 1% Arg (3.53 ± 0.10 kg). RESULTS: At day 28, compared with the IUGR group, the IUGR groups receiving NCG and Arg had 7.4% and 7.2% greater (P < 0.05) body weight, respectively. Compared with the IUGR group, the serum concentration of insulin was greater (P < 0.05) and the cortisol was lower (P < 0.05) in the IUGR groups receiving NCG and Arg. Compared with the IUGR group, the IUGR groups receiving NCG and Arg had 13.2%-62.6% greater (P < 0.05) serum concentrations of arginine, cysteine, isoleucine, and proline. Dietary NCG or Arg to IUGR lambs resulted in greater protein abundance (P < 0.05) of peptide transporter 1 (41.9% or 38.2%) in the ileum compared with the unsupplemented IUGR lambs, respectively. Furthermore, dietary NCG or Arg treatment normalized the IUGR-induced variation (P < 0.05) in the ileal ratio of phosphorylated mTOR to total mTOR protein. CONCLUSION: Both NCG and Arg can help mitigate the negative effect of IUGR on nutrient absorption in neonatal lambs.

Dietary N-carbamylglutamate and l-arginine supplementation improves intestinal energy status in intrauterine-growth-retarded suckling lambs.

This study explores the roles of l-arginine (Arg) and N-carbamylglutamate (NCG) supplementation in the diet in intestine damage, energy state, as well as the associated protein kinase signaling pathways activated by AMP in intrauterine growth retarded (IUGR) suckling lambs. A total of 48 newborn Hu lambs with a normal birth weight (CON) and those with IUGR were randomly divided into four groups, CON, IUGR, IUGR + 1% Arg, and IUGR + 0.1% NCG, with 12 animals in each group. All animals were fed for 21 days, from day 7-28, following birth. Our results indicated that the IUGR suckling Hu lambs in the Arg or NCG groups were associated with reduced (P < 0.05) plasma diamine oxidase (DAO) and d-lactic acid levels compared with IUGR suckling lambs. In addition, IUGR suckling Hu lambs in the Arg or NCG group were also linked with a higher (P < 0.05) villous height : crypt depth ratio (VCR), as well as villous height in the duodenum relative to those obtained for IUGR suckling Hu lambs. Relative to IUGR suckling Hu lambs, IUGR suckling Hu lambs in the Arg or NCG groups were found to have higher (P < 0.05) ATP, ADP and TAN contents, and AEC levels, and smaller (P < 0.05) AMP : ATP ratios in the duodenum, jejunum and ileum. Moreover, IUGR suckling Hu lambs in the Arg or NCG group were also linked with higher citrate synthase, isocitrate dehydrogenase and alpha-oxoglutarate dehydrogenase complex activities in the duodenum, jejunum and ileum compared with those found for IUGR suckling Hu lambs (P < 0.05), except for the activity of isocitrate dehydrogenase in the ileum. IUGR suckling Hu lambs in the Arg or NCG group were linked with a lower ratio of pAMPKα/tAMPKα and protein expression of Sirt1 and PGC1α in the ileum relative to those of the IUGR suckling Hu lambs (P < 0.05). Taken together, these findings show that supplementation of NCG and Arg in the diet can ameliorate intestinal injury, improve energy status, motivate key enzyme activities in the tricarboxylic acid (TCA) cycle, and also inhibit the AMP-activated protein kinase signaling pathways in IUGR suckling Hu lambs.

l-Arginine Protects Ovine Intestinal Epithelial Cells from Lipopolysaccharide-Induced Apoptosis through Alleviating Oxidative Stress.

This research aims to explore the effect of l-arginine (Arg) upon lipopolysaccharide (LPS)-induced induction of the oxidative stress as well as subsequent apoptosis within ovine intestinal epithelial cells (IOECs). Through a 16 h incubation, cells were divided into four groups and the medium was replaced with different medium as follows: (1) control (Con), Arg-free Dulbecco's modified Eagle's F12 Ham medium (DMEM); (2) Arg treatment, Arg-free DMEM supplemented with 100 µM Arg; (3) LPS treatment, Arg-free DMEM supplemented with 10 µg/mL LPS; (4) LPS with Arg treatment, Arg-free DMEM supplemented with both 10 µg/mL LPS and 100 µM Arg. After culturing for 24 h in different mediums, some characteristics of cells in the four groups were measured. Addition of Arg increased cell viability induced with LPS compared with the LPS group ( p < 0.05). Arg significantly decreased the release of dehydrogenase (LDH) and the production of malonaldehyde (MDA) ( p < 0.05) within IOECs challenged by the LPS. Compared with the LPS group, cells treated with Arg and Arg + LPS increased ( p < 0.05) mRNA as well as protein expression of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase 2 (SOD2), B-cell lymphoma 2 (Bcl2), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). IOEC treatment with Arg reduced significantly ( p < 0.05) apoptosis induced by the LPS (12.58 ± 0.79%). The results showed that Arg promoted the protein expression of Nrf2, up-regulated expression of the phase II metabolizing enzymes (NQO1 and HO-1), as well as antioxidative enzymes (GPx1, CAT, and SOD2) for alleviating oxidative injury and protected IOECs from LPS-induced apoptosis.

Jugular infusion of arginine has a positive effect on antioxidant mechanisms in lactating dairy cows challenged intravenously with lipopolysaccharide1.

The main purpose of this work was to evaluate the effects of jugular l-arginine infusion on antioxidant mechanisms in lactating dairy cows challenged intravenously with lipopolysaccharide (LPS). Eight multiparous Holstein cows (609 ± 32 kg) at midlactation were randomly assigned to 5-d jugular infusions of Control (saline), Arginine (Arg, 18 g/d), LPS (0.2 µg/kg BW per day), and LPS + Arginine (0.2 µg/kg BW per day of LPS and 18 g/d of Arg) in a replicated 4 × 4 Latin square design with 4 infusion periods separated by 10-d. Jugular solutions of saline, Arg, LPS, and LPS + Arg were continuously infused using peristaltic pumps for approximately 6 h/d. Jugular vein serum samples were obtained on the last day of each infusion period before infusion (0 h) and at 3- and 6-h postinfusion. Compared with LPS treatment, Arg infusion increased the total antioxidant capacity and activity of glutathione peroxidase, but decreased malondialdehyde concentration (P < 0.05). The concentration of nitric oxide in serum and the activity of nitric oxide synthase were greater in LPS treatment compared with saline and Arg (P < 0.05). The Arg treatment significantly increased the serum insulin concentration at 3-h postinfusion compared with the saline treatment (P < 0.05), and that of LPS and LPS + Arg treatments were in between Arg and LPS treatments. No treatment effect was observed on the activities of superoxide dismutase and catalase (P > 0.05). In conclusion, enhancing the supply of Arg during an inflammatory challenge enhances antioxidant mechanisms in lactating dairy cows.

Effects of Dietary l-Arginine and N-Carbamylglutamate Supplementation on Intestinal Integrity, Immune Function, and Oxidative Status in Intrauterine-Growth-Retarded Suckling Lambs.

This study investigated the effects of dietary l-arginine (Arg) and N-carbamylglutamate (NCG) supplementation on intestinal integrity, immune function, and oxidative status in intrauterine-growth-retarded (IUGR) suckling lambs. A total of 48 newborn Hu lambs of normal birth weight (CON) and IUGR were allocated randomly into four groups of 12 animals each: CON, IUGR, IUGR + 1% Arg, or IUGR + 0.1% NCG. All lambs were raised for a period of 21 days from 7 to 28 days after birth. The Arg or NCG group exhibited improved ( p < 0.05) final body weights compared to that of the IUGR group. In comparison to the IUGR lambs, the apoptotic percentage was lower ( p < 0.05) in the ileum of IUGR lambs supplemented with Arg and NCG. In addition, in comparison to IUGR, the concentrations of protein carbonyl and malondialdehyde were lower ( p < 0.05) and the reduced glutathione (GSH) concentration and ratio of GSH/oxidized glutathione were greater ( p < 0.05) in the jejunum, duodenum, and ileum of IUGR + 1% Arg or 0.1% NCG lambs. In comparison to the IUGR group, the mRNA abundance of myeloid differentiation factor 88, toll-like receptor 9, toll-like receptor 4, interleukin 6, and fuclear factor-κB was lower ( p < 0.05) and the mRNA abundance of superoxide dismutase 1, B-cell lymphoma/leukaemia 2, zonula occludens-1 (ZO-1), and occludin was greater in the ileum of the IUGR lambs supplemented with Arg or NCG. Furthermore, the protein abundance of ZO-1 and claudin-1 in the ileum was greater ( p < 0.05) in the IUGR + 1% Arg or 0.1% NCG lambs. The results show that Arg or NCG supplementation improves the growth, intestinal integrity, immune function, and oxidative status in IUGR Hu suckling lambs.