Increase in matrix metalloproteinase-9 (MMP-9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP-9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP-1). The binding of AP-1, a heterodimer of c-Jun and c-Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim, is to investigate the molecular mechanism of MMP-9 transcriptional regulation in diabetes. Binding of AP-1 (c-Jun, c-Fos) at the MMP-9 promoter, and AP-1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin-immunoprecipitation and co-immunoprecipitation respectively. Role of AP-1 in MMP-9 regulation was confirmed by c-Jun or c-Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP-1 binding was increased at the proximal and distal sites of the MMP-9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP-1, or overexpression of Sirt1 ameliorated glucose-induced increase in MMP-9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP-1 is hyperacetylated, which increases its binding at MMP-9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP-9-mediated mitochondrial damage. J. Cell. Physiol. 231: 1709-1718, 2016. © 2015 Wiley Periodicals, Inc.
Two trials were conducted to determine the effect of sodium butyrate microencapsulated within triglyceride matrix (Na-butyrate) in the close-up period on performance of dairy cows and rumen papillae development. In trial 1, 26 Holstein-Friesian cows were randomly allocated to 2 groups (13 cows/group) and fed prepartum a total mixed ration (TMR) without or with 300g of Na-butyrate/d from 30 d before expecting calving to parturition. After calving, the same lactational TMR without Na-butyrate was offered to both treatments. Dry matter intake and milk yield were monitored daily to 60 d in milk, and body condition of cows was scored on d 30, 21, and 4 before parturition and d 14, 31, and 60 after parturition. On d 15, 10, and 5 before parturition blood samples were collected from 6 cows randomly chosen from each group and analyzed for plasma β-hydroxybutyrate and nonesterified fatty acids concentrations. No differences in dry matter (DM) intake, milk yield, body condition score, or plasma β-hydroxybutyrate and nonesterified fatty acids concentrations was observed between treatments; however, in the last 5 d before parturition the cows receiving Na-butyrate ate 1.7kg of DM/d more, on average, as compared with control cows. In trial 2, 12 Holstein-Friesian growing bulls (404±48; body weight ± SD) were used to determine the effect of Na-butyrate inclusion in the diet on rumen papillae development. Bulls were randomly allocated to 2 groups (6 bulls/group) and fed TMR without or with 2% (on a dry matter basis) of Na-butyrate for 21 d. At the end of the study, bulls were killed and rumen fluid and rumen tissue samples from dorsal and ventral sac of the rumen were collected. No effect of Na-butyrate supplementation on BW of bulls and DMI during the trial period was observed. Sodium butyrate supplementation increased total short-chain fatty acid concentration in the rumen but had no effect on rumen pH, molar proportions of short-chain fatty acids, and NH3-N concentration. In dorsal sac of the rumen, papillae length and papillae cross-section surface area were increased as a result of Na-butyrate supplementation, whereas in the ventral sac a reverse effect was observed (significant treatment × location in the rumen interaction). Both in the dorsal and ventral sac of the rumen, dietary Na-butyrate increased rumen muscle layer thickness. Altogether, results of this study suggest that Na-butyrate supplementation in the close-up diet may have a potential to enhance rumen papillae growth and rumen adaptation to postpartum diet but lactation performance was not affected under conditions of the current study.
The aim of the study was to determine the effect of butyrate infusion into the rumen on butyrate flow to the duodenum, expression of short-chain fatty acid (SCFA) transporters (monocarboxylate transporter-1, -2, and -4) and receptors (G protein coupled receptor-41 and -43) in the duodenal epithelium and nutrient digestion in sheep. Eight wethers (39.0 ± 3.00 kg; mean ± SD) with ruminal and T-shape duodenal cannulas were allocated to 4 × 4 replicated Latin square design with each experimental period lasting for 21 d (12 d of adaptation and 9 d for data and sample collection). Experimental treatments were: 1) distilled water infusion into the rumen (CONT); 2) 15 g/d of butyric acid infusion into the rumen (BUT15); 3) 30 g/d of butyric acid infusion into the rumen (BUT30); and 4) 45 g/d of butyric acid infusion into the rumen (BUT45). The daily dose of butyrate was infused into the rumen via the rumen cannula, with 200 mL of solution of butyric acid and distilled water, at a constant rate (0.1389 mL/min) throughout the day using a peristaltic pump. Correspondingly, 200 mL/d of distilled water was infused into the rumen of CONT. The wethers were fed daily 900 g of chopped meadow hay and 200 g of concentrate in two equal meals at 0600 and 1800 h. Butyrate infusion into the rumen did not affect total SCFA concentration in the rumen fluid ( > 0.11). Molar proportion of butyrate in total SCFA linearly increased, and molar proportion of acetate and isovalerate linearly decreased ( ≤ 0.02) with an increasing amount of butyrate infused into the rumen. The molar proportion of butyrate in total SCFA in the duodenal digesta linearly increased ( < 0.01), and butyrate flow to duodenum tended to linearly increase ( = 0.06) with an increasing dose of exogenous butyrate delivered to the rumen. Butyrate infusion into the rumen did not affect ( ≥ 0.14) the mRNA expression of monocarboxylate transporter-2 and -4 and G protein coupled receptor-43 in the duodenal epithelium. The G protein coupled receptor-41 and monocarboxylate transporter-1 mRNA expression in the duodenal epithelium was very low in many of the analyzed samples. Digestibility of organic matter, neutral detergent fiber, and acid detergent fiber in the stomach (forestomach and abomasum) decreased for BUT15 and BUT30 and then increased for BUT45 (quadratic, ≤ 0.04); however, neither digestibility in the intestine nor total tract digestibility differed between treatments ( ≥ 0.10).
The aim of this study was to determine the effect of docosahexaenoic acid-rich algae (DHA-RA) supplementation in milk replacer (MR) on performance, selected cytokine expression in lymphocytes, and blood immunoglobulin concentration in newborn dairy calves. Forty female Holstein-Friesian calves (8.6 ± 0.8 d old and 41.1 ± 4.3 kg; mean ± standard deviation) were blocked by date of birth and allocated into 4 experimental groups (10 animals/group): ( 1) not supplemented with DHA-RA, (2) supplemented with 9 g of DHA-RA/d in MR, (3) supplemented with 18 g of DHA-RA/d in MR, and (4) supplemented with 27 g of DHA-RA/d in MR. Milk replacer was fed in an amount equal to 900 g of MR powder/d (as fed), 2 times a d, for 49 d. Starter mixture (SM) was fed ad libitum beginning on d 15 of the study.Each calf was in the study over a period of 49 d. The MR and SM intake and fecal score were recorded daily and body weight was recorded weekly. Blood samples were collected before the morning feeding, at the beginning of the study, every consecutive week, and at the end of the study for morphology and smear analysis, serum immunoglobulin level (IgG, IgA, and IgM), and lymphocyte isolation. The mRNA isolated from lymphocytes was checked for TNFα, IL-1β, IL-6, and p65 expression. Average daily gain between d 1 to 14 of the study increased quadratically with increasing dose of DHA-RA. However, average daily gain between d 15 to 49 of the study tended to linearly decrease and over the whole study linearly decreased with increasing dose of DHA-RA. The MR intake decreased linearly between d 1 to 14 of the study and over the whole study, and mean SM intake decreased quadratically with increasing dose of DHA-RA. Feed efficiency increased quadratically and fecal score decreased quadratically during the first 14 d of the study. Increasing dose of DHA-RA led to cubic changes in feed efficiency and fecal score between d 15 and 49 of the study. Overall, over the whole study period a tendency was observed for lower fecal score for the DHA-RA supplemented groups. Interleukin-1β mRNA expression decreased linearly, whereas the mRNA expression of p65 and TNFα as well as serum IgG concentration tended to decrease linearly with increasing dose of supplemental DHA-RA. No effect of group was found on IgA and IgM serum level and the majority of blood parameters. Altogether, treatment worsened production variables but seemed to have a beneficial effect on the immune system of calves.
The aim of this study was to determine the effect of exogenous butyrate on the structure and selected functions of the stomach in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were allocated to the study and fed a diet for 14 d without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Neither DMI nor initial BW differed between treatments (P ≥ 0.61), but final BW was greater for BUT compared with CTRL (P = 0.03). Butyrate concentration in the reticuloruminal fluid and abomasal digesta was greater for BUT compared with CTRL (P ≤ 0.01), but total short-chain fatty acids (SCFA) concentration, as well as concentration of other SCFA, did not differ between treatments (P ≥ 0.07). Relative to BW, reticuloruminal tissue mass tended (P = 0.09) to be greater and omasal digesta was less (P = 0.02) for BUT compared with CTRL. Dietary butyrate did not affect ruminal papillae length, width, and density nor did it affect ruminal epithelium thickness (P ≥ 0.12) in the ventral sac of the rumen. However, the DM of ruminal epithelium (mg/cm 2) tended (P = 0.06) to be greater for BUT compared with CTRL. Omasal and abomasal epithelium thicknesses were greater (P ≤ 0.05) for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the abomasal epithelium was less for BUT compared with CTRL (P = 0.04). Finally, the mRNA expression of peptide transporter 1 in the omasal epithelium was less (P = 0.02) and mRNA expression of monocarboxylate transporter 1 in the abomasal epithelium tended (P = 0.07) to be greater for BUT compared with CTRL. It can be concluded that exogenous butyrate supplementation affected not only the rumen but also omasum and abomasum in sheep.
In this study, we aimed to investigate the effect of xylanase (XYL), emulsifier (EMU), and a combination of both (XYL + EMU) in wheat diet with a high level of tallow on gastrointestinal tract microbiota activity, excretion of sialic acids, and selected gut segments morphology of 480 one-day-old male ROSS 308 broiler chickens. The activities of bacterial enzymes in the ileal digesta were lower in experimental groups compared to the control (CON) group. Enzyme activity in the cecum was significantly higher than in the ileum. The additives did not affect the excretion of sialic acid. The number of duodenum goblet cells on the villi decreased in all of the experimental groups (p < 0.05). The simultaneous use of XYL + EMU deepened the ileum crypts (p < 0.05). The total short-chain fatty acid (SCFA) concentration in the cecal digesta was higher in experimental groups. The abundance of Bifidobacterium, Lactobacillus, and Escherichia coli did not change among experimental groups. The relative abundance of Clostridium was significantly (p < 0.05) lower in groups with emulsifier addition. In conclusion, the simultaneous usage of EMU and XYL in wheat-based diets with beef tallow reduces the ileum microbiota activity and enhances cecum microbiota activity. Presumably, the addition of both additives results in a cumulative effect on the gut microbiota activity.
The aim of the present study was to investigate whether besides age and solid feed intake, monocarboxylic acid transporter type 1 (MCT1) expression in the rumen epithelium of calves is affected by liquid feed type [whole milk (WM) or milk replacer (MR)]. Thirty bull calves at the mean age of 5 days were randomly allocated to five experimental groups (six calves/group). Six calves were slaughtered immediately after allocation to the trial (5 days of life), eighteen calves were fed MR and slaughtered at week intervals (on 12, 19, 26 days of life respectively), and six calves were fed WM and slaughtered at the 26 days of life. MCT1 protein abundance and the MCT1 mRNA level were investigated in the dorsal and ventral sack of the rumen. Solid feed intake and short-chain fatty acids (SCFA) concentration in the rumen fluid increased linearly with calves' age. The amount of the MCT1 protein and mRNA in the dorsal sac of rumen as well as the amount of MCT1 protein in the cranial ventral sac of rumen also increased linearly with calves' age. Calves fed WM had greater solid feed intake in the last week of the study as compared to calves fed MR, but SCFA concentration in the rumen fluid was not different. MCT1 mRNA expression in the cranial dorsal sac of rumen and protein MCT1 expression in both dorsal and ventral cranial sack of the rumen were higher in calves fed WM as compared to calves fed MR. This study confirmed age-dependent changes of MCT1 expression in the rumen epithelium of newborn calves and showed that its expression might be affected by liquid feed type.
Two studies were conducted to assess the effect of protein source and microencapsulated sodium butyrate (MSB) inclusion in pelleted starter mixtures on growth performance, gain to feed (G:F) ratio, nutrient digestibility, and selected blood metabolites in calves.In study 1, 28 Holstein bull calves (8.7 ± 0.8 d of age and 43.0 ± 4.4 kg; mean ± SD) were allocated to 1 of 4 treatments in a 2 × 2 factorial arrangement and fed a pelleted starter mixture containing canola meal (CM, 35% as fed) or soybean meal (SM, 24% as fed) as the main source of protein, with or without supplemental MSB (0.3% as fed). Starter mixtures were formulated to be similar for crude protein, Lys, and Met, and were fed ad libitum. Calves were weaned after 42 d of milk replacer feeding (51.7 ± 0.8 d of age) and observed for another 21 d. Furthermore, selected blood metabolites were measured on d 21, 42, and 63 of the study, and nutrient digestibility was measured after weaning. In study 2, 60 Holstein heifer calves (9.1 ± 0.8 d of age and 43.2 ± 4.2 kg) were assigned to the same treatments as in study 1. The calves were weaned after 49 d of milk replacer feeding (59.1 ± 0.8 d of age) and observed for an additional 14 d. Milk replacer and starter mixture intake and fecal score were recorded daily, whereas body weight (BW) was recorded weekly. In study 1, calves fed starter mixtures containing CM had or tended to have lesser preweaning starter intake, weaning average daily gain (ADG), weaning and overall G:F ratio, and postweaning total-tract dry matter digestibility, as opposed to those fed starter mixtures with SM. However, these differences did not affect overall starter intake, overall ADG, or final BW. Supplementation with MSB only tended to increase the preweaning starter mixture intake. In study 2, heifer calves that were fed starter mixtures with CM had greater cumulative starter intake after weaning, but the protein source in the starter mixture had no effect on ADG, BW, or G:F ratio. Inclusion of MSB in starter mixtures for calves tended to decrease postweaning starter mixture intake. In conclusion, use of CM or SM as the main source of protein in starter mixture resulted in similar growth performance of bull and heifer calves; however, CM use in starter mixtures reduced starter intake, ADG, and G:F ratio at least at some points of rearing. Supplementation of MSB had minor effects on the growth performance of calves.
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