Milk growth factors are thought to contribute to postnatal gastrointestinal growth. The roles of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) in the neonatal piglet intestine were investigated. In the first study, piglets were infected with rotavirus on d 4 postpartum and received formula containing 0, 500 or 1000 micrograms/l of EGF for 8 days. A non-infected control group received no EGF. Infected piglets developed severe diarrhea and gained 60% less weight than controls. Rotaviral infection caused a 37% decrease in villus height and 40% decreases in intestinal enzyme activities compared to control. Oral EGF increased villus height and lactase activity in a linear dose-response fashion. Our results suggest that supplementation of formulas with high physiological levels of EGF may aid in small intestinal recovery. A second study investigated absorption of orally administered IGF-I. Newborn piglets were fitted with catheters via the umbilical artery and vein. Piglets were given formula containing 25 microCi of [125I]-IGF-I and blood samples were drawn for 24O min. Total radioactivity, protein bound counts, and counts immunoprecipitable with an antibody to IGF-I were determined in plasma. Radioactivity was detected in portal and arterial plasma within 15 min and rose throughout the study, however, protein bound counts were stable at 20-30% of total counts between 30 and 180 min postgavage. Approximately 10% of the counts were immunoprecipitable by a polyclonal antibody to IGF-I, suggesting that up to 10% of orally administered IGF-I may be absorbed intact.
ABSTRACT Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.
An experiment was conducted to define the lysine requirement of neonatal pigs fed a liquid diet up to 5.5 kg bodyweight (BW). Neonatal pigs, 1–2 days old, with an initial bodyweight of 1.63 ± 0.04 kg, were randomly allotted to 10 isocaloric diets varying in lysine concentration from 0.76 to 1.62 g lysine/MJ gross energy (GE). Diets were formulated using whey protein concentrate and casein as protein sources and contained similar balance of indispensable amino acids. On day 1 of the experiment, pigs were fed 350 g liquid diet/kg metabolic bodyweight (BW0.75) according to the average BW of all pigs. On day 2, feeding rate was increased to 400 g/kg BW0.75. Increments were 100 g/kg BW0.75 per day for the subsequent 3 days until pigs reached 700 g/kg BW0.75 on day 5. Thereafter, feed was offered to pigs at a common feeding level of 700 g/kg BW0.75 each day until they reached 5.5 kg BW. Feed intake and BW were measured daily. Concentration of fat in the carcass decreased (P < 0.05) and the ratio of crude protein (CP) to fat in the carcass increased (P < 0.05) linearly as lysine inclusion increased. Both average daily gain and CP accretion increased (quadratic, P < 0.05), whereas fat accretion decreased (quadratic, P < 0.05) as lysine inclusion increased. Using the maximum point of the quadratic function, the estimated dietary lysine required for maximal growth (271 g/day) and CP accretion (45.2 g/day) was 1.41 and 1.32 g lysine/MJ GE, respectively. The dietary lysine required, estimating the requirement at the lower limit of the 95% confidence interval for CP accretion of 42.9 g/day, was 1.12 g lysine/MJ GE. Gross efficiency of CP deposition (CP deposition/CP intake) achieved a maximum of 0.85 at 1.01 g lysine/MJ GE.
We previously showed that intestinal cell migration, the initial event in epithelial restitution, is accelerated by L-arginine (ARG). We also found that cycloheximide, rapamycin, wortmannin, and intracellular Ca++ chelation inhibited the response to ARG. In this study, we investigated if the ARG response is mediated through the mammalian target of rapamycin (mTOR), which regulates translation of mRNAs bearing a 5′-oligopyrimidine sequence and acts as an amino acid sensor. Methods: We used amino acids as prototypic activators of mTOR in IEC-6 cells and we used serum as a recognized stimulator of intestinal cell migration. Assays included razor wounding-stimulated migration, western blotting, and immunohistochemistry. Results: (1) ARG activates mTOR/p70s6k, NO production, and migration. However, neither leucine (LEU, which activates mTOR/p70s6k but does not produce NO) nor the nitric oxide (NO) donor detaNONOate (which does not activate mTOR) enhances cell migration. (2) The combination of LEU (4 mM) + NONOate (25 uM) enhances migration similarly to ARG (P < 0.01). (3) ARG activates p70s6k immunocytochemical staining in cytoplasm, phospho-p70s6k in the nucleus, and phospho-ribosomal protein S6 in the cytoplasm. Also, phosphorylated p70s6k migrates from nucleus to the cytoplasm after ARG stimulation. We conclude that stimulators of the mTOR pathway stimulate intestinal cell migration; inhibitors of the mTOR pathway inhibit intestinal cell migration; p70s6k is activated in the nucleus; and ribosomal protein S6 is activated in the cytosol. These results are consistent with translational regulation via mTOR/p70s6k playing an important role in arginine-stimulated intestinal epithelial restitution.
The objective of this study was to evaluate the effects of peroxisome proliferator-activated receptor α (PPARα) activation by clofibrate on both mitochondrial and peroxisomal fatty acid oxidation in the developing kidney. Ten newborn pigs from 5 litters were randomly assigned to two groups and fed either 5 mL of a control vehicle (2% Tween 80) or a vehicle containing clofibrate (75 mg/kg body weight, treatment). The pigs received oral gavage daily for three days. In vitro fatty acid oxidation was then measured in kidneys with and without mitochondria inhibitors (antimycin A and rotenone) using [1-14C]-labeled oleic acid (C18:1) and erucic acid (C22:1) as substrates. Clofibrate significantly stimulated C18:1 and C22:1 oxidation in mitochondria (p < 0.001) but not in peroxisomes. In addition, the oxidation rate of C18:1 was greater in mitochondria than peroxisomes, while the oxidation of C22:1 was higher in peroxisomes than mitochondria (p < 0.001). Consistent with the increase in fatty acid oxidation, the mRNA abundance and enzyme activity of carnitine palmitoyltransferase I (CPT I) in mitochondria were increased. Although mRNA of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (mHMGCS) was increased, the β-hydroxybutyrate concentration measured in kidneys did not increase in pigs treated with clofibrate. These findings indicate that PPARα activation stimulates renal fatty acid oxidation but not ketogenesis.
To evaluate the effects of maternal feeding of clofibrate, a pharmaceutical PPARα agonist, on the development of intestinal fatty acid metabolism in offspring using the pig as a model. A total of 27 pregnant sows were randomly assigned into three treatment groups. Each group was fed a standard diet (3265 kcal ME/kg) supplemented with either 0, 0.25% or 0.5% clofibrate (w/w) from d 107 of gestation to d 7 of lactation. A piglet from each litter was sampled on 1, 7, 14 and 19 d of age. Fatty acid oxidation was examined in homogenates of ileum mucosa using 1 mM [1–14C] oleic acid as a substrate in the absence or presence of in vitro supplemented L-carnitine (1 mM), malonate (10 mM) or carnitine + malonate. The metabolism of [1–14C] oleic acid was modified by the maternal clofibrate, and the effects varied with the dose of clofibrate and postnatal age (p < 0.001). The 14C accumulation in 14CO2 increased by 2.4 fold at d7 (vs d1), but showed no difference after d7. Maternal clofibrate stimulated the 14CO2 accumulation from d1 to d19, but the stimulation decreased with postnatal age. The 14C accumulation in acid soluble products (14C-ASP) was reduced by 8.4 fold at d7 (vs d1), and no difference was observed after d7. Maternal clofibrate increased 14C-ASP at d1, and the effect was greater as maternal clofibrate was incremented from 0.25 to 0.5%. The 14C accumulation in esterified products (14C-ESP) was greater at d7 than d1, 14 and 19. Maternal clofibrate reduced the 14C-ESP and, again the reduction was stronger with increasing clofibrate dose. In vitro supplementation of carnitine increased 14CO2 by 60% and 14C-ASP by 120%, but reduced 14C-ESP by 39% compared to control incubations. Supplementation of malonate reduced 14CO2 by 95% and 14C-ESP by 44%, but no effect on 14C-ASP was detected. Maternal dietary supplementation of clofibrate increases intestinal fatty acid oxidation in suckling offspring, but the increase is attenuated after one week. Inhibition of the TCA cycle reduces CO2 production without affecting ASP. The availability of carnitine could be a key element to maintain a high rate of intestinal fatty acid oxidation during the neonatal period. Supported by Program of Animal Nutrition, Growth and Lactation [grant no. 2015–67015-23245/project accession no. 1005855] from the USDA NIFA.
Abstract This study was conducted to evaluate the effects of using plasma protein (PP) as a highly digestible, functional protein source in nursery pig diets on growth performance and intestinal health. Pigs (n = 64; 6.53 ± 0.12 kg BW) weaned at 21 d of age were blocked by litter and BW and randomly assigned within blocks to 1 of 2 dietary treatments in a 35-d study. Four pigs were housed per pen, using a total of 16 pens (8 replicate pens per treatment). Treatments were: 1) control diet (without growth-promoting minerals or antibiotics) without PP, and 2) control diet with 4% of PP replacing SBM on a 1:1 basis. Diets were fed in 3 feeding phases (7, 14, and 14 days, respectively) and PP was included in all phases. Supplementation of PP tended (P = 0.07) to increase ADG (204 vs. 150 g/d) and improved (P = 0.02) gain:feed (812 vs 572 g/kg) during wk 2 only. Individual diarrhea scores (scale from 0=very hard to 6=watery diarrhea) determined using fecal loops changed over time (P < 0.001; 2.1, 2.4, 4.1 and 3.5 for d 1, 3, 7, and 14 respectively), but were not affected by PP. No differences were detected for intestinal morphology measured on d 35 in duodenum, jejunum and ileum. Cell proliferation using Ki-67 staining was increased by PP (86.6 vs. 72.2 positive cells) only in the ileum (P = 0.03). No differences in mucosal concentrations of IL-10, TNF-α, and IFN-γ in the duodenum, jejunum or ileum were detected. Number of eosinophils in blood decreased (14.1 vs. 34.8 cells/µL; P = 0.004), while red blood cell counts (5.9 vs 6.2 x 106 cells/µL; P = 0.09) and neutrophil counts (529 vs. 695 cells/µL; P = 0.07) tended to decrease with PP. Results suggest that PP was not effective in improving overall growth performance and had minimal effects on intestinal health of weanling pigs.
Effects of dietary calcium (Ca) deficiency on skeletal integrity and endocrine parameters are well characterized in growing and mature mammals; however, little work has examined Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine parameters, and in vivo mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6h post‐partum) were pair‐fed either a Ca adequate or a 30% Ca deficient liquid formula diet for 18 days. There were no differences in growth rate or feed conversion efficiency based on dietary Ca level and all pigs grew at a rate similar to sow‐reared pigs. As anticipated, dietary Ca deficiency reduced (P < 0.05) both BMD and bone flexural strength. The anticipated increase (P < 0.05) in plasma PTH levels in pigs fed the Ca deficeint diet was not evident until the end of the study. Surprisingly, dietary Ca level did not affect plasma Ca or 1,25(OH) 2 vitamin D concentrations throughout the study. Calcium deficiency reduced (P < 0.05) the in vivo proliferation of MSC isolated from bone marrow by approximately 50%. These results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early life Ca restriction may have long‐term effects on bone integrity via its effects on MSC activity.
This study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role for enteric glial cell (EGC) activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.
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