EXPLORING THE IMPACT OF BIOACTIVE COMPOUNDS ON NECROTIZING ENTEROCOLITIS (NEC) IN VIVO (GALLUS GALLUS)

291 pagesNecrotizing enterocolitis (NEC) is a severe gastrointestinal disease in premature infants and a leading cause of death in neonates (1-7% in the US). NEC is caused by opportunistic bacteria, which cause gut dysbiosis and inflammation, ultimately resulting in intestinal necrosis. Current NEC interventions include broad-spectrum antibiotic treatment and/or small intestine surgical removal, which can have harmful effects. Even with surgical intervention, NEC global mortality rates are 6-9% (1-7% in the US) [3]. Various potential animal NEC models have been utilized to study the disease; however, the disease, sensitive in vivo NEC biomarkers, and possible interventions remain undefined. Crop-based bioactive compounds have previously been shown to possess anti-inflammatory properties and positively modulate the gut microbiome, providing a viable alternative for NEC treatment. Chapter 1 summarizes NEC pathology and essential background on the disease. Chapter 2 systematically reviews the literature on NEC dietary bioactive compounds utilized in vivo from the last six years. Chapter 3 discusses a new in vivo model for NEC and the efficacy of the Gallus gallus model for NEC. Chapter 4 investigates the benefits and nutritional effects of catechin and 2 short-chain fatty acid alternatives on improving intestinal functionality and morphology and altering the gut microbiome in vivo. Chapter 5 investigates nicotinamide riboside and 2 other short-chain fatty acid derivatives on the intestinal function and morphology in ovo. To conclude, Chapter 6 highlights significant findings in chapters 2-5 and suggests future

Assessing the Interactions between Zinc and Vitamin A on Intestinal Functionality, Morphology, and the Microbiome In Vivo (<i>Gallus gallus</i>)

Dietary deficiencies in zinc (Zn) and vitamin A (VA) are among the leading micronutrient deficiencies globally and previous research has proposed a notable interaction between Zn and VA physiological status. This study aimed to assess the effects of zinc and vitamin A (isolated and combined) on intestinal functionality and morphology, and the gut microbiome (Gallus gallus). The study included nine treatment groups (n~11)—no-injection (NI); H2O; 0.5% oil; normal zinc (40 mg/kg ZnSO4) (ZN); low zinc (20 mg/kg) (ZL); normal retinoid (1500 IU/kg retinyl palmitate) (RN); low retinoid (100 IU/kg) (RL); normal zinc and retinoid (40 mg/kg; 1500 IU/kg) (ZNRN); low zinc and retinoid (ZLRL) (20 mg/kg; 100 IU/kg). Samples were injected into the amniotic fluid of the fertile broiler eggs. Tissue samples were collected upon hatch to target biomarkers. ZLRL reduced ZIP4 gene expression and upregulated ZnT1 gene expression (p p p p p Bifidobacterium and Clostridium genera (p < 0.05). These results suggest a potentially improved intestinal epithelium proceeding with Zn and VA intra-amniotic administration. Intestinal functionality and gut bacteria were modulated. Further research should characterize long-term responses and the microbiome profile

Intra-Amniotic Administration (Gallus gallus) of Cicer arietinum and Lens culinaris Prebiotics Extracts and Duck Egg White Peptides Affects Calcium Status and Intestinal Functionality

Calcium (Ca) is one of the most abundant inorganic elements in the human body and has many important physiological roles. Prebiotics and bioactive peptides are two important substances used to promote calcium uptake. However, the difference in mechanisms of the calcium uptake from these two supplements is not clear. By using the Gallus gallus model and the intra-amniotic administration procedure, the aim of this study was to investigate whether Ca status, intestinal functionality, and health-promoting bacterial populations were affected by prebiotics extracted from chickpea and lentil, and duck egg white peptides (DPs). Eleven groups (non-injected; 18 MΩ H2O; 4 mmol/L CaCl2; 50 mg/mL chickpea + 4 mmol/L CaCl2; 50 mg/mL lentil + 4 mmol/L CaCl2; 40 mg/mL DPs + 4 mmol/L CaCl2; 5 mg/mL Val-Ser-Glu-Glu (VSEE) + 4 mmol/L CaCl2; 50 mg/mL chickpea; 50 mg/mL lentil; 40 mg/mL DPs; 5 mg/mL VSEE) were utilized. Upon hatch, blood, cecum, small intestine, liver and bone were collected for assessment of serum bone alkaline phosphate level (BALP), the relative abundance of intestinal microflora, expression of Ca-related genes, brush border membrane (BBM) functional genes, and liver and bone mineral levels, respectively. The BALP level increased in the presence of lentil, DPs and VSEE (p &lt; 0.05). The relative abundance of probiotics increased significantly (p &lt; 0.05) by VSEE + Ca and chickpea. The expression of CalbindinD9k (Ca transporter) increased (p &lt; 0.05) in Ca, chickpea + Ca and lentil + Ca groups. In addition, the brush border membrane functionality genes expressions increased (p &lt; 0.05) by the chickpea or lentil extracts. Prebiotics and DPs beneficially affected the intestinal microflora and duodenal villus surface area. This research expands the understanding of the prebiotics’ properties of chickpea and lentil extracts, and peptides’ effects on calcium metabolism and gut health

Intra-Amniotic Administration&mdash;An Emerging Method to Investigate Necrotizing Enterocolitis, In Vivo (Gallus gallus)

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease in premature infants and a leading cause of death in neonates (1&ndash;7% in the US). NEC is caused by opportunistic bacteria, which cause gut dysbiosis and inflammation and ultimately result in intestinal necrosis. Previous studies have utilized the rodent and pig models to mimic NEC, whereas the current study uses the in vivo (Gallus gallus) intra-amniotic administration approach to investigate NEC. On incubation day 17, broiler chicken (Gallus gallus) viable embryos were injected intra-amniotically with 1 mL dextran sodium sulfate (DSS) in H2O. Four treatment groups (0.1%, 0.25%, 0.5%, and 0.75% DSS) and two controls (H2O/non-injected controls) were administered. We observed a significant increase in intestinal permeability and negative intestinal morphological changes, specifically, decreased villus surface area and goblet cell diameter in the 0.50% and 0.75% DSS groups. Furthermore, there was a significant increase in pathogenic bacterial (E. coli spp. and Klebsiella spp.) abundances in the 0.75% DSS group compared to the control groups, demonstrating cecal microbiota dysbiosis. These results demonstrate significant physiopathology of NEC and negative bacterial&ndash;host interactions within a premature gastrointestinal system. Our present study demonstrates a novel model of NEC through intra-amniotic administration to study the effects of NEC on intestinal functionality, morphology, and gut microbiota in vivo

Saffron (Crocus sativus L.) Flower Water Extract Disrupts the Cecal Microbiome, Brush Border Membrane Functionality, and Morphology In Vivo (Gallus gallus)

Saffron (Crocus sativus L.) is known as the most expensive spice. C. sativus dried red stigmas, called threads, are used for culinary, cosmetic, and medicinal purposes. The rest of the flower is often discarded, but is now being used in teas, as coloring agents, and fodder. Previous studies have attributed antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, anti-depressant, and anticancer properties to C. sativus floral bio-residues. The aim of this study is to assess C. sativus flower water extract (CFWE) for its effects on hemoglobin, brush boarder membrane (BBM) functionality, morphology, intestinal gene expression, and cecal microbiome in vivo (Gallus gallus), a clinically validated model. For this, Gallus gallus eggs were divided into six treatment groups (non-injected, 18 &Omega; H2O, 1% CFWE, 2% CFWE, 5% CFWE, and 10% CFWE) with n~10 for each group. On day 17 of incubation, 1 mL of the extracts/control were administered in the amnion of the eggs. The amniotic fluid along with the administered extracts are orally consumed by the developing embryo over the course of the next few days. On day 21, the hatchlings were euthanized, the blood, duodenum, and cecum were harvested for assessment. The results showed a significant dose-dependent decrease in hemoglobin concentration, villus surface area, goblet cell number, and diameter. Furthermore, we observed a significant increase in Paneth cell number and Mucin 2 (MUC2) gene expression proportional to the increase in CFWE concentration. Additionally, the cecum microbiome analysis revealed C. sativus flower water extract altered the bacterial populations. There was a significant dose-dependent reduction in Lactobacillus and Clostridium sp., suggesting an antibacterial effect of the extract on the gut in the given model. These results suggest that the dietary consumption of C. sativus flower may have negative effects on BBM functionality, morphology, mineral absorption, microbial populations, and iron status

Desalted Duck Egg White Peptides Promote Calcium Uptake and Modulate Bone Formation in the Retinoic Acid-Induced Bone Loss Rat and Caco-2 Cell Model

Desalted duck egg white peptides (DPs) have been proven to promote calcium uptake in Caco-2 cells and rats treated with a calcium-deficient diet. The retinoic acid-induced bone loss model was used to evaluate the effect of DPs on calcium absorption and bone formation. Three-month-old Wistar female rats were treated with 0.9% saline, DPs (800 mg/kg), or alendronate (5 mg/kg) for three weeks immediately after retinoic acid treatment (80 mg/kg) once daily for two weeks. The model group was significantly higher in serum bone alkaline phosphatase than the other three groups (p &lt; 0.05), but lower in calcium absorption rate, serum osteocalcin, bone weight index, bone calcium content, bone mineral density, and bone max load. After treatment with DPs or alendronate, the absorption rate increased and some serum and bone indices recovered. The morphology results indicated bone tissue form were ameliorated and numbers of osteoclasts decreased after supplementation with DPs or alendronate. The in vitro study showed that the transient receptor potential vanilloid 6 (TRPV6) calcium channel was the main transport pathway of both DPs and Val-Ser-Glu-Glu peptitde (VSEE), which was identified from DPs. Our results indicated that DPs could be a promising alternative to current therapeutic agents for bone loss because of the promotion of calcium uptake and regulation of bone formation

Empire Apple (Malus domestica) Juice, Pomace, and Pulp Modulate Intestinal Functionality, Morphology, and Bacterial Populations In Vivo (Gallus gallus)

Approximately $20 billion of apple sales are generated annually in the United States. With an estimated 5 million tons produced yearly in the U.S. within the last decade, apple consumption is considered ubiquitous. Apples are comprised of bioactive constituents such as phytochemicals and prebiotics that may potentiate intestinal health and the gut microbiome. This study aimed to evaluate the effects of Empire apple juice, pomace, and pulp soluble extracts on intestinal functionality, morphology, and the microbiome in vivo (Gallus gallus). There were five treatment groups: non-injected (NI); 18 M&#8486; H2O (H2O); 6% apple juice (AJ); 6% apple pomace (APo); 6% apple pulp (APu). The eggs were treated by intra-amniotic administration of the samples on day 17 of incubation. After hatching, the blood, tissue, and cecum samples were collected for further analyses&mdash;including duodenal histomorphology, hepatic and duodenal mRNA expression, and cecal bacterial populations. Crypt depth was significantly (p &lt; 0.5) shortest in AJ when compared to APo and APu. APo and APu soluble extracts significantly improved villi surface area compared to NI and H2O control groups. The highest count of Paneth cells per crypt was observed in APo as compared to all groups. In addition, the expression of brush border membrane micronutrient metabolism and functional proteins varied between treatments. Lastly, Lactobacillus cecal microbial populations increased significantly in the AJ group, while AJ, APu, and APu increased the abundance of Clostridium (p &lt; 0.5). Ultimately, these results indicate the potential of Empire apple pomace to improve host intestinal health and the gut microbiome

Quinoa Soluble Fiber and Quercetin Alter the Composition of the Gut Microbiome and Improve Brush Border Membrane Morphology In Vivo (<i>Gallus gallus</i>)

Quinoa (Chenopodium quinoa Willd.), a gluten-free pseudo-cereal, has gained popularity over the last decade due to its high nutritional value. Quinoa is a rich source of proteins, carbohydrates, fibers, tocopherols (Vitamin E), unsaturated fatty acids and a wide range of polyphenols. The study used Gallus gallus intra-amniotic feeding, a clinically validated method, to assess the effects of quinoa soluble fiber (QSF) and quercetin 3-glucoside (Q3G) versus control. Quercetin is a pharmacologically active polyphenol found in quinoa. Six groups (no injection, 18 Ω H2O, 5% inulin, 1% Q3G, 5% QSF, 1% Q3G + 5% QSF) were assessed for their effect on the brush border membrane (BBM) functionality, intestinal morphology and cecal bacterial populations. Our results showed a significant (p L. plantarum populations compared to the control (H2O). In conclusion, quercetin and quinoa fiber consumption has the potential to improve intestinal morphology and modulate the microbiome

Food-Grade Metal Oxide Nanoparticles Exposure Alters Intestinal Microbial Populations, Brush Border Membrane Functionality and Morphology, In Vivo (<i>Gallus gallus</i>)

Among food additive metal oxide nanoparticles (NP), titanium dioxide (TiO₂) and silicon dioxide (SiO₂) are commonly used as food coloring or anti-caking agents, while zinc oxide (ZnO) and iron oxide (Fe₂O₃) are added as antimicrobials and coloring agents, respectively, and can be used as micronutrient supplements. To elucidate potential perturbations associated with NP consumption on gastrointestinal health and development, this in vivo study utilized the Gallus gallus (broiler chicken) intraamniotic administration to assess the effects of physiologically relevant concentrations of food-grade metal oxide NP on brush border membrane (BBM) functionality, intestinal morphology and intestinal microbial populations in vivo. Six groups with 1 mL injection of the following treatments were utilized: non-injected, 18 MΩ DI H2O; 1.4 × 10−6 mg TiO2 NP/mL, 2.0 × 10−5 mg SiO2 NP/mL, 9.7 × 10−6 mg ZnO NP/mL, and 3.8 × 10−4 mg Fe2O3 NP/mL (n = 10 per group). Upon hatch, blood, cecum, and duodenum were collected to assess mineral (iron and zinc) metabolism, BBM functional, and pro-inflammatory-related protein gene expression, BBM morphometric analysis, and the relative abundance of intestinal microflora. Food additive NP altered mineral transporter, BBM functionality, and pro-inflammatory cytokine gene expression, affected intestinal BBM development and led to compositional shifts in intestinal bacterial populations. Our results suggest that food-grade TiO₂ and SiO₂ NP have the potential to negatively affect intestinal functionality; food-grade ZnO NP exposure effects were associated with supporting intestinal development or compensatory mechanisms due to intestinal damage, and food-grade Fe₂O₃ NP was found to be a possible option for iron fortification, though with potential alterations in intestinal functionality and health