Effect of vitamin E and alpha lipoic acid on intestinal development associated with wooden breast myopathy in broilers.

Intestinal development is closely associated with inflammatory wooden breast (WB) myopathy. Vitamin E (VE) and alpha lipoic acid (ALA) with antioxidant and anti-inflammatory effects were used independently and in combination to evaluate their effects on intestinal developmental changes in ileal morphology and expression of genes related with gut nutrient transport, structure, and inflammation in broilers during the first 3 wk posthatch. A total of 160 newly hatched Ross 708 broiler chicks were randomly assigned into a control and 3 dietary treatments with 10 replicates of 4 birds each. Supplementation of VE (160 mg/kg) and ALA (500 mg/kg) independently and in combination were fed during the first 3 wk. At 1, 2, and 3 wk of age, one chick from each pen was harvested. Plasma VE concentration and ileal morphology were determined. Gene expression was measured by real-time quantitative PCR. Broilers in VE and combination of ALA and VE group had higher plasma VE concentration than the control and ALA group at 1, 2, and 3 wk of age (P < 0.01). All dietary treatments increased ileal villus height at 1 wk of age (P < 0.01) and decreased intraepithelial lymphocytes at 3 wk of age compared to the control (P ≤ 0.05). Combination of VE and ALA increased collagen type IV alpha 1 chain expression (P ≤ 0.05) and improved basement membrane structure indicating increased gut basement membrane integrity at 2 and 3 wk of age compared to the control. Expression of lipopolysaccharide-induced tumor necrosis factor-alpha factor associated with inflammation was decreased in all dietary treatments at 3 wk of age compared to the control (P < 0.01). Ileal morphology and gene expression were closely correlated with breast muscle morphology and gene expression. These results suggest that VE and ALA especially when they were combined in the diet had positive effects on mitigating intestinal inflammation and improving nutrient transport beginning at 1 wk of age, which is likely critical in reducing the severity of WB.

Supplementation of vitamin E and omega-3 fatty acids during the early posthatch period on intestinal morphology and gene expression differentiation in broilers.

Early posthatch nutrition is important for gut health. Vitamin E (VE) and omega-3 (n-3) fatty acids can improve gut health through antioxidative and anti-inflammatory effects. The objective of this study was to identify the effects of VE, n-3 fatty acids, and combination of both during the starter phase (0-10 d) or grower phase (11-24 d) on intestinal morphology and expression of genes associated with gut health. A total of 210 Ross 708 broilers were randomly assigned into 7 treatments with 10 replicates of 3 birds each. The control group was fed a corn-soybean meal-basal diet during the entire study (0-58 d). Supplementation of VE (200 IU/kg), n-3 fatty acids (n-6/n-3 ratio of 3.2:1), and combination of both were fed during the starter phase (0-10 d) or grower phase (11-24 d). All of the broilers were harvested at 58 d of age. Villus height, crypt depth, villus width, distance between villi, and number of intraepithelial lymphocytes were obtained. Expression of 21 genes was measured using NanoString analysis. Expression of solute carrier family 15 member 1 (P = 0.01) associated with peptide transport and mucin 2 (P = 0.03) related with intestinal mucus barrier was increased in the broilers supplemented with n-3 fatty acids in the grower diet compared with the control. Expression of solute carrier family 7 member 1 associated with amino acid transport was decreased in the group supplemented with n-3 fatty acids during the starter phase compared with the group supplemented with n-3 fatty acids (P = 0.01) or VE and n-3 fatty acids during the grower phase (P = 0.03). These data suggest that VE and n-3 fatty acids supplemented during the grower phase have a positive effect on improving nutrient transport with n-3 fatty acids supplementation in the grower diet showing the most beneficial effect. These findings can be used in the development of nutritional management strategies to improve broiler growth performance and meat quality.

Effect of early posthatch supplementation of vitamin E and omega-3 fatty acids on the severity of wooden breast, breast muscle morphological structure, and gene expression in the broiler breast muscle.

Wooden breast (WB) has arisen primarily in the breast muscle of commercial broilers. It is characterized by palpation of a rigid pectoralis major (p. major) muscle and is under severe oxidative stress and inflammation. Previous studies have shown that vitamin E (VE) has antioxidant properties and omega-3 (n-3) fatty acids have an anti-inflammatory effect. The objectives of this study were to identify the effects of VE and n-3 fatty acids on the severity of WB, morphological structure of the p. major muscle, expression of genes likely associated with WB and to determine the most beneficial supplementation period. A total of 210 Ross 708 broilers were randomly assigned into 7 treatments with 10 replicates of 3 birds each. The control group received a corn-soybean meal basal diet during the entire study (0-58 d). Supplementation of VE (200 IU/kg), n-3 fatty acids (n-6/n-3 ratio of 3.2:1), or combination of both were fed during the starter phase (0-10 d) or grower phase (11-24 d). All broilers were harvested at 58 d of age. Morphological assessment of the p. major muscle included myofiber width, perimysial and endomysial connective tissue space, overall morphological structure, and scoring of WB microscopically. Gene expression was measured using nanostring analysis. Genes associated with muscle development and growth factors, inflammation, extracellular matrix, and glucose metabolism were differentially expressed in the p. major muscle of the broilers supplemented with VE in the grower diet. Greater than 2 times more giant myofibers (≥70 µm) were found in the group supplemented with VE and n-3 fatty acids in the starter diet compared with the group fed VE in the grower diet (P = 0.02). Microscopic evaluation showed that VE supplementation in the grower diet had a 16.19% increase in muscle with no WB compared with the control group (P = 0.05). These data suggest that supplementation of VE during the grower phase may reduce the severity of WB in broilers.

Effect of vitamin E and omega-3 fatty acids early posthatch supplementation on reducing the severity of wooden breast myopathy in broilers.

The wooden breast (WB) myopathy is identified by the palpation of a rigid pectoralis major (P. major) muscle and is characterized as a fibrotic, necrotic P. major muscle disorder in broilers resulting in reduced breast meat quality. Breast muscle affected with WB is under severe oxidative stress and inflammation. The objectives were to identify the effects of dietary vitamin E (VE) and omega-3 (n-3) fatty acids independently or in combination when fed during the starter phase (0-10 D) or grower phase (11-24 D) on growth performance, meat yield, meat quality, and severity of WB myopathy and to determine the most beneficial dietary supplementation period. A total of 210 Ross 708 broiler chicks were randomly assigned into 7 experimental groups with 10 replicates of 3 birds each. The control group was fed with corn-soybean meal basal diet with VE (10 IU/kg) and n-3 fatty acids (n-6/n-3 ratio of 30:1) at a standard level during the entire study (0-58 D). Supplementation of VE (200 IU/kg), n-3 fatty acids (n-6/n-3 ratio of 3:1), or combination of both was performed during the starter phase or grower phase. Growth performance, meat yield, meat quality, and WB scores were obtained. There was no significant difference in final body weight and meat yield when VE was increased (P > 0.05). In contrast, n-3 fatty acids supplementation in starter diets significantly decreased final body weight, hot carcass weight, and chilled carcass weight of broilers (P ≤ 0.05). The P. major muscle from broilers supplemented with VE in starter diets had lower shear force than in grower diets (P ≤ 0.05). Supplemental VE reduced the severity of WB and in starter diets showed a more beneficial effect than those fed VE in the grower diets. These data are suggestive that additional supplementation of dietary VE may reduce the severity of WB and promote breast meat quality without adversely affecting growth performance and meat yield.

Supplementation of Maternal Diets with Docosahexaenoic Acid and Methylating Vitamins Impacts Growth and Development of Fetuses from Malnourished Gilts.

BACKGROUND: Like many species, pregnant swine mobilize and repartition body nutrient stores during extreme malnutrition to support fetal development. OBJECTIVE: The objective of this study was to model chronic human maternal malnutrition and measure effects of methylating-vitamins (MVs, containing choline, folate, B-6, B-12, and riboflavin) and docosahexaenoic acid (DHA) supplementation on fetal growth and development. METHODS: Pregnant gilts (n = 24) were either fully nourished (2.0 kg/d) with a corn-plus-isolated-soy-protein basal diet (control) supplemented with MVs and DHA or nourishment was restricted throughout gestation. Basal diet fed to malnourished gilts was reduced progressively from 50% to 70% restriction (1.0 to 0.6 kg/d) and was supplemented following a 2 (±MVs) x 2 (±DHA) factorial design. Full-term c-sections were performed to assess impacts on low and normal birth weight (LBW/NBW) fetuses (n = 238). RESULTS: Body weight gain of malnourished gilts was 10% of full-fed control dams (P < 0.05), but offspring birth weight, length, girth, and percentage of LBW fetuses were not different between treatments. The number of pigs per litter was reduced by 30% in malnourished control dams. Fetal brain weights were reduced by 7% compared to positive controls (P < 0.05). Micronutrient supplementation to malnourished dams increased fetal brain weights back to full-fed control levels. Dams with DHA produced offspring with higher DHA concentrations in brain and liver (P < 0.05). Plasma choline concentration was 4-fold higher in fetuses from unsupplemented malnourished dams (P < 0.0001). Global DNA methylation status of fetuses from restricted dams was higher than in control fetuses, including brain, liver, heart, muscle, and placenta tissues (P < 0.05). Addition of DHA increased methylation in LBW fetal brains (P < 0.05). CONCLUSIONS: Despite the mobilization of maternal stores, malnourished litters displayed reduced brain development that was fully mitigated by micronutrient supplementation. Severe maternal malnutrition increased global DNA methylation in several fetal tissues that was unaltered by choline and B-vitamin supplementation.

Dietary Isomers of Sialyllactose Increase Ganglioside Sialic Acid Concentrations in the Corpus Callosum and Cerebellum and Modulate the Colonic Microbiota of Formula-Fed Piglets.

BACKGROUND: Sialyllactose is a key human milk oligosaccharide and consists of sialic acid (SA) bound to a lactose molecule. Breastfed infants have increased accumulation of ganglioside-bound SA compared with formula-fed infants. OBJECTIVE: This study aimed to determine whether different isomers of sialyllactose enrich brain SA and modulate the microbiome of developing neonatal piglets. METHODS: Day-old pigs were randomly allocated to 6 diets (control, 2 or 4 g 3'-sialyllactose/L, 2 or 4 g 6'-sialyllactose/L, or 2 g polydextrose/L + 2 g galacto-oligosaccharides/L; n = 9) and fed 3 times/d for 21 d. Pigs were killed, and the left hemisphere of the brain was dissected into cerebrum, cerebellum, corpus callosum, and hippocampus regions. SA was determined by using a modified periodic acid-resorcinol reaction. Microbial composition of the intestinal digesta was analyzed with the use of 16S ribosomal DNA Illumina sequencing. RESULTS: Dietary sialyllactose did not affect feed intake, growth, or fecal consistency. Ganglioside-bound SA in the corpus callosum of pigs fed 2 g 3'-sialyllactose or 6'-sialyllactose/L increased by 15% in comparison with control pigs. Similarly, ganglioside-bound SA in the cerebellum of pigs fed 4 g 3'-sialyllactose/L increased by 10% in comparison with control pigs. Significant (P < 0.05, Adonis Test) microbiome differences were observed in the proximal and distal colons of piglets fed control compared with 4-g 6'-sialyllactose/L formulas. Differences were attributed to an increase in bacterial taxa belonging to species Collinsella aerofaciens (phylum Actinobacteria), genera Ruminococcus and Faecalibacterium (phylum Firmicutes), and genus Prevotella (phylum Bacteroidetes) (Wald test, P < 0.05, DeSeq2) compared with piglets fed the control diet. Taxa belonging to families Enterobacteriaceae and Enterococcaceae (phylum Proteobacteria), as well as taxa belonging to family Lachnospiraceae and order Lactobacillales (phylum Firmicutes), were 2.3- and 4-fold lower, respectively, in 6'-sialyllactose-fed piglets than in controls. CONCLUSIONS: Supplementation of formula with 3'- or 6'-sialyllactose can enrich ganglioside SA in the brain and modulate gut-associated microbiota in neonatal pigs. We propose 2 potential routes by which sialyllactose may positively affect the neonate: serving as a source of SA for neurologic development and promoting beneficial microbiota.

Dietary supplementation of Bifidobacterium longum strain AH1206 increases its cecal abundance and elevates intestinal interleukin-10 expression in the neonatal piglet.

Intestinal microbiota of infants differ in response to gestational age, delivery mode and feeding regimen. Dietary supplementation of probiotic bacteria is one method of promoting healthy populations. We examined the impact of a novel probiotic strain of Bifidobacterium longum (AH1206) on the health, growth and development of neonatal pigs as a model for infants. Day-old pigs were fed milk-based formula containing AH1206 at 0, 109, or 10¹¹ CFU/d for 18 d (n=10/treatment). Differences were not detected in growth, organ weights or body temperatures (P>0.1); however pigs fed the high dose showed a small (2%) reduction in feed intake. Bacterial translocation was not affected as indicated by total anaerobic and aerobic counts (CFU) in samples of spleen, liver and mesenteric lymph nodes (P>0.1). Feeding AH1206 had no effects on fecal consistency, but increased the density of B. longum in the cecum. Ileal TNF expression tended to increase (P=0.08) while IL-10 expression increased linearly (P=0.01) with supplementation. Based upon findings in the suckling piglet model, we suggest that dietary supplementation with B. longum (AH1206) may be safe for human infants based on a lack of growth, development or deleterious immune-related effects observed in piglets.

Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge.

Long-chain (n-3) PUFA exert beneficial effects on inflammatory bowel diseases in animal models and clinical trials. In addition, pattern recognition receptors such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain proteins (NOD) play a critical role in intestinal inflammation. We hypothesized that fish oil could alleviate Escherichia coli LPS-induced intestinal injury via modulation of TLR4 and NOD signaling pathways. Twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors included a dietary treatment (5% corn oil or 5% fish oil) and immunological challenge (LPS or saline). After feeding fish oil or corn oil diets for 21 d, pigs were injected with LPS or saline. At 4 h postinjection, blood samples were collected and pigs were killed. EPA, DHA, and total (n-3) PUFA were enriched in intestinal mucosa through fish supplementation. Fish oil improved intestinal morphology, indicated by greater villus height and villus height:crypt depth ratio, and intestinal barrier function, indicated by decreased plasma diamine oxidase (DAO) activity and increased mucosal DAO activity as well as enhanced protein expression of intestinal tight junction proteins including occludin and claudin-1. Moreover, fish oil decreased intestinal TNFα and PGE(2) concentrations and caspase-3 and heat shock protein 70 protein expression. Finally, fish oil downregulated the mRNA expression of intestinal TLR4 and its downstream signals myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNFα receptor-associated factor 6, and NOD2, and its adaptor molecule, receptor-interacting serine/threonine-protein kinase 2. Fish oil decreased the protein expression of intestinal NFκB p65. These results indicate that fish oil supplementation is associated with inhibition of TLR4 and NOD2 signaling pathways and concomitant improvement of intestinal integrity under an inflammatory condition.

Dietary long-chain PUFA enhance acute repair of ischemia-injured intestine of suckling pigs.

Infant formula companies have been fortifying formulas with long-chain PUFA for 10 y. Long-chain PUFA are precursors of prostanoids, which stimulate recovery of intestinal barrier function. Supplementation of milk with PUFA increases the content of arachidonic acid (ARA) in enterocyte membranes; however, the effect of this enrichment on intestinal repair is not known. The objective of these experiments was to investigate the effect of supplemental ARA on intestinal barrier repair in ischemia-injured porcine ileum. One-day-old pigs (n = 24) were fed a milk-based formula for 10 d. Diets contained no PUFA (0% ARA), 0.5% ARA, 5% ARA, or 5% EPA of total fatty acids. Following dietary enrichment, ilea were subjected to in vivo ischemic injury by clamping the local mesenteric blood supply for 45 min. Following the ischemic period, control (nonischemic) and ischemic loops were mounted on Ussing chambers. Transepithelial electrical resistance (TER) was measured over a 240-min recovery period. Ischemia-injured ileum from piglets fed 5% ARA (61.0 ± 14%) exhibited enhanced recovery compared with 0% ARA (16 ± 14) and 0.5% ARA (22.1 ± 14)-fed pigs. Additionally, ischemia-injured ileum from 5% EPA (51.3 ± 14)-fed pigs had enhanced recovery compared with 0% ARA-fed pigs (P < 0.05). The enhanced TER recovery response observed with ischemia-injured 5% ARA supplementation was supported by a significant reduction in mucosal-to-serosal flux of (3)H-mannitol and (14)C-inulin compared with all other ischemia-injured dietary groups (P < 0.05). A histological evaluation of ischemic ilea from piglets fed the 5% ARA showed reduced histological lesions after ischemia compared with the other dietary groups (P < 0.05). These data demonstrate that feeding elevated levels of long-chain PUFA enhances acute recovery of ischemia-injured porcine ileum.

Dietary conjugated linoleic acid alters long chain polyunsaturated fatty acid metabolism in brain and liver of neonatal pigs.

Effects of dietary conjugated linoleic acid (CLA, 1% mixed isomers) on n-6 long-chain polyunsaturated fatty acid (LCPUFA) oxidation and biosynthesis were investigated in liver and brain tissues of neonatal piglets. Fatty acid ß-oxidation was measured in tissue homogenates using [1-(14)C]linoleic acid (LA) and -arachidonic acid (ARA) substrates, while fatty acid desaturation and elongation were traced using [U-(13)C]LA and GC-MS. Dietary CLA had no effect on fatty acid ß-oxidation, but significantly decreased n-6 LCPUFA biosynthesis by inhibition of LA elongation and desaturation. Differences were noted between our (13)C tracer assessment of desaturation/elongation and simple precursor-product indices computed from fatty acid composition data, indicating that caution should be exercised when employing the later. The inhibitory effects of CLA on elongation/desaturation were more pronounced in pigs fed a low fat diet (3% fat) than a high fat diet (25% fat). Direct elongation of linoleic acid to C20:2n-6 via the alternate elongation pathway might play an important role in n-6 LCPUFA synthesis because more than 40% of the synthetic products of [U-(13)C]LA accumulated in [(13)C]20:2n-6. Overall, the data show that dietary CLA shifted the distribution of the synthetic products of [U-(13)C]LA between elongation and desaturation in liver and decreased the total synthetic products of [U-(13)C]LA in brain by inhibiting LA elongation to C20:2n-6. The impact of CLA on brain LCPUFA metabolism of the developing neonate merits consideration and further investigation.

Enrichment of intestinal mucosal phospholipids with arachidonic and eicosapentaenoic acids fed to suckling piglets is dose and time dependent.

Infant formula companies began fortifying formulas with long-chain PUFA in 2002, including arachidonic acid (ARA) at approximately 0.5% of total fatty acids. The primary objective of this study was to determine the time-specific effects of feeding formula enriched with supra-physiologic ARA on fatty acid composition of intestinal mucosal phospholipids. One-day-old pigs (n = 96) were fed a milk-based formula for 4, 8, or 16 d. Diets contained either no PUFA (0% ARA, negative control), 0.5% ARA, 2.5% ARA, 5% ARA, or 5% eicosapentaenoic acid (EPA) of total fatty acids (wt:wt). Growth (299 +/- 21 g/d) and clinical hematology were unaffected by treatment (P > 0.6). Although minimal on d 4, concentrations of ARA in jejunal mucosa were enriched 47, 272 and 428% by d 8 and 144, 356, and 415% by d 16 in pigs fed the 0.5% ARA, 2.5% ARA, and 5% ARA diets, respectively, compared with the 0% ARA control pigs (P < 0.01). On d 16, ARA enrichment increased progressively with increasing dietary ARA supplementation from 0 to 2.5% but plateaued as dietary ARA rose to 5%. A similar pattern of ARA enrichment was observed in ileal mucosal phospholipids, but maximal enrichment in the ileum exceed that in the jejunum by >50%. As ARA increased, linoleic acid content decreased reciprocally. Although maximal enterocyte enrichment with EPA approached 20-fold by d 8, concentrations were only approximately 50% of those attained for ARA. Negligible effects on gross villus/crypt morphology were observed. These data demonstrate a dose-dependent response of intestinal mucosal phospholipid ARA concentration to dietary ARA with nearly full enrichment attained within 8 d of feeding formula containing ARA at 2.5% of total fatty acids and that supra-physiologic supplementation of ARA is not detrimental to growth.