Feeding Mode, but Not Prebiotics, Affects Colonic Microbiota Composition and Volatile Fatty Acid Concentrations in Sow-Reared, Formula-Fed, and Combination-Fed Piglets.

BACKGROUND: Many infants consume both human milk and infant formula (combination-fed); however, little is known about how combination-feeding affects the gut microbiota or prebiotic fermentation compared to formula feeding. OBJECTIVES: We investigated the impact of feeding mode and prebiotics on bacterial colonization and volatile fatty acid (VFA) concentrations. METHODS: Newborn piglets (Large White and Landrace) were randomly assigned to 5 groups (n = 6/group): formula-fed (FF), formula-fed with prebiotics (FP), sow-reared (SR), combination-fed (CF), and combination-fed with prebiotics (CP). SR piglets remained with the sows 24 h/d. FF and FP were fed formula or formula with galactooligosaccharide and inulin (4 g/L in a 4:1 ratio). CF and CP were sow-reared for 5 d and then rotated between the sow and formula-feeding every 12 h. Ascending colon contents were collected at day 21. The microbiota was analyzed by pyrosequencing and denaturing gradient gel electrophoresis (DGGE). VFAs were determined by gas chromatography. RESULTS: Distance-based redundancy analysis of DGGE and pyrosequencing data separated microbiota of FF from CF and SR. CF differed from SR by DGGE, but only a trend (P = 0.09) by pyrosequencing. Bacterial composition of CF was more similar to SR than FF. No bacterial genera in CF significantly differed from SR; however, 9 genera differed between CF and FF, including Lactobacillus, Clostridium XIVa, and Fusobacterium. VFA concentrations were similar between CF and SR, while isovalerate and isobutyrate were 2-fold greater (P < 0.05) in CF than FF. Neither microbiota nor VFA profile was affected by prebiotic supplementation. CONCLUSIONS: Microbial colonization patterns and VFA profiles of CF piglets were more similar to SR piglets than FF piglets. Prebiotics did not affect piglet bacterial composition and/or VFA concentrations relative to the main feeding modes (FF and CF). Thus, partial exposure to breast milk can be beneficial for microbiota development of FF neonates.

Herring roe oil supplementation alters microglial cell gene expression and reduces peripheral inflammation after immune activation in a neonatal piglet model.

Neonatal brain development can be disrupted by infection that results in microglial cell activation and neuroinflammation. Studies indicate that polyunsaturated fatty acids (PUFAs) and their metabolites can resolve inflammation. It is not known if dietary PUFA increases lipid metabolites in brain or reduces neuroinflammation in neonates. We hypothesized that dietary PUFAs might suppress neuroinflammation by inhibiting pro-inflammatory cytokine over-production and promoting inflammatory resolution in the periphery and brain. Piglets were obtained on postnatal day (PD) 2 and randomly assigned to herring roe oil (HRO) or control (CON) diet. HRO was included at 2 g/kg powdered diet. HRO increased DHA levels in occipital lobe and the DHA to arachidonic acid (ARA) ratio in hippocampal tissue. HRO decreased ARA metabolites in occipital lobe. HRO failed to attenuate microglial pro-inflammatory cytokine production ex vivo. HRO did not affect fever or circulating resolvin D1 levels. HRO decreased circulating neutrophils and liver inflammatory gene expression, but increased resolution marker gene expression in liver post LPS. HRO upregulated CXCL16, TGFBR1, and C1QA in microglial cells. HRO supplementation exerted beneficial effects on inflammation in the periphery, but further studies are needed to evaluate the specific effects of omega-3 supplementation on microglial cell physiology in the neonate.

Modeling the Impact of Adding a Serving of Dairy Foods to the Healthy Mediterranean-Style Eating Pattern Recommended by the 2015-2020 Dietary Guidelines for Americans.

OBJECTIVE: The Healthy Mediterranean-Style Eating Pattern (HMEP) in the 2015 Dietary Guidelines for Americans (DGA) recommends that adults eating less than 2400 kcal a day consume only two daily servings (or cup-equivalents) of low-fat or fat-free dairy foods like milk, cheese, and yogurt, which does not provide enough calcium, potassium, and vitamin D to meet dietary reference intakes (DRIs). Our objective was to assess the impact of additional servings of dairy foods on the nutrient adequacy of the 1600, 2000, and 2400 kcal HMEP in the 2015 DGA. METHODS: Using the same food pattern modeling procedures as the 2015 DGA, we assessed the nutrient composition of three alternative models of the 1600, 2000, and 2400 kcal HMEP. For Model 1, we increased servings of dairy foods (77 kcal/serving). For Model 2, we added one serving of dairy foods and removed one serving of refined grains (85 kcal/serving), and for Model 3 (2400 kcal HMEP only), we added one-half serving of dairy foods and removed one-half serving of refined grains. We then assessed these models for nutrient adequacy and compared them to the Healthy U.S.-Style Eating Pattern and the HMEP. RESULTS: The changes to the HMEP with these models increased the amounts of several nutrients to encourage, including calcium, vitamin D, potassium, vitamin A, phosphorus, riboflavin, vitamin B12, zinc, and magnesium. For instance, Model 1 increased the calcium (by 295 mg), vitamin D (by 59.3 IU), potassium (by 235 mg), vitamin A (by 98 mcg), and phosphorus (by 232 mg) content of the original HMEP, and Model 3 increased the amounts of these nutrients by half of those amounts. Model 2 increased the calcium content by 266 mg, vitamin D by 58 IU, potassium by 202 mg, vitamin A by 88 mcg, and phosphorus by 193 mg. Notably, Models 1 and 2 increased the vitamin D content of the HMEP to about 62% of the DRI (average across all calorie levels) and the potassium content to 78% of the DRI (average across all calorie levels), from 52% and 73%, respectively, in the original HMEP. Most of our models increased the saturated fat (0.5 g in Model 1 and 0.2 g in Model 2) and sodium (202 mg in Model 1 and 101 mg in Model 2) content as well. The amounts of these nutrients to limit remained within the ranges recommended in the 2015 DGA. CONCLUSIONS: The addition of a dairy food serving to the 1600, 2000, and 2400 kcal HMEP brings their nutrient profiles closer to the DRIs for several nutrients to encourage, including calcium, vitamin D, and potassium.

Altered Hippocampal Gene Expression and Morphology in Fetal Piglets following Maternal Respiratory Viral Infection.

Maternal infection during pregnancy increases the risk of neurobehavioral problems in offspring. Evidence from rodent models indicates that the maternal immune response to infection can alter fetal brain development, particularly in the hippocampus. However, information on the effects of maternal viral infection on fetal brain development in gyrencephalic species is limited. Thus, the objective of this study was to assess several effects of maternal viral infection in the last one-third of gestation on hippocampal gene expression and development in fetal piglets. Pregnant gilts were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) at gestational day (GD) 76 and the fetuses were removed by cesarean section at GD 111 (3 days before anticipated parturition). The gilts infected with PRRSV had elevated plasma interleukin-6 levels and developed transient febrile and anorectic responses lasting approximately 21 days. Despite having a similar overall body weight, fetuses from the PRRSV-infected gilts had a decreased brain weight and altered hippocampal gene expression compared to fetuses from control gilts. Notably, maternal infection caused a reduction in estimated neuronal numbers in the fetal dentate gyrus and subiculum. The number of proliferative Ki-67+ cells was not altered, but the relative integrated density of GFAP+ staining was increased, in addition to an increase in GFAP gene expression, indicating astrocyte-specific gliosis. Maternal viral infection caused an increase in fetal hippocampal gene expression of the inflammatory cytokines TNF-α and IFN-γ and the myelination marker myelin basic protein. MHCII protein, a classic monocyte activation marker, was reduced in microglia, while expression of the MHCII gene was decreased in hippocampal tissue of the fetuses from PRRSV-infected gilts. Together, these data suggest that maternal viral infection at the beginning of the last trimester results in a reduction in fetal hippocampal neurons that is evident 5 weeks after infection, when fetal piglets are near full term. The neuronal reduction was not accompanied by pronounced neuroinflammation at GD 111, indicating that any activation of classic neuroinflammatory pathways by maternal viral infection, if present, is mostly resolved by parturition.

Maternal viral infection during pregnancy elicits anti-social behavior in neonatal piglet offspring independent of postnatal microglial cell activation.

Maternal infection during pregnancy increases risk for neurodevelopmental disorders and reduced stress resilience in offspring, but the mechanisms are not fully understood. We hypothesized that piglets born from gilts infected with a respiratory virus during late gestation would exhibit aberrant microglia activity, cognitive deficits and reduced sociability. Pregnant gilts were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV; 5×105 TCID50 of live PRRSV) or saline at gestational day 76. Gilts infected with PRRSV exhibited fever (p<0.01) and reduced appetite (p<0.001) for 2weekspost-inoculation and were PRRSV-positive at parturition. Piglets born from infected and control gilts were weaned at postnatal day (PD) 1 and assigned to two groups. Group 1 was challenged with lipopolysaccharide (LPS, 5µg/kg body weight i.p.) or saline on PD 14 and tissues were collected. Group 2 was tested in a T-maze task to assess spatial learning and in a 3-chamber arena with unfamiliar conspecifics to assess social behavior from PD 14-27. Microglia (CD11b+ CD45low) isolated from Group 2 piglets at PD 28 were challenged ex vivo with LPS; a subset of cells was analyzed for MHCII expression. Maternal infection did not affect offspring circulating TNFα, IL-10, or cortisol levels basally or 4h post-LPS challenge. While performance in the T-maze task was not affected by maternal infection, both sociability and preference for social novelty were decreased in piglets from infected gilts. There was no effect of maternal infection on microglial MHCII expression or LPS-induced cytokine production. Taken together, these results suggest the reduced social behavior elicited by maternal infection is not due to aberrant microglia activity postnatally.

Postnatal Iron Deficiency Alters Brain Development in Piglets.

BACKGROUND: Cognitive deficits associated with postnatal iron deficiency (ID) suggest abnormal brain development, but little is known about animals with gyrencephalic brains. OBJECTIVE: The objective was to assess the impact of ID on brain development in piglets. METHODS: Male and female Yorkshire piglets were reared from postnatal day (PD) 2 until PD 29 or 30 by using milk replacer adequate [control (CON)] or deficient (100 compared with 10 mg/kg) in iron and subjected to MRI to assess brain macrostructure, microstructure, and metabolites in the dorsal hippocampi and intervening space. After MRI, brains were collected for histology. Hematocrit, hemoglobin, and liver iron were measured to determine iron status. RESULTS: Hematocrit and hemoglobin in ID piglets were less than CON after PD 14 (P < 0.001), and at the study end liver iron in ID piglets was less than CON (P < 0.001). Brain region volumes were not affected by ID, but changes in brain composition were evident. ID piglets had less white matter in 78,305 voxels, with large clusters in the hippocampus and cortex. ID piglets had less gray matter in 13,625 voxels primarily in cortical areas and more gray matter in 28,017 voxels, most notably in olfactory bulbs and hippocampus. The major effect of ID on white matter was supported by lower fractional anisotropy values in the corpus callosum (0.300 compared with 0.284, P = 0.006) and in whole brain white matter (0.313 compared with 0.307, P = 0.002) in ID piglets. In coronal brain sections, corpus callosum width was less (P = 0.043) in ID piglets. Inositol was lower (P = 0.01) and phosphocholine was higher (P = 0.03) in hippocampus of ID piglets. CONCLUSIONS: Postnatal ID in piglets affects brain development, especially white matter. If the effects of ID persist, it might explain the lasting detrimental effects on cognition.

Early supplementation of phospholipids and gangliosides affects brain and cognitive development in neonatal piglets.

BACKGROUND: Because human breast milk is a rich source of phospholipids and gangliosides and breastfed infants have improved learning compared with formula-fed infants, the importance of dietary phospholipids and gangliosides for brain development is of interest. OBJECTIVE: We sought to determine the effects of phospholipids and gangliosides on brain and cognitive development. METHODS: Male and female piglets from multiple litters were artificially reared and fed formula containing 0% (control), 0.8%, or 2.5% Lacprodan PL-20 (PL-20; Arla Foods Ingredients), a phospholipid/ganglioside supplement, from postnatal day (PD) 2 to PD28. Beginning on PD14, performance in a spatial T-maze task was assessed. At PD28, brain MRI data were acquired and piglets were killed to obtain hippocampal tissue for metabolic profiling. RESULTS: Diet affected maze performance, with piglets that were fed 0.8% and 2.5% PL-20 making fewer errors than control piglets (80% vs. 75% correct on average; P < 0.05) and taking less time to make a choice (3 vs. 5 s/trial; P < 0.01). Mean brain weight was 5% higher for piglets fed 0.8% and 2.5% PL-20 (P < 0.05) than control piglets, and voxel-based morphometry revealed multiple brain areas with greater volumes and more gray and white matter in piglets fed 0.8% and 2.5% PL-20 than in control piglets. Metabolic profiling of hippocampal tissue revealed that multiple phosphatidylcholine-related metabolites were altered by diet. CONCLUSION: In summary, dietary phospholipids and gangliosides improved spatial learning and affected brain growth and composition in neonatal piglets.

Mode of delivery and early nutrition modulate microbial colonization and fermentation products in neonatal piglets.

Colonization of the intestinal microbiota after birth plays an important role in development of the neonatal gastrointestinal and immune systems. Two key environmental factors that influence the colonization pattern are delivery mode and nutrition. In this study, the impact of delivery mode and nutrition on microbial colonization and metabolic activity was investigated in the pig model. Vaginally (VD) or caesarean- (CD) delivered piglets were sow-reared (SR) or fed formula alone (FF) or with 4 g/L prebiotics [1:1 ratio of short-chain fructo-oligosaccharides (scFOS) and polydextrose (PDX); FP]. Intestinal contents were collected on d 7 and 14. SR piglets harbored different microbial populations from FF and FP piglets in ileum and ascending colon (AC). On d 7, FF piglets had a greater abundance of Clostridium XIVa in AC, but lower total bacteria, Clostridium XIVa, and Lactobacillus spp. in ileum and Fecalibacterium prausnitzii in AC compared with FP piglets. On d 14, total bacteria were more abundant in FP than FF piglets. Butyrate, isobutyrate, valerate, and isovalerate concentrations in AC were greater in SR piglets compared with FF or FP piglets. At both sampling days, acetate concentrations in AC were similar between the SR and FF groups, whereas propionate was higher in the SR compared with FF group. Delivery mode also significantly affected microbial populations. Bacterial densities differed in AC for Bacteroides-Prevotella at d 7 and Clostridium XIVa at d 14, being higher in VD piglets. Correspondingly, VD piglets had higher propionate in ileum and propionate and butyrate in AC compared with CD piglets. Our results indicate that both delivery mode and nutrition affect microbial composition and metabolic activity. Supplementation of scFOS/PDX to formula modulates microbial colonization and produces a SCFA pattern closer to that of SR piglets.

Combination-Feeding Causes Differences in Aspects of Systemic and Mucosal Immune Cell Phenotypes and Functions Compared to Exclusive Sow-Rearing or Formula-Feeding in Piglets.

Combination feeding (human milk and formula) is common and influences immune development compared to exclusive breastfeeding. Infant formulas contain prebiotics, which influence immune development. Herein, immune development of combination-fed (CF), sow-reared (SR) and formula-fed (FF) piglets, and the effect of prebiotics was tested. Piglets (n = 47) were randomized to: SR, FF, CF, FF+prebiotic (FP), and CF+prebiotic (CP). FP and CP received formula with galactooligosaccharides and inulin (4 g/L in a 4:1 ratio). CF and CP piglets were sow-reared for until d5 and then rotated between a sow and formula every 12 h. On day 21, piglets received an intraperitoneal injection of lipopolysaccharide 2 h prior to necropsy. Immune cells from blood, mesenteric lymph nodes (MLN), and spleen were phenotyped. Classical (nitric oxide synthase) and alternative (arginase activity) activation pathways were measured in isolated macrophages. Serum IL-6 and TNF-α were measured by ELISA. SR piglets had lower (p < 0.0001) CD4+ T-helper cells and higher (p < 0.0001) B-cells in PBMC than all other groups. CP piglets had higher (p < 0.0001) arginase activity compared to all other groups. FF piglets had higher (p < 0.05) IL-6 compared to both CF and SR, but were similar to FP and CP. Thus, CF, with or without prebiotics, differentially affected immunity compared to exclusively fed groups.

Hydrolyzed Fat Formula Increases Brain White Matter in Small for Gestational Age and Appropriate for Gestational Age Neonatal Piglets.

Background: Intrauterine growth restriction is a common cause of small for gestational age (SGA) infants worldwide. SGA infants are deficient in digestive enzymes required for fat digestion and absorption compared to appropriate for gestational age (AGA) infants, putting them at risk for impaired neurocognitive development. Objective: The objective was to determine if a hydrolyzed fat (HF) infant formula containing soy free fatty acids, 2-monoacylglycerolpalmitate, cholesterol, and soy lecithin could increase brain tissue incorporation of essential fatty acids or white matter to enhance brain development in SGA and AGA neonatal piglet models. Methods: Sex-matched, littermate pairs of SGA (0.5-0.9 kg) and AGA (1.2-1.8 kg) 2 days old piglets (N = 60) were randomly assigned to control (CON) or HF formula diets in a 2 × 2 factorial design. On day 14, 24 piglets were used for hippocampal RNA-sequencing; the rest began a spatial learning task. On days 26-29, brain structure was assessed by magnetic resonance imaging (MRI). Cerebellum and hippocampus were analyzed for fatty acid content. Results: SGA piglets grew more slowly than AGA piglets, with no effect of diet on daily weight gain or weight at MRI. HF diet did not affect brain weight. HF diet increased relative volumes of 7 brain regions and white matter (WM) volume in both SGA and AGA piglets. However, HF did not ameliorate SGA total WM integrity deficits. RNA sequencing revealed SGA piglets had increased gene expression of synapse and cell signaling pathways and decreased expression of ribosome pathways in the hippocampus compared to AGA. HF decreased expression of immune response related genes in the hippocampus of AGA and SGA piglets, but did not correct gene expression patterns in SGA piglets. Piglets learned the T-maze task at the same rate, but SGA HF, SGA CON, and AGA HF piglets had more accurate performance than AGA CON piglets on reversal day 2. HF increased arachidonic acid (ARA) percentage in the cerebellum and total ARA in the hippocampus. Conclusions: HF enhanced brain development in the neonatal piglet measured by brain volume and WM volume in specific brain regions; however, more studies are needed to assess long-term outcomes.

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.

A neonatal piglet model for investigating brain and cognitive development in small for gestational age human infants.

The piglet was investigated as a potential model for studying brain and cognitive deficits associated with being born small for gestational age (SGA). Naturally farrowed SGA (0.7-1.0 kg BW) and average for gestational age (AGA, 1.3-1.6 kg BW) piglets were obtained on postnatal day (PD) 2, placed in individual cages, and provided a nutritionally adequate milk replacer diet (285 ml/kg/d). Beginning at PD14, performance in a spatial T-maze task was assessed. At PD28, piglets were anesthetized for magnetic resonance (MR) imaging to assess brain structure (voxel-based morphometry), connectivity (diffusion-tensor imaging) and metabolites in the hippocampus and corpus callosum (proton MR spectroscopy). Piglets born SGA showed compensatory growth such that BW of SGA and AGA piglets was similar (P>0.05), by PD15. Birth weight affected maze performance, with SGA piglets taking longer to reach criterion than AGA piglets (p<0.01). Total brain volume of SGA and AGA piglets was similar (P<0.05), but overall, SGA piglets had less gray matter than AGA piglets (p<0.01) and tended to have a smaller internal capsule (p = 0.07). Group comparisons between SGA and AGA piglets defined 9 areas (≥ 20 clusters) where SGA piglets had less white matter (p<0.01); 2 areas where SGA piglets had more white matter (p<0.01); and 3 areas where SGA piglets had more gray matter (p<0.01). The impact of being born SGA on white matter was supported by a lower (p<0.04) fractional anisotropy value for SGA piglets, suggesting reduced white matter development and connectivity. None of the metabolites measured were different between groups. Collectively, the results show that SGA piglets have spatial learning deficits and abnormal development of white matter. As learning deficits and abnormalities in white matter are common in SGA human infants, the piglet is a tractable translational model that can be used to investigate SGA-associated cognitive deficits and potential interventions.

Perinatal iron deficiency and neurocognitive development.

Iron deficiency is the most common form of nutrient deficiency worldwide. It is highly prevalent due to the limited availability of high quality food in developing countries and poor dietary habits in industrialized countries. According to the World Health Organization, it affects nearly 2 billion people and up to 50% of women who are pregnant. Maternal anemia during pregnancy is especially burdensome to healthy neurodevelopment in the fetus because iron is needed for proper neurogenesis, development, and myelination. Maternal anemia also increases the risk of low birth weight, either due to premature birth or fetal growth restriction, which is associated with delayed neurocognitive development and even psychiatric illness. As rapid neurodevelopment continues after birth infants that received sufficient iron in utero, but that receive a low iron diet after 6 months of age, also show deficits in neurocognitive development, including impairments in learning and memory. Unfortunately, the neurocognitive complications of iron deficiency during critical pre- and postnatal periods of brain development are difficult to remedy, persisting into adulthood. Thus, preventing iron deficiency in the pre- and postnatal periods is critical as is devising new means to recapture cognitive function in individuals who experienced early iron deficiency. This review will discuss the prevalence of pre- and postnatal iron deficiency, the mechanism, and effects of iron deficiency on brain and cognitive development.