Formula-fed Piglets Research Articles

323 Advances in using membrane vesicles for studying intestinal functions

Abstract Enterocytes are the major cell lineage of the small intestinal epithelia along the crypt-villus axis in gut mucosa. Enterocytes participate in the gut terminal nutrient digestion and absorption; and have essential roles in nutrient and metabolite recycling and efflux, nutrient and hormonal sensing, interactions with symbiotic microbiome and binding to infectious viruses, thus influencing the host homeostasis and whole-body physiological status. Intestinal membrane vesicles have been widely used for studying various gut functions in the past several decades. Villus enterocytic brush border membrane and the apical membrane vesicles along the crypt-villus axis in gut mucosa can be prepared and isolated by Mg2+- and Ca2+-precipitation and differential centrifugation procedures. Whereas, gut mucosal basolateral membrane vesicles can be prepared, fractionated and differentiated from other intracellular organelles such as the mitochondrial inner membrane by the Percoll gradient ultracentrifugation. Nutrient transport and exchange activities in the membrane vesicles in vitro are conducted often by rapid hand filtration, and in excess, in an automated fast sampling apparatus. Nutrient transport and exchange activity kinetics are derived according to the well-established classic Michaelis-Menten and the tracer inhibitory kinetic models with a simple linear diffusional component. Physiological and biochemical properties associated with various nutrient uptake systems such as pH optimum, requirements for co-transported ions (e.g., Na+, K+, Cl-), transporter affinity and substrate specificity can be effectively investigated in vitro with the membrane vesicles by altering the compositions of uptake buffers and intra-vesicular pre-loading buffers. Some of the fundamental gut functions are established via using the membrane vesicles in different mammalian species. And these include H+-peptide cotransport, Na+-cotransport and Na+-independent systems of hexose uptake across the apical membrane and the low-affinity Na+-independent hexose bidirectional uptake across the basolateral membrane that were characterized in rodents; Na+-dependent cotransport and Na+-independent systems of amino acid (AA) transport across the apical membrane that were revealed in rodents and pigs; as well as the Na+-hexose and AA cotransport and the co-expression of the coronavirus SARS-CoV-2 host epithelial apical membrane binding receptor, i.e., the angiotensin-converting enzyme-2 (ACE2), along the gut crypt-villus axis that were identified in liquid formula-fed piglets. In the post genomic and metagenomic sequencing era, further research efforts need to pursue understanding of expression and various functionality of the gut terminal hydrolases, nutrient transporters, nutrient exchangers, nutrient sensors, substrate and particle receptors, taste receptors, viral binding receptors such as ACE2 and hormonal receptors and their interactions with luminal factors such as nutrient, metabolites, symbiotic bacterial outer membrane vesicles etc., along the longitudinal and crypt-villus axes, through using the mammalian primary enterocytic apical and the basolateral membrane vesicles under various research settings.

Gut Microbiota-Derived 5-Hydroxyindoleacetic Acid Alleviates Diarrhea in Piglets via the Aryl Hydrocarbon Receptor Pathway.

With the improvement in sow prolificacy, formula feeding has been increasingly used in the pig industry. Diarrhea remains a serious health concern in formula-fed (FF) piglets. Fecal microbiota transplantation (FMT) is an efficacious strategy to reshape gut microbiota and the metabolic profile for treating diarrhea. This study aims to investigate whether FMT from breast-fed piglets could alleviate diarrhea in FF piglets. The piglets were randomly assigned to the control (CON) group, FF group, and FMT group. Our results showed that FF piglets exhibited a higher diarrhea incidence, damaged colonic morphology, and disrupted barrier function. In contrast, FMT treatment normalized the morphology and barrier function. FMT suppressed the JNK/MAPK pathway and production of proinflammatory cytokines. Additionally, FF piglets had a lower abundance of the beneficial bacterial genus Bifidobacterium compared to CON piglets. Following FMT administration, Bifidobacterium was restored. Meanwhile, 5-HIAA, a metabolite of tryptophan, and AHR-responsive CYP1A1 and CYP1B1 were upregulated. Importantly, integrated multiomics analysis revealed a strong positive correlation between Bifidobacterium and 5-HIAA. In vitro, 5-HIAA supplementation reversed the LPS-induced disruption of tight junctions and production of proinflammatory cytokines in IPEC-J2 cells. In conclusion, FMT reduced diarrhea incidence and improved growth performance. The alleviative effect of FMT on diarrhea was associated with Bifidobacterium and 5-HIAA.

Evaluation of a Plant-Based Infant Formula Containing Almonds and Buckwheat on Gut Microbiota Composition, Intestine Morphology, Metabolic and Immune Markers in a Neonatal Piglet Model.

A controlled-neonatal piglet trial was conducted to evaluate the impact of a plant-based infant formula containing buckwheat and almonds as the main source of protein compared to a commercially available dairy-based formula on the gut health parameters. Two day old piglets were fed either a plant-based or a dairy-based formula until day 21. Gut microbiome, cytokines, growth and metabolism related outcomes, and intestinal morphology were evaluated to determine the safety of the plant-based infant formula. This study reported that the plant-based formula-fed piglets had a similar intestinal microbiota composition relative to the dairy-based formula-fed group. However, differential abundance of specific microbiota species was detected within each diet group in the small and large intestinal regions and fecal samples. Lactobacillus delbrueckii, Lactobacillus crispatus, and Fusobacterium sp. had higher abundance in the small intestine of plant-based formula-fed piglets compared to the dairy-based group. Bacteroides nordii, Enterococcus sp., Lactobacillus crispatus, Prevotella sp., Ruminococcus lactaris, Bacteroides nordii, Eisenbergiella sp., Lactobacillus crispatus, Prevotella sp., and Akkermansia muciniphila had greater abundance in the large intestine of the plant based diet fed piglets relative to the dairy-based diet group. In the feces, Clostridiales, Bacteroides uniformis, Butyricimonasvirosa, Cloacibacillus porcorum, Clostridium clostridioforme, and Fusobacterium sp. were abundant in dairy-based group relative to the plant-based group. Lachnospiraceae, Clostridium scindens, Lactobacillus coleohominis, and Prevetolla sp. had greater abundance in the feces of the plant-based group in comparison to the dairy-based group. Gut morphology was similar between the plant and the dairy-based formula-fed piglets. Circulatory cytokines, magnesium, triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH), vitamin D, vitamin K, and IgE levels were similar among all piglets independent of dietary group. Overall, the present study demonstrated that a plant-based formula with buckwheat and almonds as the primary source of protein can support similar gut microbiota growth and health outcomes compared to a dairy-based infant formula.

Distinct Gut Microbiome Induced by Different Feeding Regimes in Weaned Piglets.

It is well accepted that the gut microbiota of breast-fed (BF) and formula-fed (FF) infants are significantly different. However, there is still a limited number of studies comparing the gut microbiota of BF and FF piglets, despite increasing numbers of FF piglets in the modern pig industry. The present study identified the differences in gut microbiota composition between BF- and FF-weaned Rongchang piglets at 30 days old, using pair-end sequencing on the Illumina HiSeq 2500 platform. The BF piglets had lower microbiota diversities than FF piglets (p < 0.05), and the community structures were well clustered as a result of each feeding pattern. Firmicutes and Bacteroidetes represented the most dominant phyla, and Ruminococcus, Prevotella, and Gemmiger were prominent genera in all piglets. Ruminococcus, Prevotella, Oscillospira, Eubacterium, Gemmiger, Dorea, and Lactobacillus populations were significantly higher, while Treponema and Coprococcus were significantly lower in BF piglets compared to FF piglets (p < 0.05). The metabolism pathways in the BF piglets were significantly different from FF piglets, which included carbohydrate and amino acid metabolism (p < 0.05). In addition, the top 10 abundance of microbiota were more or less significantly associated with the two phenotypes (p < 0.05). Collectively, these findings provide probable explanations for the importance of BF in neonates and support a theoretical basis for feeding regimes in indigenous Chinese piglets.

Blood transcriptomic markers of necrotizing enterocolitis in preterm pigs.

Necrotizing enterocolitis (NEC), a severe gut disorder in preterm infants, is difficult to predict due to poor specificity and sensitivity of clinical signs and biomarkers. Using preterm piglets as a model, we hypothesized that early development of NEC affects blood gene expression, potentially related to early systemic immune responses. A retrospective analysis of clinical, tissue, and blood data was performed on 129 formula-fed piglets with NEC diagnosis at necropsy on day 5. Subgroups of NEC (n = 20) and control piglets (CON, n = 19) were analyzed for whole-blood transcriptome. Preterm piglets had variable NEC lesions, especially in the colon region, without severe clinical signs (e.g. normal growth, activity, hematology, digestion, few piglets with bloody stools). Transcriptome analysis showed 344 differentially expressed genes (DEGs) between NEC and CON piglets. Validation experiment showed that AOAH, ARG2, FKBP5, PAK2, and STAT3 were among the genes affected by severe lesions on day 5, when analyzed in whole blood and in dried blood spots (DBS). Whole-blood gene expressions may be affected in preterm pigs before clinical signs of NEC get severe. Blood gene expression analysis, potentially using DBS samples, is a novel tool to help identify new early biomarkers of NEC. Preterm pig model was used to investigate if blood transcriptomics could be used to identify new early blood biomarkers of NEC progression. Whole-blood transcriptome revealed upregulation of target genes in NEC cases when clinical symptoms are subtle, and mainly colon regions were affected. Differential NEC-associated gene expressions could be detected also in dried blood spots, potentially allowing easy collection of small blood volumes in infants.

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.

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

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. We investigated the impact of feeding mode and prebiotics on bacterial colonization and volatile fatty acid (VFA) concentrations. 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. 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. 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.

Effect of milk protein composition and amount of β-casein on growth performance, gut hormones, and inflammatory cytokines in an in vivo piglet model

The objective of this work was to better understand the effect of differences in milk protein composition, and specifically, a change in β-casein to total casein in a milk-based matrix, on growth performance and metabolic and inflammatory responses using a piglet model. Three formulas were optimized for piglets, with similar metabolizable energy, total protein content, and other essential nutrients. Only the protein type and ratio varied between the treatments: the protein fraction of the control diet contained only whey proteins, whereas 2 other matrices contained a whey protein to casein ratio of 60:40, and differed in the amount of β-casein (12.5 and 17.1% of total protein). Piglets fed formula containing whey proteins and caseins, regardless of the concentration of β-casein, showed a significantly higher average daily gain, average daily feed intake, and feed efficiency compared with piglets consuming the formula with only whey protein. Consumption of the formula containing only whey protein showed higher levels of plasma glucagon-like peptide-1 and ghrelin compared with the consumption of formula containing casein and whey protein. A positive correlation was observed between postprandial time and glucagon-like peptide-1 response. The intestinal pro-inflammatory cytokine tumor necrosis factor α increased significantly in piglets fed the whey protein/casein diet compared with those fed whey protein formula. All formula-fed piglets showed a lower level of IL-6 cytokine compared with the ad libitum sow-fed piglets, regardless of composition. No significant differences in the anti-inflammatory IL-10 concentration were observed between treatment groups. Milk protein composition contributed to the regulation of piglets' metabolic and physiological responses, with whey protein/casein formula promoting growth performance and a different immune regulatory balance compared with a formula containing only whey protein. Results indicated no differences between treatments containing different levels of β-casein.

Serum lactoferrin concentration of primiparous sow during gestation and lactation, and comparison between sow-fed and formula-fed piglets.

Lactoferrin (LF) is a sialylated iron-binding glycoprotein, occurring in several biological secretions like milk, saliva, and seminal fluids and is a major component of a mammalian innate immune system. It plays multiple protective roles against large group of microorganisms and performs anti-inflammatory and anti-cancer activities. The concentration of serum LF in gilt (primiparous sow) and their piglets remains unknown. We determined serum LF concentration in gilts during gestation and lactation to that of 19-d-old piglets, including sow-fed and formula-fed piglets using enzyme-linked immunosorbent assay (ELISA). We found that the concentration of serum LF in gilts varied during gestation (0.77 ± 0.10 µg/mL) and lactation (0.62 ± 0.11 µg/mL). The mean concentration of serum LF in gilts (0.72 ± 0.06 µg/mL) was significantly higher than that of piglets (0.42 ± 0.07 µg/mL, P = 0.004). Additionally, a marginal significant difference (P =0.06) was observed for serum LF concentration in sow-fed piglets (0.42 ± 0.03 µg/mL) at 19 d old compared to that of formula-fed piglets (0.33 ± 0.04 µg/mL) at 37 d old. This study provides noble information regarding the serum LF concentration in the healthy gilts and piglets and thereby the data can be used as a standard reference point for future studies on the role of LF in pig reproduction.

Formula diet alters small intestine morphology, microbial abundance and reduces VE-cadherin and IL-10 expression in neonatal porcine model.

BackgroundBreastfeeding is associated with a variety of positive health outcomes in children and is recommended exclusively for the first 6 months of life; however, 50–70 % of infants in the US are formula-fed. To test the hypothesis that immune system development and function in neonates and infants are significantly influenced by diet, 2-day old piglets were fed soy or milk formula (n = 6/group/gender) until day 21 and compared to a sow-fed group (n = 6/gender).MethodsHistomorphometric analyses of ileum, jejunum and Peyer’s patches were carried out, to determine the inflammation status, mRNA and protein expression of pro-inflammatory, anti-inflammatory and growth-related chemokines and cytokines.ResultsIn formula-fed animals, increases in ileum and jejunum villus height and crypt depth were observed in comparison to sow-fed animals (jejunum, p < 0.01 villus height, p < 0.04 crypt depth; ileum p < 0.001 villus height, p < 0.002 crypt depth). In formula-fed the lymphoid follicle size (p < 0.01) and germinal centers (p < 0.01) with in the Peyer’s patch were significantly decreased in comparison to sow-fed, indicating less immune education. In ileum, formula diet induced significant up-regulation of AMCFII, IL-8, IL-15, VEGFA, LIF, FASL, CXCL11, CCL4, CCL25 and down-regulation of IL-6, IL-9, IL-10, IL-27, IFNA4, CSF3, LOC100152038, and LOC100736831 at the transcript level. We have confirmed some of the mRNA data by measuring protein, and significant down-regulation of anti-inflammatory molecule IL-10 in comparison to sow-fed piglets was observed. To further determine the membrane protein expression in the ileum, VE-cadherin, occludin, and claudin-3, Western blot analyses were conducted. Sow fed piglets showed significantly more VE-Cadherin, which associated with levels of calcium, and putrescine measured. It is possible that differences in GI tract and immune development are related to shifts in the microbiome; notably, there were 5-fold higher amounts of Lactobacillaceae spp and 3 fold higher Clostridia spp in the sow fed group in comparison to milk formula-fed piglets, whereas in milk formula-fed pigs Enterobacteriaceae spp was 5-fold higher.ConclusionIn conclusion, formula diet alters GI morphology, microbial abundance, intestinal barrier protein VE-cadherin and anti-inflammatory molecule IL-10 expression. Further characterization of formula effects could lead to modification of infant formula to improve immune function, reduce inflammation and prevent conditions such as allergies and infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-016-0456-x) contains supplementary material, which is available to authorized users.

Bovine milk-based formula leads to early maturation-like morphological, immunological, and functional changes in the jejunum of neonatal piglets.

Artificial rearing and formula feeding is coming more into the focus due to increasing litter sizes and limited nursing capacity of sows. The formula composition is important to effectively support the development of the gut and prevent intestinal dysfunction in neonatal piglets. In this study, newborn piglets ( = 8 per group) were fed a bovine milk-based formula (FO), containing skimmed milk and whey as the sole protein and carbohydrate sources, or were suckled by the sow (sow milk [SM]). After 2 wk, tissue from the jejunum was analyzed for structural (i.e., morphometry) and functional (i.e., disaccharidase activity, glucose transport, permeability toward macromolecules, and immune cell presence) changes and concomitant expression of related genes. Formula-fed piglets had more liquid feces ( < 0.05) over the entire experimental period. Although FO contained twice as much lactose (46% on a DM basis) as SM (21%) and no maltose or starch, the lactase activity was lower ( < 0.05) and glucose transport capacity was higher ( < 0.05) in FO-fed pigs. The relative proportion of intraepithelial natural killer cells and proinflammatory cytokine gene expression (, , and ) was higher in FO-fed pigs ( < 0.05). Piglets fed FO had deeper crypts, larger villus area, and higher expression of caspase 3 and proliferating cell nuclear antigen ( < 0.05). Epithelial permeability toward fluorescein isothiocyanate-dextran was higher and expression of claudin-4 was lower in FO-fed piglets ( < 0.05). The data suggest an early response to bovine milk-based compounds in the FO accompanied with early onset of functional maturation and impaired barrier function. Whether lactose, absence of species-specific protective factors, or antigenicity of foreign proteins lead to to the observed intestinal reactions requires further clarification.

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.

Intestinal immune cell quantification and gram type classification of the adherent microbiota in conventionally and artificially reared, normal and low birth weight piglets

To be able to raise larger litters, the influences of alternative rearing strategies on piglets are currently assessed. This study compared the predominant Gram type of the adherent intestinal microbiota between conventionally and artificially reared piglets and quantified their major mucosal immune cell populations. In addition, the potential influence of the piglets' birth weights was examined. To this purpose, 40 neonatal piglets consisting of 20 normal birth weight (NBW) and 20 low birth weight (LBW) piglets suckled the sow for three days after which 20 piglets (10 NBW and 10 LBW) continued to suckle the sow and 20 piglets (10 NBW and 10 LBW) were transferred to brooders and raised on milk formula. At the age of 10 or 28 days, five piglets of each group (birth weight×rearing strategy) were euthanized and the jejunum was sampled. The presence of adherent Gram+ and Gram− bacteria was scored and the volume densities of CD8+ cells, CD4+ cells and CD172a+ myloid cells were determined. Irrespective of birth weight, sow-fed piglets possessed a predominant Gram+ microbiome at 10 days of age, whereas formula-fed animals had more Gram- microbiota. With increasing age, however, Gram+ microbiota took the upper hand in these animals. The volume densities of CD8+ and CD4+ cells rose with increasing age and were consistently lower in LBW piglets. Both rearing strategies had a similar influence on the volume densities of these cell populations. In contrast, the volume densities of CD172a+ myloid cells did not differ significantly between the birth weight, rearing strategy and age groups. Three observations allow to conclude that artificial rearing could be a valuable alternative for suckling the sow. First, within each birth weight category, the mean weight of the 28-day-old piglets was similar for both rearing groups. Secondly, the effect of milk formula on the composition of the intestinal microbiome was only temporary since formula-fed piglets restored the more beneficial Gram+ microbiome by the end of the artificial rearing period. Finally, artificial rearing did not influence, either positively or negatively, the volume densities of CD8+, CD4+ and CD172a+ immune cells when compared to conventional rearing.

Formula-derived advanced glycation end products are involved in the development of long-term inflammation and oxidative stress in kidney of IUGR piglets.

Formula-derived dietary advanced glycation end products (AGEs) may promote programming of inflammation and oxidative stress in the kidney of intrauterine growth retardation (IUGR) piglets. IUGR piglets received either a low temperature heated formula (n = 8) or a high temperature heated formula (HHF: n = 8) or suckled naturally for 3 wk postnatally. Then they were fed with normal ad libitum regular diet. N(ε)-carboxymethyllysine (CML) was measured in plasma, feces, and formula by HPLC/MS-MS. CML was detected by immunofluorescence in kidney cells. Target renin-angiotensin-apoptotic, pro-inflammatory genes-p62 NF-κB, and soluble receptor of AGE (sRAGE) levels were quantified. Compared with that in controls, free CML and plasma urea increased significantly in the HHF-fed group at PND36 (p < 0.05). CML was detected in the nuclei of renal tubular cells of formula-fed piglets but not in suckled ones. This presence of CML was associated with the activation of the soluble receptor of AGE. AT1, AT2, caspase 3, caspase 8, NF-κB, p62 NF-κB, and total protein oxidation in kidney were higher in HHF-fed group as compared to LHF-fed group (p < 0.05). Food processes aimed at reducing the concentration of AGEs in infant formula are urgently needed and may be therapeutically relevant for premature and/or IUGR babies.

Trace element status and zinc homeostasis differ in breast and formula-fed piglets.

Differences in trace element composition and bioavailability between breast milk and infant formulas may affect metal homeostasis in neonates. However, there is a paucity of controlled studies in this area. Here, piglets were fed soy infant formula (soy), cow's milk formula (milk), or were allowed to suckle from the sow from PND2 to PND21. Serum iron concentrations were higher in formula-fed compared to breastfed piglets (P < 0.05). Serum zinc values were higher in milk compared to breastfed or soy groups (P < 0.05). Zinc transporter Zip4 mRNA was elevated in small intestine of the soy compared to breastfed group (P < 0.05). Transporter Znt1 mRNA was greater in small intestine of both formula-fed groups and in liver of the milk compared to the breastfed group (P < 0.05). Metallothionein Mt1 mRNA expression was higher in small intestine and liver of milk compared to breastfed and soy groups (P < 0.05). In liver, metallothionein protein levels and protein bound zinc were also highly elevated in the milk compared to other groups (P < 0.05). mRNA encoding the hepatic zinc-regulated gene Gclc was higher in the milk than soy group (P < 0.05). ChIP assay revealed increased binding of the zinc-regulated transcription factor MTF1 to the promoters of hepatic Mt3 and Gclc genes in the milk compared to the soy group. These data provide evidence that trace element status differs in breastfed, milk-fed, and soy-fed piglets and that despite similar levels of dietary supplementation, allows strong causal inference that significant differences in serum zinc after cow's milk formula compared to soy formula consumption result in compensatory changes in expression of zinc transporters, binding proteins, and zinc-regulated genes.

Artificial rearing of piglets: Effects on small intestinal morphology and digestion capacity

The use of hyper-prolific sows results in large litters but also leads to an increasing number of supernumerary and underprivileged (e.g. low birth weight (LBW)) piglets. The effects of artificial rearing on the growth, small intestinal morphology and digestion capacity of these piglets remain unclear. Therefore, the aim of this study was to assess the effect of sow-feeding versus formula-feeding on piglets' structural and functional gut maturation. To this purpose, pairs of LBW and normal birth weight (NBW) piglets (n=40) were allocated to four treatment groups. Groups 1 and 2 contained piglets that suckled until either d 10 or 28 of age, respectively. Groups 3 and 4 contained animals that suckled until 3d of age and were then formula-fed until either d 10 or 28. During d 3–10, formula-fed piglets showed reduced average daily gain (ADG; −112gd−1) and lactase activities (−4.50Ug−1 tissue) compared to suckling piglets (P<0.01). In contrast, animals that were formula-fed until d 28 had a comparable ADG compared to sow-fed pigs. In addition, formula-fed piglets had a greater absorptive area (P<0.01; +59.1μm2), maltase and sucrase activities (P<0.05; +0.97 and +0.23Ug−1 tissue) and deeper crypts (P<0.03; +42.5μm) compared to suckling piglets. In general, the differences between LBW and NBW piglets were scarce. These results suggest that the combination of ad libitum access to formulated milk and an increased capacity to absorb nutrients makes artificial rearing a good alternative to raise supernumerary and/or LBW piglets.

Natural killer cell populations and cytotoxic activity in pigs fed mother’s milk, formula, or formula supplemented with bovine lactoferrin

Natural killer (NK) cells are components of the innate immune defense system, and their levels differ between breast and formula-fed (FF) infants. Lactoferrin (Lf) modulates NK cell cytotoxicity ex vivo. We hypothesized that dietary bovine Lf (bLf) would increase NK cell populations and cytotoxicity. Piglets were sow-reared (SR), FF, or 1 g/l bLf-fed (LF) for 21 d. NK cells (CD3(-)CD4(-)CD8(+)) in blood (peripheral blood mononuclear cells (PBMCs)), spleen, and mesenteric lymph node (MLN) were determined by flow cytometry. PBMC NK cells were tested for cytotoxic activity against target K562 cells ex vivo in the presence of media (unstimulated), interleukin-2, or bLf. NK cell mRNA expression was determined by reverse transcription-quantitative PCR. SR and LF piglets had more NK cells in MLN (P = 0.0097) and spleen (P = 0.0980) than FF piglets. In PBMCs, SR piglets had more NK cells than FF piglets (P = 0.0072); LF piglets were intermediate and not different from FF or SR piglets. NK cell intelectin-2 mRNA expression was 2.5-fold higher (P = 0.0095) in LF than SR or FF piglets. NK cells in SR piglets exhibited greater (P < 0.0001) cytotoxic activity than those in LF or FF piglets, which was supported by greater perforin mRNA expression. Dietary bLf increased blood NK cell populations and NK Lf receptor expression but not NK cell cytotoxicity.

Increased IGF-1 serum levels and discordant protein and mRNA IGF-1 receptor expression in the small intestine of formula-fed piglets

The present investigation addresses the question whether feeding piglets on formulated milk alters the insulin-like growth factor-1 (IGF-1) system differently in normal versus low birth weight piglets. In this study, both types of piglets were either sow-fed until 28 days of age or until 3 days of age and subsequently formula-fed until day 28. Formula-fed piglets had higher serum IGF-1 levels compared to suckled piglets, whereas low birth weight piglets had lower IGF-1 levels in comparison with normal birth weight piglets. In contrast, the mRNA expression of IGF-1 receptor (IGF-1R) showed lower expression in formula-fed piglets versus suckled piglets. However, the opposite effect was observed for IGF-1R protein abundance. Moreover, birth weight did not markedly affect IGF-1R mRNA or protein abundance. In conclusion, formula-fed piglets have higher IGF-1 serum levels and altered gene and protein expression of IGF-1R in the small intestine, irrespective of their birth weight.

Human milk oligosaccharides inhibit rotavirus infectivityin vitroand in acutely infected piglets

Human milk (HM) is rich in oligosaccharides (HMO) that exert prebiotic and anti-infective activities. HM feeding reduces the incidence of rotavirus (RV) infection in infants. Herein, the anti-RV activity of oligosaccharides was tested in an established in vitro system for assessing cellular binding and viral infectivity/replication, and also tested in a newly developed, acute RV infection, in situ piglet model. For the in vitro work, crude HMO isolated from pooled HM, neutral HMO (lacto-N-neotetraose, LNnT; 2'-fucosyllactose) and acidic HMO (aHMO, '-sialyllactose, 3'-SL; -sialyllactose, -SL) were tested against the porcine OSU strain and human RV Wa strain. The RV Wa strain was not inhibited by any oligosaccharides. However, the RV OSU strain infectivity was dose-dependently inhibited by sialic acid (SA)-containing HMO. 3'-SL and 6'-SL concordantly inhibited (125)I-radiolabelled RV cellular binding and infectivity/replication. For the in situ study, a midline laparotomy was performed on 21-d-old formula-fed piglets and six 10 cm loops of ileum were isolated in situ. Briefly, 2 mg/ml of LNnT, aHMO mixture (40% 6'-SL/10 % 3'-SL/50 % SA) or media with or without the RV OSU strain (1 x 10(7) focus-forming units)were injected into the loops and maintained for 6 h. The loops treated with HMO treatments þ RV had lower RV replication, as assessed by non-structural protein-4 (NSP4) mRNA expression, than RV-treated loops alone. In conclusion, SA-containing HMO inhibited RV infectivity in vitro; however, both neutral HMO and SA with aHMO decreased NSP4 replication during acute RV infection in situ.

Increased intestinal barrier function in the small intestine of formula-fed neonatal piglets

Within-litter birth weight variation is adversely correlated to piglet survival and postnatal growth. A less efficient epithelial barrier function in light piglets may partly explain this inverse relationship between birth weight and zootechnical performance. A compromised epithelial barrier increases paracellular permeability; consequently, toxins, allergenic compounds, or bacteria may enter systemic circulation and induce inflammatory responses. Dietary effects on function of gut epithelium of piglet are largely unknown. This study investigated epithelial barrier function of the small intestine of normal birth weight (NBW) piglets (1.46 ± 0.10 kg) and low birth weight (LBW) piglets (<1 kg at birth) in relation to their diet. Sixteen pairs of 3-d-old LBW and NBW piglets were randomly assigned to 3 groups: a sow-fed control group euthanized at day 3 of age (SOW3), piglets sow fed until day 10 (SOW10), and formula-fed piglets fed formula from day 3 until day 10 (FOR10). To measure gut permeability, piglets were dosed intragastrically with 0.75 g lactulose/kg BW and 0.3 g mannitol/kg BW 4 h before euthanasia. Urinary sugar excretion was measured using enzymatic spectrophotometry. Irrespective of birth weight, lactulose levels of FOR10 (4.4 ± 2.3 mmol/L) tended to be lower (P = 0.07) than SOW10 (26.4 ± 10.2 mmol/L) indicating a reduced paracellular intestinal permeability in FOR10. This reduction was associated with a 6-fold elevated (P < 0.01) protein expression of occludin, an important tight junction protein, in FOR10 compared to SOW10. Mannitol levels in FOR10 (31.0 ± 18.2 mmol/L) did not differ (P = 0.28) from SOW10 (61.1 ± 10.2 mmol/L). However, shorter villi (P < 0.01) in FOR10 indicated a reduced absorptive capacity. In conclusion, formula feeding caused minor symptoms of gastrointestinal dysfunction compared to sow-fed piglets irrespective of their birth weight.