Early effects on the intestinal barrier and pancreatic function after enteral stimulation with protease or kidney bean lectin in neonatal rats.

Gut maturation naturally accelerates at weaning in altricial mammalian species, such as the rat. Mimicking this, gut development can also be induced precociously, 3-4 d earlier than it would occur naturally, by enteral exposure to phytohaemagglutinin (PHA), or various proteases. We investigated the early effects of gut provocation on intestinal barrier and pancreatic functions, to get a better understanding of the mechanisms that initiate gut maturation. The effects of oral administration of protease (trypsin) or PHA to 14-d-old suckling rats were studied during 24 h in comparison with water-fed controls. Intestinal in vivo permeability was assessed by oral administration of different-sized marker molecules and measuring their passage into the blood or urine 3 h later. A period of 24 h following oral administration, both PHA and protease provocation stimulated small intestinal (SI) growth and pancreatic secretion, as indicated by decreased pancreatic trypsin and increased luminal enzyme content. Within 1 h of oral administration, both treatments prevented the absorption of macromolecules to blood that was observed in controls. PHA treatment hindered the passage of fluorescein isothiocyanate-dextran (FD) 4 to blood, whereas protease treatment temporarily increased plasma levels of FD4, and the urine lactulose:mannitol ratio, indicating increased intestinal leakiness. Following protease treatment, fluorescence microscopy showed decreased vesicular uptake of FD70 in the proximal SI and increased epithelial fluorescence in the distal SI. In conclusion, PHA and protease differed in their early effects on the intestinal barrier; both exerted a blocking effect on epithelial endocytosis, whereas protease treatment alone temporarily increased epithelial leakiness, which seemed to be confined to the distal SI.

Exocrine Pancreatic Maturation in Pre-term and Term Piglets Supplemented With Bovine Colostrum.

Pre-term infants have an immature digestive system predisposing to short- and long-term complications including feeding intolerance, maldigestion and necrotizing enterocolitis (NEC). Optimal feeding strategies are required to promote maturation of the gut including the exocrine pancreas. Little is known about age- and diet-related development of pancreatic exocrine enzymes following pre-term birth. Currently, bovine colostrum supplementation is investigated in clinical trials on pre-term infants. Using pigs as models for infants, we hypothesized that pancreatic enzyme content is (1) immature following pre-term birth, (2) stimulated by early colostrum supplementation, and (3) stimulated by later colostrum fortification. Thus, using piglets as models for infants, we measured trypsin, amylase, lipase and total protein in pancreatic tissue collected from piglets delivered by cesarean section either pre-term (90% gestation) or close to term. Experiment 1:Pre-term and term pigs were compared at birth and 11 days. Experiment 2: Pre-term and term pigs were either enterally supplemented with bovine colostrum or fed total parenteral nutrition for 5 days, followed by exclusive milk feeding until day 26. Experiment 3: Pre-term pigs were fed bovine's milk with or without colostrum fortification until 19 days. The results showed that pancreatic trypsin, amylase and total protein contents were reduced in pre-term vs. term pigs. Trypsin mainly increased with advancing post-conceptional age (2-fold), while amylase was affected predominantly by advancing post-natal age, and mostly in pre-term pigs from birth to 11 or 26 days. Colostrum feeding in both term and pre-term piglets decreased trypsin and increased amylase contents. Lipase activity decreased with advancing gestational age at birth and post-natal age, with no consistent responses to colostrum feeding, with lipase activities decreasing relative to total pancreatic protein content. In summary, key pancreatic enzymes, amylase and trypsin, are immature following pre-term birth, potentially contributing to reduced digestive capacity in pre-term neonates. Rapid post-natal increases occurs within few weeks of pre-term birth, partly stimulated by enteral colostrum intake, reflecting a marked adaptation capacity. Alternatively, lipase is less affected by pre-/post-natal age and feeding. Thus, there is a highly enzyme-specific and asymmetric perinatal development of the exocrine pancreas.

Pre-Weaning Inulin Supplementation Alters the Ileal Transcriptome in Pigs Regarding Lipid Metabolism.

Prebiotics, such as inulin, are non-digestible compounds that stimulate the growth of beneficial microbiota, which results in improved gut and overall health. In this study, we were interested to see if, and how, the ileal transcriptome altered after inulin administration in the pre-weaning period in pigs. Seventy-two Piétrain-Landrace newborn piglets were divided into three groups: (a) a control (CON) group (n = 24), (b) an inulin (IN)-0.5 group (n = 24), and (c) an IN-0.75 group (n = 24). Inulin was provided as a solution and administered twice a day. At week 4, eight piglets per group, those closest to the average in body weight, were sacrificed, and ileal scrapings were collected and analyzed using 3' mRNA massively parallel sequencing. Only minor differences were found, and three genes were differentially expressed between the CON and IN-0.5 group, at an FDR of 10%. All three genes were downregulated in the IN-0.5 group. When comparing the CON group with the IN-0.75 group, five genes were downregulated in the IN-0.75 group, including the three genes seen earlier as differentially expressed between CON and IN-0.5. No genes were found to be differential expressed between IN-0.5 and IN-0.75. Validation of a selection of these genes was done using qRT-PCR. Among the downregulated genes were Angiopoietin-like protein 4 (ANGPTL4), Aquaporin 7 (AQP7), and Apolipoprotein A1 (APOA1). Thus, although only a few genes were found to be differentially expressed, several of them were involved in lipid metabolism, belonging to the peroxisome proliferator-activated receptor (PPAR) signaling pathway and known to promote lipolysis. We, therefore, conclude that these lipid metabolism genes expressed in the ileum may play an important role when supplementing piglets with inulin early in life, before weaning.

The Impact of Maternal and Piglet Low Protein Diet and Their Interaction on the Porcine Liver Transcriptome around the Time of Weaning.

Maternal diet during early gestation affects offspring phenotype, but it is unclear whether maternal diet during late gestation influences piglet metabolism. We evaluated the impact of two dietary protein levels in sow late gestation diet and piglet nursery diet on piglet metabolism. Diets met or exceeded the crude protein and amino acid requirements. Sows received either 12% (Lower, L) or 17% (Higher, H) crude protein (CP) during the last five weeks of gestation, and piglets received 16.5% (L) or 21% (H) CP from weaning at age 3.5 weeks. This resulted in a 2 × 2 factorial design with four sow/piglet diet treatment groups: HH and LL (match), HL and LH (mismatch). Piglet hepatic tissues were sampled and differentially expressed genes (DEGs) were determined by RNA sequencing. At age 4.5 weeks, 25 genes were downregulated and 22 genes were upregulated in the mismatch compared to match groups. Several genes involved in catabolic pathways were upregulated in the mismatch compared to match groups, as were genes involved in lipid metabolism and inflammation. The results show a distinct interaction effect between maternal and nursery diets, implying that sow late gestation diet could be used to optimize piglet metabolism.

Impact of Citrus Pulp or Inulin on Intestinal Microbiota and Metabolites, Barrier, and Immune Function of Weaned Piglets.

We investigated the use of citrus pulp (CP) as a novel prebiotic capable of exerting microbiota and immunomodulating capacities to alleviate weaning stress. Inulin (IN), a well-known prebiotic, was used for comparison. Hundred and 28 male weaned piglets of 21 days old were assigned to 32 pens of 4 piglets each. Piglets were assigned to one of the four treatments, i.e., control, IN supplemented at 0.2% (IN0.2%), and CP supplemented either at 0.2% (CP0.2%) or at 2% (CP2%). On d10-11 and d31-32 post-weaning, one pig per pen was euthanized for intestinal sampling to evaluate the growth performance, chyme characteristics, small intestinal morphology, colonic inflammatory response and barrier integrity, metabolite profiles [gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS)], and microbial populations. The IN treatment and the two CP treatments induced higher small intestinal villus height to crypt depth ratios in comparison with the control diet at both sampling times. All treatments decreased acidic goblet cell absolute counts in the crypts in comparison to the control diet of the duodenum on d10-11 and d31-32. The gene expression of ß-defensin 2 was downregulated in colonic tissues following the IN and CP2% inclusion on d31-32. On d31-32, piglets fed with IN and CP0.2% showed lower mRNA levels of occludin and claudin-3, respectively. Not surprisingly, flavonoids were observed in the colon in the CP treatments. Increased colonic acetate proportions on d10-11, at the expense of branched-chain fatty acid (BCFA) levels, were observed following the CP2% supplementation compared to the control diet, inferring a reduction of proteolytic fermentation in the hindgut. The beneficial microbial community Faecalibacterium spp. was promoted in the colon of piglets fed with CP2% on d10-11 (p = 0.04; false discovery rate (FDR) non-significant) and on d31-32 (p = 0.03; FDR non-significant) in comparison with the control diet. Additionally, on d31-32, CP2% increased the relative abundance of Megasphaera spp. compared to control values (p = 0.03; FDR non-significant). In conclusion, CP2% promoted the growth of beneficial bacterial communities in both post-weaning time points, modulating colonic fermentation patterns in the colon. The effects of CP supplementation were similar to those of IN and showed the potential as a beneficial feed supplement to alleviate weaning stress.

Isoquinoline Alkaloids in Sows' Diet Reduce Body Weight Loss during Lactation and Increase IgG in Colostrum.

Isoquinoline alkaloids (IQ) exert beneficial antimicrobial and anti-inflammatory effects in livestock. Therefore, we hypothesized that supplementing sows' diets with IQ during gestation would decrease farrowing stress, affecting the piglets' development and performance. Sows were divided into: IQ1, supplemented with IQ from gestation day 80 (G80) to weaning; IQ2, supplemented from gestation day 110 (G110) to weaning, and a non-supplemented (NC) group. Sow body weight (BW), feed intake, back-fat thickness and back-muscle thickness were monitored. Cortisol, glucose and insulin were measured in sows' blood collected 5 d before, during, and after 7 d farrowing. Protein, fat, IgA and IgG were analyzed in the colostrum and milk. Piglets were monitored for weight and diarrhea score, and for ileum histology and gene expression 5 d post-weaning. IQ-supplemented sows lost less BW during lactation. Glucose and insulin levels were lower in the IQ groups compared to NC-sows 5 d before farrowing and had higher levels of protein and IgG in their colostrum. No other differences were observed in sows, nor in the measured parameters in piglets. In conclusion, IQ supplementation affected sows' metabolism, reducing body weight loss during lactation. Providing IQ to sows from their entrance into the maternity barn might be sufficient to induce these effects. IQ improved colostrum quality, increasing the protein and IgG content, improving passive immunity for piglets.

The Immature Gut Barrier and Its Importance in Establishing Immunity in Newborn Mammals.

The gut is an efficient barrier which protects against the passage of pathogenic microorganisms and potential harmful macromolecules into the body, in addition to its primary function of nutrient digestion and absorption. Contrary to the restricted macromolecular passage in adulthood, enhanced transfer takes place across the intestines during early life, due to the high endocytic capacity of the immature intestinal epithelial cells during the fetal and/or neonatal periods. The timing and extent of this enhanced endocytic capacity is dependent on animal species, with a prominent non-selective intestinal macromolecular transfer in newborn ungulates, e.g., pigs, during the first few days of life, and a selective transfer of mainly immunoglobulin G (IgG), mediated by the FcRn receptor, in suckling rodents, e.g., rats and mice. In primates, maternal IgG is transferred during fetal life via the placenta, and intestinal macromolecular transfer is largely restricted in human neonates. The period of intestinal macromolecular transmission provides passive immune protection through the transfer of IgG antibodies from an immune competent mother; and may even have extra-immune beneficial effects on organ maturation in the offspring. Moreover, intestinal transfer during the fetal/neonatal periods results in increased exposure to microbial and food antigens which are then presented to the underlying immune system, which is both naïve and immature. This likely stimulates the maturation of the immune system and shifts the response toward tolerance induction instead of activation or inflammation, as usually seen in adulthood. Ingestion of mother's milk and the dietary transition to complex food at weaning, as well as the transient changes in the gut microbiota during the neonatal period, are also involved in the resulting immune response. Any disturbances in timing and/or balance of these parallel processes, i.e., intestinal epithelial maturation, luminal microbial colonization and mucosal immune maturation due to, e.g., preterm birth, infection, antibiotic use or nutrient changes during the neonatal period, might affect the establishment of the immune system in the infant. This review will focus on how differing developmental processes in the intestinal epithelium affect the macromolecular passage in different species and the possible impact of such passage on the establishment of immunity during the critical perinatal period in young mammals.

Correction: Maturation of the Intestinal Epithelial Barrier in Neonatal Rats Coincides with Decreased FcRn Expression, Replacement of Vacuolated Enterocytes and Changed Blimp-1 Expression.

[This corrects the article DOI: 10.1371/journal.pone.0164775.].

Importance of neonatal immunoglobulin transfer for hippocampal development and behaviour in the newborn pig.

Neurological disorders are among the main clinical problems affecting preterm children and often result in the development of communication and learning disabilities later in life. Several factors are of importance for brain development, however the role of immunoglobulins (passive immunity transfer) has not yet been investigated. Piglets are born agammaglobulinemic, as a result of the lack of transfer of maternal immunoglobulins in utero, thus, they serve as an ideal model to mimic the condition of immunoglobulin deficiency in preterm infants. Thirty six, unsuckled newborn piglets were fed an infant formula or colostrum and supplemented orally or intravenously with either species-specific or foreign immunoglobulin and then compared to both newborn and sow-reared piglets. Two days after the piglets were born behavioural tests (novel recognition and olfactory discrimination of conspecifics scent) were performed, after which the piglets were sacrificed and blood, cerebrospinal fluid and hippocampi samples were collected for analyses. Both parameters of neuronal plasticity (neuronal maturation and synapse-associated proteins) and behavioural test parameters appeared to be improved by the appearance of species-specific porcine immunoglulin in the circulation and cerebrospinal fluid of the piglets. In conclusion, we postulate possible positive clinical effects following intravenous infusion of human immunoglobulin in terms of neuronal plasticity and cognitive function in preterm infants born with low blood immunoglobulin levels.

Maturation of the Intestinal Epithelial Barrier in Neonatal Rats Coincides with Decreased FcRn Expression, Replacement of Vacuolated Enterocytes and Changed Blimp-1 Expression.

BACKGROUND: The intestinal barrier is immature in newborn mammals allowing for transfer of bioactive macromolecules, e.g. protecting antibodies, from mother's milk to the blood circulation and in neonatal rodents lasts until weaning. This passage involves the neonatal-Fc-receptor (FcRn) binding IgG in the proximal and highly endocytic vacuolated enterocytes in the distal immature small intestine (SI). Recent studies have suggested an involvement of the transcription factor B-lymphocyte-induced maturation-protein-1 (Blimp-1) in the regulation of SI maturation in mice. Hence, the objective of the present study was to monitor the development of the intestinal barrier function, in relation to Blimp-1 expression during both natural and precociously induced intestinal maturation in rats. RESULTS: During the suckling period IgG plasma levels increased, while after gut closure it temporarily decreased. This corresponded to a high expression of FcRn in the proximal SI epithelium and the presence of vacuolated enterocytes in the distal SI. The immature foetal-type epithelium was replaced after weaning or induced precocious maturation, by an adult-type epithelium with FcRnneg cells in the proximal and by non-vacuolated enterocytes in the distal SI. In parallel to this epithelial shift, Blimp-1 expression decreased in the distal SI. CONCLUSION: The switch from foetal- to adult-type epithelium, with decreased proximal expression of FcRn and distal replacement of vacuolated enterocytes, was concurrent in the two SI regions and could be used for monitoring SI maturation in the rat. The changes in expression of Blimp-1 in the distal SI epithelium followed the maturation pattern.

Pancreatic and pancreatic-like microbial proteases accelerate gut maturation in neonatal rats.

OBJECTIVES: Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. METHODS: Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14-16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. RESULTS: Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. CONCLUSION: Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.