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Deficient levels of milk osteopontin (OPN) in infant formula may partly account for developmental differences between infants fed formula or maternal milk. We hypothesized that a milk diet supplemented with bovine milk OPN improves gut, immunity and brain development and tested this in a preterm pig model. Preterm pigs delivered by cesarean section (90% gestation) were fed raw bovine milk (CON, n = 19) or the same diet supplemented with a physiologically relevant dose of OPN (46 mg/(kg·d), n = 16). Endpoints related to clinical outcomes, systemic immunity and neurocognitive development were assessed during the study and gut tissues were collected at Day 19. Growth pattern, early motor development and most systemic immune parameters were similar between OPN and CON pigs. The OPN pigs had higher villus-to-crypt ratios than CON pigs and higher monocyte and lymphocyte counts on Day 8. Gut digestive and absorptive functions and cognitive performance (T-maze test) were similar between OPN and CON pigs. In conclusion, dietary supplementation with OPN above basal bovine milk levels induced minor improvements in gut structure and systemic immunity without any effects on cognitive performance. The minimal levels of OPN in infant formula to secure optimal adaptation in the immediate neonatal period remain to be determined.

Human milk is the optimal diet for infant development, but infant milk formula (IMF) must be available as an alternative. To develop high-quality IMF, bovine milk processing is required to ensure microbial safety and to obtain a protein composition that mimics human milk. However, processing can impact the quality of milk proteins, which can influence gastro-intestinal (GI) tolerance by changing digestion, transit time and/or absorption. The aim of this study was to evaluate the impact of structural changes of proteins due to thermal processing on gastro-intestinal tolerance in the immature GI tract. Preterm and near-term piglets received enteral nutrition based on whey protein concentrate (WPC) either mildly pasteurized (MP-WPC) or extensively heated (EH-WPC). Clinical symptoms, transit time and gastric residuals were evaluated. In addition, protein coagulation and protein composition of coagulates formed during in vitro digestion were analyzed in more detail. Characterization of MP-WPC and EH-WPC revealed that mild pasteurization maintained protein nativity and reduced aggregation of β-lactoglobulin and α-lactalbumin, relative to EH-WPC. Mild pasteurization reduced the formation of coagulates during digestion, resulting in reduced gastric residual volume and increased intestinal tract content. In addition, preterm piglets receiving MP-WPC showed reduced mucosal bacterial adherence in the proximal small intestine. Finally, in vitro digestion studies revealed less protein coagulation and lower levels of β-lactoglobulin and α-lactalbumin in the coagulates of MP-WPC compared with EH-WPC. In conclusion, minimal heat treatment of WPC compared with extensive heating promoted GI tolerance in immature piglets, implying that minimal heated WPC could improve the GI tolerance of milk formulas in infants.

Background: Extrauterine growth restriction (EUGR) in preterm infants is associated with higher morbidity and impaired neurodevelopment. Early nutrition support may prevent EUGR in preterm infants, but it is not known if this improves organ development and brain function in the short and long term., Objective: Using pigs as models for infants, we hypothesized that diet-induced EUGR impairs gut, immunity, and brain development in preterm neonates during the first weeks after birth., Methods: Forty-four preterm caesarean-delivered pigs (Danish Landrace × Large White × Duroc, birth weight 975 ± 235 g, male:female ratio 23:21) from 2 sows were fed increasing volumes [32-180 mL/(kg·d)] of dilute bovine milk (EUGR group) or the same diet fortified with powdered bovine colostrum for 19 d (CONT group, 50-100% higher protein and energy intake than the EUGR group)., Results: The EUGR pigs showed reduced body growth (-39%, P < 0.01), lower plasma albumin, phosphate, and creatine kinase concentrations (-35 to 14%, P < 0.05), increased cortisol and free iron concentrations (+130 to 700%, P < 0.05), and reduced relative weights of the intestine, liver, and spleen (-38 to 19%, all P < 0.05). The effects of EUGR on gut structure, function, microbiota, and systemic immunity were marginal, although EUGR temporarily increased type 1 helper T cell (Th1) activity (e.g. more blood T cells and higher Th1-related cytokine concentrations on day 8) and reduced colon nutrient fermentation (lower SCFA concentration; -45%, P < 0.01). Further, EUGR pigs showed increased relative brain weights (+19%, P < 0.01), however, memory and learning, as tested in a spatial T-maze, were not affected., Conclusion: Most of the measured organ growth, and digestive, immune, and brain functions showed limited effects of diet-induced EUGR in preterm pigs during the first weeks after birth. Likewise, preterm infants may show remarkable physiological adaptation to deficient nutrient supply during the first weeks of life although early life malnutrition may exert negative consequences later., (Copyright © The Author(s) 2020.)

Background: The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1-2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition., Methods: WPC was obtained by cold filtration and mildly pasteurized (73 °C, 30 s) or extensively heat treated (73 °C, 30 s + 80 °C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis., Results: A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-β, IL-8 and TNF-α expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC., Conclusions: Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.

Objectives: Preterm neonates have an immature gastrointestinal tract and show an altered bacterial colonization of the gut. However, it is not clear if such immature gut microbiota (GM) colonization is induced by specific delivery, diet, environment, and/or host factors related to preterm birth. Using piglets as models for infants, we hypothesized that both shortened gestational age (GA) and start of enteral feeding affect GM composition after caesarean delivery and rearing in identical environments., Methods: Caesarean-delivered preterm and term pigs were reared in incubators and fed total parenteral nutrition (TPN) or gradually increasing early enteral feeding (EEF) for 5 days, followed by full enteral feeding with bovine milk until day 26. GM composition was determined by 16S rRNA gene-amplicon sequencing and luminal short-chain fatty acids (SCFAs) by GC-MS., Results: Both GA and EEF feeding affected GM composition on day 5, but only the GA effect persisted until day 26. On day 5, Enterobacteriaceae were dominant, with Lachnospiraceae members also being abundant. Enterobacteriaceae still dominated the GM at day 26 but with higher Akkermansia relative abundance in term pigs. Colonic concentrations of acetate and propionate were higher, and formate lower in term pigs, relative to preterm pigs on day 26., Conclusions: Preterm and term piglets, born and reared in similar ways, show differences in GM colonization during the first 4 weeks of life, which may play a role for early and later gut dysfunction resulting from preterm birth.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1β levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Oligosaccharides support gut development and bacterial colonization in term infants, but it is unknown if they benefit preterm infants. Using preterm pigs, we investigated effects of bovine milk supplements enriched with oligosaccharides to improve gut development and colonization. Caesarean-delivered preterm pigs (n = 57) were reared for 19 days. The pigs were fed bovine milk supplemented with an oligosaccharide-enriched whey containing sialyllactose, or a heterogeneous oligosaccharide ingredient. To evaluate the influence of artificial rearing, near-term, vaginally born pigs raised by their sow (n = 12) were compared with artificially reared, caesarean-delivered near-term pigs (n = 14). In preterm pigs, the clinical outcome, gut function, gut microbiota, and systemic immunity were similar among dietary treatments. Natural rearing increased growth rates, gut functions, colon short chain fatty acid concentrations and bacterial diversity, relative to artificial rearing. In conclusion, supplements with bovine milk oligosaccharides were well tolerated, but did not improve gut maturation or clinical outcomes in artificially reared preterm piglets. Immaturity at birth, coupled with artificial rearing, may render the neonate unresponsive to the gut-protective effects of milk oligosaccharides. Whether bovine milk oligosaccharides may affect other endpoints (e.g., brain functions) in conditions of immaturity remains to be investigated.

After birth, the newborn must adapt to the acute challenges of circulatory changes, active respiration, thermoregulation, microbial colonization, and enteral nutrition. Whereas these processes normally occur without clinical complications in neonates born at term, birth at a preterm state of gestation is associated with high morbidity and mortality. In commercial pig production, perinatal mortality is higher than in any other mammalian species. Asphyxia, hypothermia, hypoglycemia, sepsis, and gut dysmotility, represent some of the most common findings. The intestine is a particularly sensitive organ after birth, as it must adapt acutely to enteral nutrition and microbial colonization. Likewise, during the weaning phase, the intestine must adapt to new diet types. Both critical phases are associated with high morbidity. This review focuses on the endocrine changes occurring around birth and weaning. There are a number of endocrine adaptations in late gestation and early postnatal life that are under influence of development stage and environmental factors such as diet. The review discusses general endocrine changes in perinatal life but specifically focuses on the role of glucagon-like peptide-2. This gut-derived hormone plays a key role in development and function of the intestine in early life., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background & Aims: Only few hours of formula feeding may induce proinflammatory responses and predispose to necrotizing enterocolitis (NEC) in preterm pigs. We hypothesized that bovine colostrum, rich in bioactive factors, would improve intestinal function in preterm pigs following an initial exposure to formula feeding after some days of total parenteral nutrition (TPN)., Methods: After receiving TPN for 2 days, preterm pigs were fed formula (FORM, n = 14), bovine colostrum (COLOS, n = 6), or formula (6 h) followed by bovine colostrum (FCOLOS, n = 14). Intestinal lesions, function, and structure, abundance and location of bacteria, and inflammation markers were investigated., Results: NEC severity and interleukins (IL)-1β and -8 protein concentrations were lower, while villus height, galactose absorption, and brush-border enzyme activities were increased in the distal small intestine in COLOS and FCOLOS pigs, relative to FORM pigs. Intestinal gene expression of serum amyloid A, IL-1β, -6 and -8, and bacterial abundance, correlated positively with NEC severity of the distal small intestine., Conclusions: Bovine colostrum restores intestinal function after initial formula-induced inflammation in preterm pigs. Further studies are required to test if bovine colostrum may also benefit preterm infants during the challenging transition from total parenteral nutrition to enteral nutrition, when human milk is unavailable., (Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

At birth, the newborn mammal undergoes a transition from a sterile uterine environment with a constant nutrient supply, to a microbe-rich environment with intermittent oral intake of complex milk nutrients via the gastrointestinal tract (GIT). These functional challenges partly explain the relatively high morbidity and mortality of neonates. Preterm birth interrupts prenatal organ maturation, including that of the GIT, and increases disease risk. Exemplary is necrotizing enterocolitis (NEC), which is associated closely with GIT immaturity, enteral feeding, and bacterial colonization. Infants with NEC may require resection of the necrotic parts of the intestine, leading to short bowel syndrome (SBS), characterized by reduced digestive capacity, fluid loss, and dependency on parenteral nutrition. This review presents the preterm pig as a translational model in pediatric gastroenterology that has provided new insights into important pediatric diseases such as NEC and SBS. We describe protocols for delivery, care, and handling of preterm pigs, and show how the immature GIT responds to delivery method and different nutritional and therapeutic interventions. The preterm pig may also provide a sensitive model for postnatal adaptation of weak term piglets showing high mortality. Attributes of the preterm pig model include close similarities with preterm infants in body size, organ development, and many clinical features, thereby providing a translational advantage relative to rodent models of GIT immaturity. On the other hand, the need for a sow surgical facility, a piglet intensive care unit, and clinically trained personnel may limit widespread use of preterm pigs. Studies on organ adaptation in preterm pigs help to identify the physiological basis of neonatal survival for hypersensitive newborns and aid in defining the optimal diet and rearing conditions during the critical neonatal period.

Preterm infants are susceptible to necrotizing enterocolitis (NEC). Using preterm pigs, we determined whether a whey-casein-based formula would be superior to a formula based on whey protein alone. Twenty cesarean-derived preterm pigs (92% gestation) were given total parenteral nutrition for 36 h followed by 30 h of enteral feeding with whey [protein fraction of milk formula based on whey (WHEY); n = 11] or casein and/or whey [protein fraction of milk formula based on a combination of casein and whey (CASEIN); n = 9]-based formulas. Sugar absorptive function was investigated at 6 and 30 h after initiation of enteral feeding using bolus feedings with galactose and mannitol. Pigs were killed after the last in vivo sugar absorption test and evaluated for NEC and the mid intestine was used for ex vivo measurements of hexose absorption. Microbiota profile and short chain fatty acid (SCFA) levels were studied in gut contents. Severity of NEC lesions was similar between diet groups but galactose absorption was markedly higher in CASEIN than in WHEY (P < 0.01) although only 6 h after the start of the enteral feeding period. There were no differences in ex vivo (14)C-D-glucose uptake, digestive enzymes, microbiota profile, or SCFA concentration. Casein may transiently stimulate intestinal sugar absorption but has limited effects on gut structure, microbiota, and NEC in preterm pigs.

The gastrointestinal inflammatory disorder, necrotizing enterocolitis (NEC), is among the most serious diseases for preterm neonates. Nutritional, microbiological and immunological dysfunctions all play a role in disease progression but the relationship among these determinants is not understood. The preterm gut is very sensitive to enteral feeding which may either promote gut adaptation and health, or induce gut dysfunction, bacterial overgrowth and inflammation. Uncontrolled inflammatory reactions may be initiated by maldigestion and impaired mucosal protection, leading to bacterial overgrowth and excessive nutrient fermentation. Tumor necrosis factor alpha, toll-like receptors and heat-shock proteins are identified among the immunological components of the early mucosal dysfunction. It remains difficult, however, to distinguish the early initiators of NEC from the later consequences of the disease pathology. To elucidate the mechanisms and identify clinical interventions, animal models showing spontaneous NEC development after preterm birth coupled with different forms of feeding may help. In this review, we summarize the literature and some recent results from studies on preterm pigs on the nutritional, microbial and immunological interactions during the early feeding-induced mucosal dysfunction and later NEC development. We show that introduction of suboptimal enteral formula diets, coupled with parenteral nutrition, predispose to disease, while advancing amounts of mother's milk from birth (particularly colostrum) protects against disease. Hence, the transition from parenteral to enteral nutrition shortly after birth plays a pivotal role to secure gut growth, digestive maturation and an appropriate response to bacterial colonization in the sensitive gut of preterm neonates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Necrotizing enterocolitis (NEC) in preterm neonates is dependent on bacterial colonization, but it remains unclear whether a particular microbiota or specific pathogens are involved. We hypothesized that gut colonization differs between preterm and term neonates and that overgrowth of Clostridium perfringens predisposes to NEC. By using terminal-RFLP and FISH, we characterized the gut microbiota of preterm, caesarean-delivered, formula-fed pigs (n = 44) with or without NEC and of formula- or colostrum-fed term, and vaginally born pigs (n = 13). A different microbiota with high C. perfringens abundance was observed in preterm pigs with NEC compared with healthy individuals. However, immunization against C. perfringens toxins did not prevent NEC, and C. perfringens inoculation (3.6 × 10 cfu/d) failed to induce NEC (n = 16), whereas prophylactic broad-spectrum antibiotics treatment prevented NEC (n = 24). Colonization in both groups of term pigs differed from preterm pigs and was dominated by Lactobacilli spp. In conclusion, gestational age (GA) and NEC influence neonatal gut colonization, whereas diet has minor effects. C. perfringens is more abundant in pigs with NEC but rather as a consequence than a cause of disease. The general bacterial load and underdeveloped gut immune responses in preterm neonates seem more important for NEC development than specific pathogens.

Abstract: Enteral nutrition coupled with bacterial colonization has been shown to have major functional and developmental effects during the postnatal period of neonates. In this study, we developed a highly sensitive premature pig model to elucidate the specific developmental impact of initial bacterial colonization on premature neonates by comparing germ-free and conventionally reared pigs. Thirty-eight preterm pigs (93% gestation) were delivered via caesarean section and reared in either germ-free or conventional isolators for 40–48 h. Pigs were fed either infant milk formula or sow''s colostrum. Enteral feeding for two days had trophic effects on gastrointestinal weights, particularly for pancreas, stomach and small intestine (SI). The absence of bacteria in formula fed pigs resulted in a mucosa that appeared more robust and had higher weights for both SI and pancreas, compared to conventional formula fed pigs (P <0.05). Colostrum fed pigs also had markedly increased mucosal SI proportions, lung and spleen weights compared to conventional formula fed pigs. Colostrum fed pigs and germ-free pigs were similar although stomach, pancreas and distal SI weights were highest for germ-free pigs. The results demonstrate that the initial bacterial colonization interacts with diet to modulate the early neonatal organ development, particularly of the GIT. [Copyright &y& Elsevier]