Neonatal Gut and Immune Responses to ß-Casein Enriched Formula in Piglets.

BACKGROUND: ß-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less ß-casein than HM, but whether different concentrations of ß-casein affect tolerability and gut and immune maturation in newborns is unknown. OBJECTIVES: Using near-term piglets as a model for newborn infants, we investigated whether increasing the ß-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation. METHODS: Three groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) standard skim milk casein (BCN-standard, 35% ß-casein of total casein, n = 18); 2) ß-casein enrichment to HM concentrations (BCN-medium, 65%, n = 19); and 3) high ß-casein enrichment (BCN-high, 91%, n = 19). A reference group was fed 100% whey protein concentrate (WPC) as protein (WPC, n = 18). Intestinal and immune parameters were assessed before and after euthanasia on day 5. RESULTS: Clinical variables (mortality, activity, body growth, and diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of ß-casein (BCN-high) had lower small intestine weight and tended to have more intestinal complications (highest gut pathology score, permeability, and interleukin-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte, and reticulocyte counts were increased with higher ß-casein, whereas eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups. CONCLUSIONS: ß-casein enrichment of bovine-based formula to HM concentrations is clinically safe, as judged from newborn, near-term pigs, whereas no additional benefits to gut maturation were observed. However, excessive ß-casein supplementation, beyond concentrations in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural ß-casein concentrations or pure whey-based formula.

Enteral plasma supports brain repair in newborn pigs after birth asphyxia.

Newborns exposed to birth asphyxia transiently experience deficient blood flow and a lack of oxygen, potentially inducing hypoxic-ischaemic encephalopathy and subsequent neurological damage. Immunomodulatory components in plasma may dampen these responses. Using caesarean-delivered pigs as a model, we hypothesized that dietary plasma supplementation improves brain outcomes in pigs exposed to birth asphyxia. Mild birth asphyxia was induced by temporary occlusion of the umbilical cord prior to caesarean delivery. Motor development was assessed in asphyxiated (ASP) and control (CON) piglets using neonatal arousal, physical activity and gait test parameters before euthanasia on Day 4. The ASP pigs exhibited increased plasma lactate at birth, deficient motor skills and increased glial fibrillary acidic protein levels in CSF and astrogliosis in the putamen. The expression of genes related to oxidative stress, inflammation and synaptic functions was transiently altered in the motor cortex and caudate nucleus. The number of apoptotic cells among CTIP2-positive neurons in the motor cortex and striatal medium spiny neurons was increased, and maturation of preoligodendrocytes in the internal capsule was delayed. Plasma supplementation improved gait performance in the beam test, attenuated neuronal apoptosis and affected gene expression related to neuroinflammation, neurotransmission and antioxidants (motor cortex, caudate). We present a new clinically relevant animal model of moderate birth asphyxia inducing structural and functional brain damage. The components in plasma that support brain repair remain to be identified but may represent a therapeutic potential for infants and animals after birth asphyxia.

Insulin-like growth factor-1 infusion in preterm piglets does not affect growth parameters of skeletal muscle or tendon tissue.

Prematurity has physical consequences, such as lower birth weight, decreased muscle mass and increased risk of adult-onset metabolic disease. Insulin-like growth factor 1 (IGF-1) has therapeutic potential to improve the growth and quality of muscle and tendon in premature births, and thus attenuate some of these sequalae. We investigated the effect of IGF-1 on extensor carpi radialis muscle and biceps brachii tendon of preterm piglets. The preterm group consisted of 19-day-old preterm (10 days early) piglets, treated with either IGF-1 or vehicle. Term controls consisted of groups of 9-day-old piglets (D9) and 19-day-old piglets (D19). Muscle samples were analysed by immunofluorescence to determine the cross-sectional area (CSA) of muscle fibres, fibre type composition, satellite cell content and central nuclei-containing fibres in the muscle. Tendon samples were analysed for CSA, collagen content and maturation, and vascularization. Gene expression of the tendon was measured by RT-qPCR. Across all endpoints, we found no significant effect of IGF-1 treatment on preterm piglets. Preterm piglets had smaller muscle fibre CSA compared to D9 and D19 control group. Satellite cell content was similar across all groups. For tendon, we found an effect of age on tendon CSA, and mRNA levels of COL1A1, tenomodulin and scleraxis. Immunoreactivity for elastin and CD31, and several markers of tendon maturation, were increased in D9 compared to the preterm piglets. Collagen content was similar across groups. IGF-1 treatment of preterm-born piglets does not influence the growth and maturation of skeletal muscle and tendon.

Insulin-like growth factor-1 effects on kidney development in preterm piglets.

BACKGROUND: Preterm birth disrupts fetal kidney development, potentially leading to postnatal acute kidney injury. Preterm infants are deficient in insulin-like growth factor 1 (IGF-1), a growth factor that stimulates organ development. By utilizing a preterm pig model, this study investigated whether IGF-1 supplementation enhances preterm kidney maturation. METHODS: Cesarean-delivered preterm pigs were treated systemically IGF-1 or vehicle control for 5, 9 or 19 days after birth. Blood, urine, and kidney tissue were collected for biochemical, histological and gene expression analyses. Age-matched term-born pigs were sacrificed at similar postnatal ages and served as the reference group. RESULTS: Compared with term pigs, preterm pigs exhibited impaired kidney maturation, as indicated by analyses of renal morphology, histopathology, and inflammatory and injury markers. Supplementation with IGF-1 reduced signs of kidney immaturity, particularly in the first week of life, as indicated by improved morphology, upregulated expression of key developmental genes, reduced severity and incidence of microscopic lesions, and decreased levels of inflammatory and injury markers. No association was seen between the symptoms of necrotizing enterocolitis and kidney defects. CONCLUSION: Preterm birth in pigs impairs kidney maturation and exogenous IGF-1 treatment partially reverses this impairment. Early IGF-1 supplementation could support the development of preterm kidneys. IMPACT: Preterm birth may disrupt kidney development in newborns, potentially leading to morphological changes, injury, and inflammation. Preterm pigs have previously been used as models for preterm infants, but not for kidney development. IGF-1 supplementation promotes kidney maturation and alleviates renal impairments in the first week of life in preterm pigs. IGF-1 may hold potential as a supportive therapy for preterm infants sensitive to acute kidney injury.

Glucose-regulatory hormones and growth in very preterm infants fed fortified human milk.

BACKGROUND: Bovine colostrum (BC) contains a range of milk bioactive components, and it is unknown how human milk fortification with BC affects glucose-regulatory hormones in very preterm infants (VPIs). This study aimed to investigate the associations between hormone concentrations and fortification type, birth weight (appropriate/small for gestational age, AGA/SGA), milk intake, postnatal age, and body growth. METHODS: 225 VPIs were randomized to fortification with BC or conventional fortifier (CF). Plasma hormones were measured before, one and two weeks after start of fortification. ΔZ-scores from birth to 35 weeks postmenstrual age were calculated. RESULTS: Compared with CF, infants fortified with BC had higher plasma GLP-1, GIP, glucagon, and leptin concentrations after start of fortification. Prior to fortification, leptin concentrations were negatively associated with growth, while IGF-1 concentrations associated positively with growth during fortification. In AGA infants, hormone concentrations generally increased after one week of fortification. Relative to AGA infants, SGA infants showed reduced IGF-1 and leptin concentrations. CONCLUSION: Fortification with BC increased the plasma concentrations of several glucose-regulatory hormones. Concentrations of IGF-1 were positively, and leptin negatively, associated with growth. Glucose-regulatory hormone levels were affected by birth weight, milk intake and postnatal age, but not closely associated with growth in VPIs. IMPACT: Little is known about the variation in glucose-regulatory hormones in the early life of very preterm infants (VPIs). This study shows that the levels of glucose-regulatory hormones in plasma of VPIs are highly variable and modified by birth weight (appropriate or small for gestational age, AGA or SGA), the type of fortifier, enteral nutritional intake, and advancing postnatal age. The results confirm that IGF-1 levels are positively associated with early postnatal growth in VPIs, yet the levels of both IGF-1 and other glucose-regulatory hormones appeared to explain only a small part of the overall variation in growth rates.

Bovine colostrum prevents formula-induced gut microbiota dysbiosis in preterm pigs.

BACKGROUND: Preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), a gut inflammatory disease known to be associated with gut microbiota (GM) changes in infants. Supplemental bovine colostrum may protect against formula-induced NEC via GM changes. We hypothesised that feeding colostrum before, after, or during formula feeding affects NEC sensitivity via changes to GM. METHODS: Colonic GM (profiled by 16S ribosomal RNA gene amplicon sequencing) was compared in preterm pigs fed colostrum for 4 days, either before, after, or together with formula feeding for 4 days. Correlations between GM and gut parameters were assessed on day 5 or 9. RESULTS: Both exclusive and partial colostrum feeding induced higher GM diversity, lower Enterococcus abundance, and improved intestinal maturation parameters (villus structure, digestive enzyme activities, permeability), relative to exclusive formula feeding (all p < 0.05). Across feeding regimens, Enterococcus abundance was inversely correlated with intestinal maturation parameters. Conversely, there was no correlation between GM changes and early NEC lesions. CONCLUSION: Bovine colostrum inhibits formula-induced Enterococcus overgrowth and gut dysfunctions just after preterm birth but these effects are not causally linked. Optimising diet-related host responses, not GM, may be critical to prevent NEC in preterm newborn pigs and infants. IMPACT: Supplement of bovine colostrum to formula feeding modified the gut microbiota by increasing species diversity and reducing Enterococcus abundance, while concurrently improving intestinal functions in preterm pigs. Diet-related changes to the gut microbiota were not clearly associated with development of necrotizing enterocolitis (NEC) in preterm pigs, suggesting that diet-related gut microbiota effects are not critical for diet-related NEC protection. The study highlights the potential to use bovine colostrum as a supplement to formula feeding for preterm infants lacking human milk.

Systemic immune markers and infection risk in preterm infants fed human milk fortified with bovine colostrum or conventional fortifier, a secondary analysis of the FortiColos trial.

BACKGROUND: For very preterm infants, human milk is often fortified with formula products based on processed bovine milk. Intact bovine colostrum (BC), rich in anti-inflammatory milk factors, is considered an alternative. We investigated if BC affects anti-inflammatory/TH2 immunity and infection risk in very preterm infants. METHODS: For a secondary analysis of a multicenter, randomized controlled trial (NCT03537365), very preterm infants (26-31 weeks gestation, 23% small for gestational age, SGA) were randomized to receive BC (ColoDan, Biofiber, Denmark, n = 113) or conventional fortifier (PreNAN, Nestlé, Switzerland, n = 116). Infection was defined as antibiotic treatment for five or more consecutive days and 29 cytokines/chemokines were measured in plasma before and after start of fortification. RESULTS: In general, infection risk after start of fortification was associated with low gestational age, SGA status and antibiotics use prior to fortification. Adjusted for confounders, infants fortified with BC showed more infection episodes (20 vs 12%, P < 0.05) and higher cumulative infection risk (hazard ratio, HR 1.9, P = 0.06), particularly for SGA infants (HR 3.6, P < 0.05). Additionally, BC-fortified infants had higher levels of TH2-related cytokines/chemokines (IL-10, MDC, MCP4) and reduced levels of cytokines related to TH1/TH17-responses (IL-15, IL-17, GM-CSF). The differences were most pronounced in SGA infants, displaying higher levels of TH2-related IL-4, IL-6, and IL-13, and lower interferon-γ and IL-1α levels in the BC group. CONCLUSION: Infants fortified with BC displayed a delayed shift from TH2- to TH1-biased systemic immunity, notably in SGA infants, possibly influenced by multiple confounding factors, alongside elevated antibiotic use, suggesting increased susceptibility to infection.

Mineral supplementation for very preterm infants fed fortified human milk.

OBJECTIVES: The safety and feasibility of human milk fortification with bovine colostrum (BC) were investigated in very preterm infants (FortiColos trial, NCT03537365). The BC product contained lower calcium, phosphate, and iron levels compared to the conventional fortifier (CF). We tested whether fortification with BC plus extra phosphate was sufficient to support the infants' mineral status assessed by blood biochemistry. METHODS: In a randomised controlled trial (FortiColos, NCT03537365), mineral status was compared after fortification with BC versus CF. Blood calcium, phosphate, and haemoglobin were determined before and up to 3 weeks after the start of fortification (at the mean age of 8-9 days). The maximum supplemental doses of calcium, phosphate, and iron given were retrieved from patient medical records. Results were adjusted for gestational age, birth weight, and enteral nutrition with the mother's own milk and/or donor human milk. RESULTS: Blood values of calcium, phosphate, and haemoglobin were similar between groups. Infants in both groups required supplementation with calcium and phosphate, but infants fed BC required higher maximum doses of phosphate and calcium (p < 0.05) to maintain acceptable blood values. Regardless of fortification groups, the most immature (<29 weeks of gestation) and small for gestational age infants showed a higher risk for requiring additional phosphate (odds ratio [OR]: 3.9, p < 0.001; OR: 2.14, p = 0.07, respectively). CONCLUSIONS: The use of BC as a fortifier for human milk requires additional phosphate and calcium relative to a CF. Regardless of the fortification product, the most immature and small infants require additional mineral supplementation.

Neonatal prophylactic antibiotics after preterm birth affect plasma proteome and immune development in pigs.

BACKGROUND: Most preterm infants receive antibiotics to prevent serious infections shortly after birth. However, prolonged antibiotic treatment predisposes to gut dysbiosis and late-onset sepsis. Using preterm pigs as model, we hypothesized that neonatal prophylactic antibiotics impair systemic immune development beyond the days of antibiotic treatment. METHODS: Preterm pigs (90% gestation) were fed formula for 9 days, treated with sterile water (CON) or enteral antibiotics from day 1 to 4. On days 5 and 9, blood was collected for haematology, in vitro LPS stimulation, and plasma proteomics. RESULTS: Antibiotic treatment altered the abundance of 21 and 47 plasma proteins on days 5 and 9, representing 6.6% and 14.8% of the total annotated proteins, respectively. Most antibiotics-induced proteome changes related to complement cascade, neutrophil degranulation, and acute phase responses. Neutrophil and lymphocyte counts were higher in antibiotics-treated pigs on day 5 but did not change from days 5-9, in contrast to increasing cell counts in CON. The antibiotics treatment suppressed TNF-alpha and IL-10 responses to in vitro LPS challenge on day 5, 7 and 9. CONCLUSION: Few days of antibiotics treatment following preterm birth alter the plasma proteome and inhibit systemic immune development, even beyond the days of treatment. IMPACT: 1. Neonatal prophylactic antibiotics alter the plasma proteome and suppress systemic immune development in preterm pigs 2. The effects of prophylactic antibiotics last beyond the days of treatment. 3. Neonatal antibiotics treatment for compromised human newborns may predispose to longer-term risks of impaired immunity and infections.

Gut development following insulin-like growth factor-1 supplementation to preterm pigs.

BACKGROUND: Reduced insulin-like growth factor-1 (IGF-1) levels may contribute to impaired organ development in preterm infants. Using preterm pigs as a model, we hypothesized that IGF-1 supplementation improves health and gut development during the first three weeks of life. METHODS: First, clinical and organ endpoints were compared between artificially-reared, cesarean-delivered preterm pigs and vaginally-delivered, sow-reared term pigs at 5, 9 and 19 days. Next, preterm pigs were treated with recombinant human IGF-1 for 19 days (2.25 mg/kg/day, systemically). RESULTS: Relative to term pigs, preterm pigs had lower body weight, fat, bone contents, relative weights of liver and spleen and a longer and thinner intestine at 19 days. Preterm birth reduced intestinal villi heights and peptidase activities, but only at 5 and 9 days. In preterm pigs, IGF-1 reduced mortality primarily occurring from gastrointestinal complications and with a tendency towards salvaging smaller pigs. IGF-1 supplementation also increased spleen and kidney weights, small intestine length and maltase to lactase activity, reflecting gut maturation. CONCLUSION: Preterm birth affects body composition and gut maturation in the first 1-2 weeks, but differences are marginal thereafter. Supplemental IGF-1 may improve gut health in pigs and infants in the first few weeks after preterm birth. IMPACT: Insulin-like growth factor 1 (IGF-1) supplementation may improve gut health and development in prematurity, but whether the effects are sustained beyond the immediate postnatal period is unclear. In preterm pigs, the prematurity effects on IGF-1 and gut health deficiencies are most pronounced during the first week of life and diminishes thereafter. In preterm pigs, IGF-1 supplementation beyond the first week of life reduced mortality. The present study provides evidence of a sustained effect of IGF-1 supplementation on the gastrointestinal tract after the immediate postnatal period.

Blood transcriptomic markers of necrotizing enterocolitis in preterm pigs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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. IMPACT: 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.

Supplementary Bovine Colostrum Feedings to Formula-Fed Preterm Pigs Improve Gut Function and Reduce Necrotizing Enterocolitis.

OBJECTIVES: Exclusive feeding with bovine colostrum (BC) protects preterm pigs against necrotizing enterocolitis (NEC) and BC has recently been tested as a supplement to a mother's own milk or formula (FOR) for very preterm infants. Using preterm pigs as a model for infants, we investigated if BC has gut- and NEC-protective effects at different proportions of the daily enteral intake given as BC. METHODS: Sixty-eight caesarean-delivered preterm piglets (90% gestation) were allocated into four groups with increasing proportions of eight daily bolus feedings as BC: BC00 (only FOR feeding), BC25 (25% BC), BC50 (50% BC), or BC75 (75% BC). On day 5, the gut was collected for biochemical analyses. RESULTS: Body growth was increased in BC50 and BC75 piglets (2-fold, P < 0.05 vs BC00). The incidence of mild NEC-like lesions was similar among groups (67-86%), but BC75 reduced severe NEC-like lesions (27% vs 79% in BC00, P < 0.05). BC50 and BC75 improved hexose absorption and mucosal structure and reduced gut permeability (P < 0.05 vs BC00), while enzyme activities (lactase, aminopeptidase N and A, dipeptidyl peptidase IV) were improved in all pigs fed BC (P < 0.05). Across the measured variables, beneficial effects were most clear for the BC75 group, including reductions in colon tissue cytokine levels (interleukin 8, interleukin 1ß, tumor necrosis factor α) and expression of immune- and apoptosis-related genes (LBP, TLR4, TLR2, IL8, STAT3, IL17, C3, all P < 0.05, relative to BC00). CONCLUSION: A proportion of 50-75% of daily enteral intake as BC is required to improve the intestinal structure, function, immunology, and NEC resistance in preterm piglets also fed formula. Further studies are required to show if and how supplementary BC may support gut development in preterm infants during the immediate postnatal period. It is challenging to translate results on optimal feeding regimens between species, and preterm infants would not receive a majority of their daily enteral intake as BC.

Nutrient Restriction has Limited Short-Term Effects on Gut, Immunity, and Brain Development in Preterm Pigs.

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.

Physical Activity and Spatial Memory Are Minimally Affected by Moderate Growth Restriction in Preterm Piglets.

BACKGROUND: Preterm birth is associated with impaired brain functions, but it is unknown whether fetal growth restriction (GR) makes these deficits worse. Using piglets as a model for preterm infants, we hypothesized that moderate GR reduces growth rate, physical activity, and spatial memory in the first weeks after preterm birth. METHODS: Preterm pigs were delivered by caesarean section and fed until 19 days (n = 830 from 55 pregnant sows) and received intensive clinical care. GR pigs were classified as animals with the lowest 5-20% percentile birth weight within each litter and were compared with litter-mate controls (21-100% percentile birth weight). Basic motor skill development, physical activity, and morbidities (e.g., necrotizing enterocolitis) were recorded within the first week. Weight of internal organs and data from a T-maze spatial memory test were noted until 19 days. RESULTS: Moderate GR and control preterm pigs (birth weights 728 ± 140 and 1,019 ± 204 g, respectively) showed similar relative weights of internal organs (relative to body), except higher adrenal gland weights in GR pigs (+20-50%, p < 0.05). This was associated with a tendency to higher plasma cortisol (p < 0.05 on day 11). GR preterm pigs showed delayed ability to stand and walk (days 2-5, p < 0.01), but physical activity and proportion of correct choices in a T-maze test (70.3 vs. 71.6%) were similar. CONCLUSION: Moderate GR has limited effect on motor function and spatial memory in the early postnatal period of preterm pigs, despite some initial delays in basic motor skills. In the postnatal period, moderately growth-restricted preterm infants may adapt well with regards to organ growth and neurodevelopment.

Preterm Birth Has Effects on Gut Colonization in Piglets Within the First 4 Weeks of Life.

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.

Brain Barrier Disruption and Region-Specific Neuronal Degeneration during Necrotizing Enterocolitis in Preterm Pigs.

Necrotizing enterocolitis (NEC) increases the risk of brain injury and impaired neurodevelopment. Rapid brain maturation prior to birth may explain why preterm brains are particularly vulnerable to serious infections. Using pigs as models, we hypothesized that preterm birth was associated with altered blood-cerebrospinal fluid (CSF) barrier (BCSFB) function and cerebral structural deficits, and that NEC was associated with systemic inflammation, BCSFB disruption, and neuroinflammation. First, cesarean-delivered preterm and term pigs (n = 43-44) were euthanized at birth to investigate BCSFB function and markers of brain structural maturation, or on day 5 to measure markers of blood-brain barrier maturation in the hippocampus and striatum (experiment 1). Next, preterm pigs (n = 162) were fed increasing volumes of infant formula to assess NEC lesions, systemic inflammation, BCSFB permeability, cerebral histopathology, hippocampal micro-glial density, and cytokine levels on day 5 (experiments 2 and 3). In experiment 1, preterm newborns had increased CSF-plasma ratios of albumin and raffinose, reduced CSF glucose levels, as well as increased cerebral hydration and reduced white matter myelination compared with term animals. We observed lower hippocampal (but not striatal) perivascular astrocyte coverage for the first 5 days after preterm birth, accompanied by altered cell junction protein levels. In experiments 2 and- 3, piglets with severe NEC lesions showed reduced blood thrombocytes and increased plasma C-reactive protein and interleukin-6 levels. NEC was associated with increased CSF-plasma albumin and raffinose ratios, reduced CSF leukocyte numbers, and increased cerebral hydration. In the hippocampus, NEC was associated with pyramidal neuron loss and increased interleukin-6 levels. In the short term, NEC did not affect cerebral myelination or microglia density. In conclusion, altered BCSFB properties and brain structural deficits were observed in pigs after preterm birth. Acute gastrointestinal NEC lesions were associated with systemic inflammation, increased BCSFB permeability and region-specific neuronal damage. The results demonstrate the importance of early interventions against NEC to prevent brain injury in preterm infants.

Rapid Postnatal Adaptation of Neurodevelopment in Pigs Born Late Preterm.

Preterm birth interrupts intrauterine brain growth and maturation and may induce a delay in postnatal neurodevelopment. Such developmental delays can result from the reduced fetal age at birth, together with the clinical compli-cations of preterm birth (e.g., hypoxia, ischemia, and inflammation). We hypothesized that late preterm birth, inducing only mild clinical complications, has minimal effects on brain-related outcomes such as motor function and behavior. Using the pig as a model for late preterm infants, piglets were cesarean delivered preterm (90%, 106 days gestation) or at full term, reared by identical procedures, and euthanized for tissue collection at birth or after 11 days (e.g., term-corrected age for preterm pigs). Clinical variables and both structural and functional brain endpoints were assessed. The preterm pigs were slow to get on their feet, gained less weight (-30%), and had a higher cerebral hydration level and blood-to-cerebrospinal fluid permeability than the term pigs. At term-corrected age (11 days), the absolute weight of the brain and the weights of its regions were similar between 11-day-old preterm and newborn term pigs, and both were lower than in 11-day-old term pigs. Postnatally, physical activity and movements in an open field were similar, except that preterm pigs showed a reduced normalized stride length and increased normalized maximum stride height. Perinatal brain growth is closely associated with advancing postconceptional age in pigs, and late preterm birth is initially associated with impaired brain growth and physical activity. Postnatally, neuromuscular functions mature rapidly and become similar to those in term pigs, even before term-corrected age. Neuromuscular functions and behavior may show rapid postnatal adaptation to late preterm birth in both pigs and infants.

Bovine Colostrum for Preterm Infants in the First Days of Life: A Randomized Controlled Pilot Trial.

OBJECTIVES: Mother's own milk (MM) is the best nutrition for newborn preterm infants, but is often limited in supply just after birth. Pasteurized human donor milk (DM), and especially preterm infant formula (IF) are less optimal diets than MM. We hypothesized that it is feasible to use bovine colostrum (BC), the first milk from cows, as a supplement to MM, during the first weeks after preterm birth. METHODS: In an open-label, randomized, controlled pilot safety trial, supplementation of MM with BC was compared with DM supplementation (Danish unit) or IF supplementation (Chinese unit). If MM was limited or lacking, BC, DM or IF were given according to local feeding guidelines during the first 14 days of life. RESULTS: Forty infants were included and randomized in Denmark and in China, with gestational ages 29.9 ±â€Š0.4 and 31.1 ±â€Š0.2 weeks, respectively. Infants supplemented with BC received more enteral protein (P < 0.05) and tended to reach full enteral feeding earlier (China only). Eight infants fed BC showed a temporary elevation in plasma tyrosine on day 7, versus 2 infants in the DM/IF groups. There were no differences between diet groups in feeding intolerance or clinical adverse events. CONCLUSIONS: Our results indicate that it is feasible to use BC as a supplement to MM during the first weeks of life to increase enteral protein intake in preterm infants. Plasma tyrosine levels may be a good marker for excessive protein intake. A larger randomized trial is required to test the safety and possible short- and long-term clinical benefits of BC supplementation during the first weeks of life for preterm infants.

Antibiotic Treatment Preventing Necrotising Enterocolitis Alters Urinary and Plasma Metabolomes in Preterm Pigs.

Necrotising enterocolitis (NEC) is a serious gut inflammatory condition in premature neonates, onset and development of which depend on the gut microbiome. Attenuation of the gut microbiome by antibiotics can reduce NEC incidence and severity. However, how the antibiotics-suppressed gut microbiome affects the whole-body metabolism in NEC-sensitive premature neonates is unknown. In formula-fed preterm pigs, used as a model for preterm infants, plasma and urinary metabolomes were investigated by LC-MS and 1H NMR, with and without antibiotic treatment immediately after birth. While it reduced the gut microbiome density and NEC lesions as previously reported, the antibiotic treatment employed in the current study affected the abundance of 44 metabolites in different metabolic pathways. In antibiotics-treated pigs, tryptophan metabolism favored the kynurenine pathway, relative to the serotonin pathway, as shown by specific metabolites. Metabolites associated with the gut microbiome, including 3-phenyllactic acid, 4-hydroxyphenylacetic acid, and phenylacetylglycine, all from phenylalanine, and three bile acids showed lower levels in the antibiotics-treated pigs where the gut microbiome was extensively attenuated. Findings in the current study warrant further investigation of metabolic and developmental consequences of antibiotic treatment in preterm neonates.

Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs.

BACKGROUND: Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome. RESULTS: We found that the MspI-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110-220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40-110 bp, 110-220 bp and 40-220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40-220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions. CONCLUSIONS: In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.