Gut response to pasteurized donor human milk in a porcine model of the premature infant.

This study investigated the tolerance and safety of pasteurized donor human milk (PDHM) given either alone or together with commercially-used supplements in a porcine model of premature infants. A porcine model, mimicking human neonates at 30-32 weeks of gestational age, was used. The 7-day experiment was performed on 20 piglets. After birth, the piglets were infused with porcine immunoglobulins via the umbilical artery and surgically fitted with a stomach port. The piglets were then randomized into five groups and fed either PDHM, different variants of fortified PDHM or 'raw' human milk (RHM). Preterm piglets fed PDHM showed signs of gastrointestinal intolerance. Four piglets across the various PDHM-fed groups died, none of them were from the group fed PDHM supplemented with long-chain polyunsaturated fatty acids (LC PUFA). In all groups fed PDHM, macroscopic features of enterocolitis were observed, however, these pathological gut changes were less manifested in piglets receiving PDHM supplemented with LC PUFA. The piglets fed RHM had no specific signs of gut damage. The poor tolerance to PDHM suggests changes in milk composition caused by the Holder pasteurization. The supplementation with LC PUFA probably improves tolerance to PDHM.

The pig as a model for premature infants - the importance of immunoglobulin supplementation for growth and development.

Preterm human neonates, contrary to preterm piglets, obtain immunoglobulins from their mothers via the placenta during intrauterine development. However, one should note that the majority of trans-placental transfer of immunoglobulins in humans takes place during the last trimester of pregnancy. It is also known that the feeding of limited amounts of colostrum or systemic infusion of small amounts of serum improves the survival of preterm and full-term piglets. Full-term piglets deprived of their mother's immunoglobulins exhibit strong apathy and develop watery diarrhoea, often resulting in death. The aim of the current study was to determine if provision of immunoglobulins using different approaches would be beneficial for survival outcomes. To reach the immunological sufficient level we infused immunoglobulins intravenously in amount mimicking the blood level in piglets fed with sow colostrum. Intravenous infusion of immunoglobulins in both preterm and full-term newborn piglets fully ensured their survival, growth and blood immunoglobulin G and protein levels similar to those observed in piglets fed colostrum. Piglets completely deprived of immunoglobulins exhibited significantly lower blood levels of immunoglobulins and protein compared to colostrum-fed animals. Piglets infused with only serum exhibited significantly lower blood immunoglobulin G level compared to those infused with immunoglobulins. In conclusion, based on the data obtained, we suggest that passive immune support provided by colostrum intake or early systemic infusion of Ig's in sufficient amounts is key to ensuring the general well-being of preterm and full-term new born piglets, used as an animal model for the human infant.

The influence of enteral obestatin administration to suckling rats on intestinal contractility.

This study investigated the effect of enteral administration of obestatin on the contractility of whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated with: C-saline solution; LO-obestatin (125nmol/kgb.wt); HO-obestatin (250nmol/kgb.wt). Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life. Sections were studied in an organ bath, for isometric recording in the presence of acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was performed in the intestinal segments. In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction compared to that observed in the C group. In the middle jejunum, the LO treatment also decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous contraction in the jejunum of LO and HO-treated animals. Compared to the C group, duodenal sections from HO animals and middle jejunum sections from LO and HO groups displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in the thickness of the muscularis layer was observed in the duodenum of LO and HO groups whereas the number ICC did not change significantly after treatment with obestatin. Moreover, the enteral administration of obestatin did not effect significantly on the cytoplasmic expression of M2 receptor in the jejunum. Our study demonstrated that enteral administration of obestatin to suckling rats influences small intestine contractility in the segment specific manner.

Age-dependent effect of obestatin on intestinal contractility in Wistar rats.

Obestatin is a 23-amino acid peptide encoded by the ghrelin gene. We have investigated the effect of obestatin on intestinal contractility in rats ranging from the suckling period till adolescence. Duodenal and middle jejunum whole-thickness preparations from neonatal and adult rats were studied in an organ bath, for isometric recording under treatment with obestatin (1µmolL(-1)) in the presence of acetylocholine (ACh), atropine and tetradotoxin (TTX). Both the EFS and ACh-stimulated contractile response, as well as spontaneous contractile activity is age-dependent and specific for the segment of jejunum. Except for the middle jejunum of 7day old rats, treatment with obestatin caused a significant TTX-sensitive increase in the amplitude of EFS-stimulated off-contraction of both intestinal segments studied. Following injection of obestatin, the amplitude of spontaneous contraction in the duodenum increased in 7day old rats. In the middle jejunum, treatment with obestatin significantly increased both the amplitude and frequency of spontaneous contraction in rats till the 28th day of life, whereas in adult rats the observed effect of obestatin was the opposite (P<0.001 and P<0.0001, respectively). The effects of treatment with obestatin on stimulation with increasing doses of ACh were only observed in the preparations from suckling rats. ACh-stimulated contractility in the duodenum was decreased while in the middle jejunum the observed effect was opposite. These results indicate the importance of peripheral obestatin in the cholinergic control of intestinal contractility in both neonatal and adult rats.

Influence of obestatin on the histological development of the small intestine in piglets during the first week of postnatal life.

Obestatin is a gastrointestinal peptide having wide-ranging effects on cell proliferation; however, its mechanism of action remains poorly understood. Thus, the aim of the study was to elucidate the effect of exogenous obestatin on the postnatal structural development of the small intestine. Seven-day-old piglets with an average BW of 1.56 ± 0.23 kg were divided into four groups (n = 10) that received intragastrically obestatin (2, 10 or 15 µg/kg BW) or vehicle. After a 6-day experimental period, morphological analysis of gastrointestinal tract and small intestine wall (mitosis and apoptosis indexes, histomorphometry of mucosa and muscularis layers) was performed. The study revealed a seemingly incoherent pattern of the histological structure of the small intestine among the experimental groups, suggesting that the effect of obestatin is both intestinal segment specific and dose dependent. Histomorphometric analysis of the small intestine showed that higher doses of obestatin seem to promote the structural development of the duodenum while simultaneously hindering the maturation of more distal parts of the intestine. Intragastric administration of obestatin increased the crypt mitotic index in all segments of the small intestine with the strongest pro-mitotic activity following the administration of obestatin at a dose of 10 and 15 µg/kg BW. The significant differences in the number of apoptotic cells in the intestinal villi among the groups were observed only in proximal jejunum and ileum. In conclusion, it seems that obestatin shows a broad-spectrum of activity in the gastrointestinal tract of newborn piglets, being able to accelerate its structural development. However, the varied effect depending on the intestinal segment or the concentration of exogenous obestatin causes that further research is needed to clarify the exact mechanism of this phenomenon.

Sodium-butyrate as a growth promoter in milk replacer formula for young calves.

In milk-fed calves, the effects of sodium-butyrate (Na-butyrate) to replace flavomycin on growth performance and some mechanisms involved were studied. Pancreatic and intestinal morphology, digestive enzyme activities, plasma gut regulatory peptide concentrations, and expression of their receptors in the gastrointestinal tract were measured. Gastrointestinal tract defense systems were examined by measuring protein levels of 2 heat-shock proteins (HSP27 and HSP70). The calves were randomly allocated into 2 groups fed the same basic diet with flavomycin as an antimicrobial growth promoter or with Na-butyrate (3 g/kg of dry matter). Sodium-butyrate disappeared quickly in the upper gut and was not found in circulating blood. Supplementation with Na-butyrate enhanced growth rate and improved feed conversion into body weight gain compared with the flavomycin group. Supplementation with Na-butyrate was likely associated with an improvement in efficacy of the gastrointestinal tract digestive capacities expressed by enhanced production of digestive enzymes and increased absorptive capacities in the upper small intestine. The effects could have been controlled by insulin-like growth factor-1 but probably not by any of the cholecystokinin/gastrin peptide family. Concentrations of HSP27 and HSP70 were increased in stomach and colon of calves receiving Na-butyrate, thereby assuring protection of cells with intensive metabolism (chaperone function). In conclusion, beneficial effects of Na-butyrate on maturation of gastrointestinal functions were shown in milk-fed calves and may be applied to young mammals of other species.

Skim milk powder with high content of Maillard reaction products affect weight gain, organ development and intestinal inflammation in early life in rats.

BACKGROUND: The intestinal tract is important for development of immune tolerance and disturbances are suggested to trigger autoimmune disorders. The aim of this study was to explore the effect of Maillard products in skim milk powder obtained after long storage, compared to fresh skim milk powder. METHODS: Young rats were weaned onto a diet based on skim milk powder with high concentration of Maillard products (HM-SM, n = 18) or low (C-SM, n = 18) for one week or four weeks. Weekly body weight and feed consumption were noted. At the end, organ weights, intestinal histology, permeability and inflammatory cytokines were evaluated. RESULTS: Rats fed with HM-SM had after one week, 15% less weight gain than controls, despite equal feed intake. After one week thymus and spleen were smaller, intestinal mucosa thickness was increased and acute inflammatory cytokines (IL-17, IL-1ß, MCP-1) were elevated. After four weeks, cytokines associated with chronic intestinal inflammation (fractalkine, IP-10, leptin, LIX, MIP-2, RANTES and VEGF) were increased in rats fed with HM-SM compared to C-SM. CONCLUSION: High content of Maillard products in stored milk powder caused an intestinal inflammation. Whether this is relevant for tolerance development and future autoimmune diseases remains to be explored.

Advances in the ultrastructural study of the implant-bone interface by backscattered electron imaging.

The biocompatibility of titanium implants in bone depends on the response shown by cells in contact with the implant surface. Several developments have been targeted at achieving successful implant treatment. The aim of this study was to develop a novel preparation procedure to evaluate the bone cell response produced at the bone-implant interface using the technique scanning electron microscopy with backscattered electron imaging (SEM-BSE). Dental prostheses with an SLA-modified or TOP-modified surface were implanted in a toothless part of the mandibula in female pigs. The animals were sacrificed 12 weeks after surgery, at which time block specimens containing the implants were obtained. These specimens were then processed for SEM-BSE by optimizing a protocol involving chemical fixation and heavy metal staining. In addition, element distribution maps for the implant-bone tissue interface were obtained using a microanalytical system based on energy-dispersive X-ray spectrometry (EDS). This novel visualisation approach enabled a comprehensive study of the extracellular matrix and cell components of the host tissues neoformed around the implant. SEM-BSE images also provided ultrastructural details of the bone cells. This technique appears to be an effective and very promising tool for detailed studies on the implant-bone tissue interface and the host response to the bone incorporation process.

Influence of preterm delivery on ghrelin and obestatin concentrations in maternal plasm, milk and their expression in mammary epithelial cells.

Ghrelin and obestatin are gastrointestinal peptides with a potential role in the programming of metabolism in newborns. The present study aimed to investigate the influence of preterm delivery on ghrelin and obestatin concentrations in the maternal blood plasma and breast milk as well as their gene expressions in the mammary epithelial cells (MECs). On the 3rd day after delivery, milk and plasma samples were collected from mothers that carried to term or gave birth prematurely (< 36 weeks of gestation) and analyzed for ghrelin and obestatin concentrations. MECs isolated from the milk were analyzed for the relative expression of GHRL splice variants. In both groups ghrelin concentrations were significantly lower in milk than in blood plasma. In the preterm group obestatin concentrations were significantly higher in milk than in blood plasma but significantly lower in comparison to that of the control mothers. The expression of GHRL mRNAs was higher (P < 0.05) in MECs isolated from the preterm group as compared to those isolated from control mothers. The concentration of obestatin (but not ghrelin) in the breast milk is dependent on the term of pregnancy. Moreover, the lactating mammary gland is one of the sources of ghrelin and obestatin.

Maternal high-fat diet during pregnancy and lactation had gender difference effect on adiponectin in rat offspring.

This study investigated the effect of a high-fat (HF) diet on plasma adiponectin and steroid hormones levels, and the protein expression of adiponectin and its receptors, in the gonads and gonadal (periovarian and epididymal) white adipose tissue (WAT) of dams and their offspring. Female Wistar rats were fed a HF diet (30% fat) or a standard breeding (BD) diet (5% fat) during pregnancy and lactation. At 21 days of lactation, mothers and both sexes of prepubertal offspring were killed by decapitation. Plasma adiponectin, testosterone (T) and oestradiol (E2) levels were analyzed by ELISA. The protein expression of adiponectin and its receptors 1 (AdipoR1) and 2 (AdipoR2) was assayed by Western blot and immunohistochemistry. Plasma adiponectin levels in HF dams were lower compared to BD dams, and correlated with protein expression of adiponectin and its receptors, but not with steroid hormone levels. Female HF offspring had lower plasma adiponectin levels, reduced intensity of adiponectin and AdipoR1 in the ovary, and decreased E2 in parallel with increased T. In contrast, male HF offspring had higher plasma adiponectin levels, increased intensity of adiponectin and AdipoR1 in the testis, and decreased T in parallel with increased E2. In conclusion, feeding a HF diet to dams during pregnancy and lactation disturbs plasma adiponectin levels and protein expression, both in female and male offspring; it lowers adiponectin secretion and protein expression in the female whereas in male it is increased. As a consequence, there is disruption of steroid secretion in offspring, towards T in females, and E2 in males.

Exogenous leptin controls the development of the small intestine in neonatal piglets.

Leptin, a hormone produced and secreted by adipose tIssue, muscles and stomach, is involved in the regulation of adipose tIssue mass, food intake and body weight in neonatal animals. It is also produced in the mammary glands and secreted into the colostrum and milk. Since leptin receptors are widely distributed in the small intestine mucosa, the aim of the present study was to investigate the effect of exogenous leptin on the development of the small intestine in neonatal piglets. Male neonatal piglets were fed with sow's milk or artificial milk formula. Every 8 h the latter received either vehicle or leptin (2 or 10 microg/kg body weight). The animals were either killed after 6 days of treatment and the small intestine sampled for histology and brush border enzyme activities or were tested for marker molecule (Na-fluorescein and BSA) absorption in vivo. Feeding milk formula slowed the maturation of small intestinal mucosa compared with feeding sow's milk. However, after leptin treatment the length of the small intestine was increased, and intestinal villi length, but not crypt size, was reduced compared with controls. The mitotic index was increased and the percentage of vacuolated enterocytes was reduced in the entire small intestine. Enterocyte brush border protease and lactase activities were reduced in the jejunum. Na-fluorescein marker molecule absorption did not change but that of BSA was reduced 3.8-fold. In conclusion, exogenous leptin administered in physiological doses reversed the maturation of the small intestinal mucosa to the range found in sow-reared piglets.

Physiological and chemical characteristics of antibacterial activity of pancreatic juice.

Attempts were made to find and characterize an antibacterial activity (ABA) factor in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor was found to be heat resistant, the first step was heating for 30 min at 65 degrees C. Afterwards column chromatography, ethanol precipitation and polyacrylamide gel electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with antibacterial activity against Escherichia coli strain AB1157. In the presence of this fraction the number of living bacterial cells in overnight culture decreased about 10,000 fold and a spot-test gave clearly positive results. The results of analysis suggest that the antibacterial factor is a polypeptide active in a pH range 8.0-8.5, that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass spectroscopy analysis of active fraction showed high concentration of porcine pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling bacterial homeostasis has been found in the porcine pancreatic juice.

Enteral leptin administration affects intestinal autophagy in suckling piglets.

Leptin has been shown to play an integral role in the endocrine regulation of metabolism. Moreover, a substantial amount of this peptide has been found in colostrum and milk. The aim of the study was to investigate the effects of exogenous leptin, administered intragastrically, on the process of autophagy and the changes in cell hyperplasia and hypertrophy in the small intestine mucosa. Three groups (n = 6) of neonatal piglets were used in the study. The pigs were fed either by their sows (sow-reared piglets) or with only milk formula, or with milk formula together with leptin administered via a stomach tube (10 µg/kg BW) every 8 h for 6 d. We have shown that pure milk formula feeding significantly elevates (P < 0.05) autophagy compared with that observed in sow-reared piglets. Compared with the control group, feeding milk formula supplemented with leptin resulted in a significant decrease (P < 0.05) in immunodetection of microtubule-associated protein 1 light chain 3, as well as significantly accelerated epithelial cell renewal (P < 0.05). We demonstrated that autophagy is involved in the remodeling of the small intestine mucosa and that leptin, when administered enterally, may be an important factor for its regulation.

Stimulating effect of pancreatic-like enzymes on the development of the gastrointestinal tract in piglets.

Use of nutritional components from the milk and eventually from the solid feed relates to the growth and development of gastrointestinal tract (GIT). We studied the effect of pancreatic-like enzymes [porcine pancreatic enzymes (Creon) or microbial-derived amylase, protease, and lipase] on GIT morphology and lipid absorption in suckling piglets. Both enzyme preparations, in low or high dose, were fed via a stomach tube twice a day for 7 d starting at 8 d of age and controls received vehicle, n = 6. The day after treatments ended, lipid absorption was tested after which pigs were euthanized and GIT was examined. Enzyme cocktails, irrespective of their origin, increased (P < 0.001) triglyceride level in blood. Enzyme preparation affected (P < 0.001) small intestinal mucosal thickness, villi length, and crypt depth and (P < 0.01) mitotic division of enterocytes. In addition, the external administration of pancreatic enzymes stimulated pancreatic growth as observed by increased (P < 0.05) mitotic division of pancreatic cells. The study revealed that pancreatic or pancreatic-like enzymes of microbial origin administrated in the early postperinatal period enhance GIT development and may be used to better prepare the GIT of piglets for milk use and weaning.

Effect of feeding colostrum versus exogenous immunoglobulin G on gastrointestinal structure and enteric nervous system in newborn pigs.

Colostrum is an indispensable source of antibodies (IgG) protecting the newborn pig against infection. We studied the effect of feeding colostrum and purified IgG on early structure and development of the gastrointestinal tract (GIT). Newborn littermate pigs were fed either colostrum, an elemental diet (ED), or an ED supplemented with purified serum IgG (ED + IgG) for 24 h or then only ED up to 72 h. Afterwards, pigs were slaughtered. Colostrum-fed pigs or ED supplemented with IgG (ED + IgG) increased thickness (P < 0.001) of stomach mucosa and muscularis (P < 0.05) compared to the ED group not receiving IgG. Feeding an ED supplemented with IgG improved morphology of the GIT towards that of colostrum-fed piglets and indicates a beneficial effect of IgG on GIT development in neonatal pigs. Immunohistochemical studies indicate that ED feeding may influence the expression of nitric oxide synthase in jejunal myenteric (but not submucous) neurons of newborn pigs.