Jarosław Woliński

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.

Haemolytic anaemia and alterations in hepatic iron metabolism in aged mice lacking Cu,Zn-superoxide dismutase.

The continuous recycling of haem iron following phagocytosis and catabolism of senescent and damaged red blood cells by macrophages is a crucial process in the maintenance of systemic iron homoeostasis. However, little is known about macrophage iron handling in haemolytic states resulting from a deficiency in antioxidant defences. Our observations indicate that the recently described chronic, but moderate regenerative, haemolytic anaemia of aged SOD1 (superoxide dismutase 1)-knockout mice is associated with red blood cell modifications and sensitivity to both intra- and extra-vascular haemolysis. In the present study, we have characterized the molecular pathways of iron turnover in the liver of Sod1-deficient mice. Despite iron accumulation in liver macrophages, namely Kupffer cells, we did not measure any significant change in non-haem liver iron. Interestingly, in Kupffer cells, expression of the rate-limiting enzyme in haem degradation, haem oxygenase-1, and expression of the iron exporter ferroportin were both up-regulated, whereas the hepcidin mRNA level in the liver was decreased in Sod1-/- mice. These results suggest that concerted changes in the hepatic expression of iron- and haem-related genes in response to haemolytic anaemia in Sod1-/- mice act to reduce toxic iron accumulation in the liver and respond to the needs of erythropoiesis.

The effects of enteral ghrelin administration on the remodeling of the small intestinal mucosa in neonatal piglets.

Ghrelin is a multifunctional peptide produced predominantly in the stomach, however substantial amounts have also been found in colostrum and milk. The aim of the study was to investigate the effect of exogenous ghrelin, administered intra-gastrically, on the processes of mitosis, apoptosis, autophagy, crypt fission and changes in histometry of the small intestine mucosa in neonatal pigs, fed with a milk formula. Three groups (n=6) of piglets were used in the study. The pigs were fed either milk formula (C7) or milk formula together with ghrelin, administered via a stomach tube (7.5 μg/kg body weight (BW), (LG)) and 15 μg/kg BW (HG), every 8h for 6 days. Compared to the control group (C7), feeding milk formula supplemented with ghrelin resulted in significant changes in the small intestinal morphometry and mucosa histometry. The observed changes were dependent on the dosage of hormone and the part of intestine investigated. Administration of ghrelin via the stomach tube (HG) significantly influenced epithelial cell renewal. Moreover, we demonstrated that autophagy is involved in the small intestine mucosa remodeling and ghrelin may be an important factor for its regulation. In conclusion, we found that enteral ghrelin influences the gut mucosa remodeling in a dose-related manner in the early postnatal period. Moreover in neonates, stomach activity does not interfere with the action of ghrelin in the small intestine.

Hepatic iron content corresponds with the susceptibility of lymphocytes to oxidative stress in neonatal pigs.

The pig is born with limited iron supplies. If not supplemented, piglets dramatically loose their body iron stores during the first few days of postnatal life. The aim of this study was to investigate the influence of hepatic iron content on susceptibility of blood cells to oxidative stress. Four 1-day-old and three 7-days-old animals were used in this study. The alkaline version of the comet assay was used to measure DNA damage. As expected, iron body stores of non-supplemented animals decrease significantly during the first 4 days of life. However, no difference in background DNA damage was found between untreated lymphocytes from these two groups of animals, despite the difference in their hepatic iron content. Interestingly, DNA damage induced by H2O2 and X-radiation in lymphocytes taken from 1-day-old piglets was significantly higher than in those taken from 7-days-old animals. In contrast, NaOCl or tert-butyl-hydroxide also induced significant amounts of DNA damage, but no differences between the two groups of piglets were found. Our data show that decreased hepatic iron content corresponds with decreased susceptibility of blood lymphocytes to oxidative stressors.

Roux‐en‐Y or ‘uncut’ Roux procedure? Relation of intestinal migrating motor complex recovery to the preservation of the network of interstitial cells of Cajal in pigs

We designed a conscious pig model to investigate myoelectric activity and the number of interstitial cells of Cajal (ICC) in the proximal jejunum following the Roux‐en‐Y and ‘uncut’ Roux procedures in relation to clinical outcomes. Twelve male Polish White pigs (8 weeks old, 10–13 kg) underwent surgery under general anaesthesia first to implant bipolar electrodes and telemetry transmitters for continuous electromyography recordings and then, after 1 week recovery, to create Roux‐en‐Y (n= 6) and ‘uncut’ Roux loops (n= 6). Upper gut tissue specimens were studied for the expression of c‐kit staining procedure to quantitatively identify the presence of interstitial cells of Cajal. The intestinal migrating motor complex was restored within 10.5 and 37 h in ‘uncut’ Roux and Roux‐en‐Y pigs, respectively (P < 0.05). During 2 weeks, the ‘uncut’ Roux piglets increased their body weight by 18.0%, whereas the Roux‐en‐Y piglets increased their body weight by only 7.3% (P≤ 0.05). Two weeks after surgery, the number of ICC located in the region of Auerbachs plexus was higher and adhesions in the abdominal cavity lower in the ‘uncut’ Roux group. In conclusion, in the pig model, preservation of smooth muscle and ICC network continuity in the proximal jejunum may play an important role in early postsurgical recovery.

Leptin and ghrelin levels in colostrum, milk and blood plasma of sows and pig neonates during the first week of lactation

Radioimmunology was used to determine leptin and ghrelin levels in sow colostrum and milk in relation to those in sow and neonatal pig blood plasma and to the body weight of piglets during the first week of lactation. The highest concentration of leptin was found in colostrum on the second day of lactation (69.3 ± 6.3 ng/mL). Leptin concentrations in sow plasma were significantly lower than in colostrum/milk (2.19 ± 0.9 ng/mL, P = 0.7692) and were stable in the first 7 days of lactation. Total and active ghrelin concentrations in colostrum/milk were stable in the measured time points (6734 ± 261 pg/mL, P = 0.3397; 831 ± 242 pg/mL, P = 0.3988, respectively). Total ghrelin concentrations in sow plasma were lower than in colostrum/milk. These results indicate that pigs follow a unique species-specific pattern of leptin and ghrelin synthesis, release and existence, and that the mammary gland is an important source of leptin and ghrelin contained in colostrum/milk.

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.

Maternal High-Fat Diet during Pregnancy and Lactation Influences Obestatin and Ghrelin Concentrations in Milk and Plasma of Wistar Rat Dams and Their Offspring

The study aims to establish the effect of a maternal high-fat diet on obestatin concentration, total ghrelin, and ghrelin/obestatin ratio during pregnancy and lactation of Wistar rats and their offspring in the first 21 days of life. On the mating day, females were randomly allocated and fed either a high-fat diet (30% of fat; HF) or breeding diet (5% fat; BD) till the 21st day of lactation. Hormones were analyzed in the blood plasma and milk of rat dams as well as in the blood plasma of their offspring. HF resulted in a significant decrease in obestatin level on the 14th day of lactation and elevation on the 21st day. Plasma obestatin in HFD offspring was significantly higher than in BD ones. HF diet did not significantly affect dam plasma ghrelin until the 21st day of lactation. The ghrelin concentrations in milk after both diets were significantly lower than in blood plasma. Milk ghrelin in HF dams was significantly higher than in the BD ones. Plasma ghrelin from HF offspring was significantly higher than that from BD dams. Our results demonstrate that a maternal HF diet during pregnancy and lactation influences ghrelin and obestatin level in both dams and their offspring.

Enteral Pancreatic-like Enzymes of Microbial Origin affect Insulin Release during an Intravenous Glucose Tolerance Test

We have previously shown that the presence of pancreatic enzymes in the gut lumen of exocrine pancreatic insufficient pigs influences blood glucose and insulin levels during an intravenous glucose tolerance test (IVGTT). The present study aims to highlight the effects of orally applied pancreatic-like enzymes on blood glucose and plasma insulin levels during an IVGTT in young intact pigs. Five, 7-week old pigs were fed with pancreatic-like enzymes of microbial origin, a proteinase (from Aspergillus melleus), α-amylase (from Aspergillus oryzae) or lipase (from Burkholderia cepacia) alone or in combination with the Ca/Na salts of α-ketoglutaric acid (AKG). One hour following administration of the various supplements an IVGTT was performed. Blood samples were withdrawn during the 2 hours of IVGTT for glucose and insulin analyses. Blood glucose during the IVGTT was identical following administration of all combinations of the various enzymes or enzyme mixtures. Enteral loading of amylase or any amylase containing mixture resulted in reduced insulin secretion while administration of proteinase or any proteinase containing mixture resulted in enhanced insulin secretion during IVGTT, as compared to the control water vehicle. Lipase or AKG and lipase or AKG containing mixtures did not affect insulin secretion. Thus, it can be suggested that host amylase/protease ratio and their amount in pancreatic juice can participate in regulation of insulin release, thus, possibly affecting development of obesity and diabetes type 2.

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.