**1. Introduction**

374 New Advances in the Basic and Clinical Gastroenterology

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The health quality of human population is strongly connected to the decrease of environmental burden and increase of quality and safety of food. The production of highquality and safe food and materials of animal origin is conditioned by the good health of raised animals. Diseases of the gastrointestinal tract can be considered the most important health and economic problem of rearing young animals, since they may cause extremely high losses due to morbidity, mortality, cost of treatment and weight loss. At an early age, diseases debilitate the animal organism and cause delays in development which can subsequently become evident as health problems and decreased productivity. For this reason, it is extremely important to ensure optimum development of the digestive tract in young animals. These relations are determined by digestive juice and enzyme secretion, morphological development and microbial colonization of the digestive tract as well as by absorption capacity of the latter. The pig gut is exposed to a variety of stress factors particularly in the early postnatal period and just after weaning. This is the period of significant growth, morphological changes and maturation of the gastrointestinal tract (Godlewski et al., 2005; Trahair & Sanglid, 2002; Xu, 1996). Prior to birth, the alimentary tract is exposed to substances from the ingested amniotic fluid which seems to be of importance to its development (Trahair & Sanglid, 2002). The colostrum, however, differs from the amniotic fluid by the density of nutrients and high immunoglobulin, enzyme, hormone, growth factor and neuroendocrine peptide levels. Widdowson & Crabb (1976) were the first to demonstrate the effect of the colostrum upon development of the alimentary tract by comparing the colostrums-suckling piglets with watered animals. Maternal colostrums contained high levels of several hormones and growth promoting peptides like insulin, epidermal growth factor (EGF), insulin-like growth factor-I and II (IGF-I and II), transforming growth factor-β (TGF- β), glucagon-like peptide-2 (GLP-2) and leptin. It was proved that colostral growth factors play an important role in the postnatal development of the digestive tract in newborn animals (Guilloteau et al., 2002; Xu, 1996). During the several initial days of life of newborns, their small intestine increases its weight by about 70%, length by approx. 20%, diameter by 15%. Its absorption area increases by about 50% during the first postnatal day and by 100% during the first 10 postnatal days (Marion et al., 2003; Xu, 1996). A large luminal surface area with optimal enterocyte functional maturity is

Differences in the Development of the Small Intestine

**2. Materials and methods** 

**2.1 Animal, housing and diets** 

70 kPa and air flow 8-30 m3.

**2.1.1 Gnotobiotic piglets - 1st experiment** 

piglets fed on milk replacement, as well as in gnotobiotic piglets.

Between Gnotobiotic and Conventionally Bred Piglets 377

disaccharidase enzymes activity in the small intestine in piglets reared under the sow,

The experiments on growing and weaned piglets were carried out at the Institute of Microbiology and Gnotobiology, University of Veterinary Medicine and Pharmacy, Košice, Slovakia. The State Veterinary and Food Administration of the Slovak Republic approved the experimental protocols and the animals were handled and sacrificed in a humane

The experiment was carried out in 4 gnotobiotic units, each consisting of reserve, waste and rearing isolator (Velaz s.r.o., Prague, Czech Republic). All experimental materials, including milk substitute, distilled water, saline solution and glass and metal materials were sterilised by autoclaving at 121oC and pressure 1.3 MPa for 30 minutes and cellulose wadding and other sanitary material was gamma-irradiated (Bioster, Veverská Bitýška, Czech Republic). The isolators were sterilized with a 2% solution of peracetic acid (36%, Merci s.r.o., Brno, Czech Republic), sealed for 24 hrs, and vented for a minimum of 72 hrs prior to placing pigs inside. Isolators were maintained under positive pressure, the filtering unit consisting of a fan with preliminary EU 3 filter and two-stage filtering chamber (Velaz s.r.o., Prague, Czech Republic). The first stage of filtration consisted of a frame filter type KS-W, filtration class F 7, the second stage used a KS MIKRO S filter, filtration class H 13, for removing of microparticles. The vented air passed through a frame filter KS W/48, filtration class F 5. The filtration unit assured a minimum of 10 exchanges of air per hour at overpressure of 50-

The experiment was carried out on 18 gnotobiotic piglets of Slovak white × Landrace breed. Gnotobiotic sucklings were obtained using the method of open hysterotomy on day 112 of pregnancy. After opening the abdominal cavity and uterus the piglets were immediately transferred through a disinfectant bath containing 2% Incidur® (Ecolab GmbH & Co. OHG, Düsseldorf, Germany) into a hysterectomy box were they were subjected to preliminary treatment and then were placed into 1 of 4 gnotobotic rearing isolators. The floor of isolators was heated by electric underfloor heating system to ensure floor temperature of 34oC for new born piglets and 30oC for 7-14 days old piglets. The piglets were non-colostral and were fed autoclaved milk substitute (Sanolac Ferkel, Germany, in 1 kg dry matter: fat 18.0%, Nfree extract 20.0%, lysine 1.7%, Ca 0.9%, P 0.7%, Na 1.0%, Mg 0.2%, fibre 1.5%, ash 10.0%, ME 17.5 MJ, vitamin A 50 000 IU, vitamin D3 5 000 IU, vitamin E 100 mg, biotin 200 μg, Fe 100 mg, vitamin B1 4 mg, vitamin B2 4 mg, vitamin B6 2 mg, vitamin B12 20 μg, calcium pantothenate 10 mg, nicotinic acid 20 mg, folic acid 1 mg, vitamin C 100 mg, choline chloride 250 mg), diluted 1 : 5 with distilled water. The milk substitute was fed to piglets individually from a glass bottle six times daily (2, 6, 10, 14, 18, 22 h), *ad libitum.* A total of 18 gnotobiotic animals derived from 2 litters were divided into 4 isolators. From the first day of life, a probiotic strain of *Enterococcus faecium* isolated from non autoclaved milk substitute (Sanolac Ferkel, Germany) was administered continuously at a dose of 2 ml of inoculum;

manner in accordance with the guidelines established by the relevant commission.

important to young growing pigs so they may attain maximum digestive and absorptive capability. Consequently, suboptimal or adverse environmental factors, influencing the morphological development of intestinal tissue, may have critical functional consequences for the young growing pig. The marked and abrupt morphological responses to weaning in the small intestine, characterized by the transformation from a dense finger-like villi population to a smooth, compact, tongue-shaped luminal villi surface may indicate critical consequences for the young pig digestive capacity and subsequent use of nutrients during the starter phase (Skrzypek et al., 2005). The changes at weaning which include shortening of villi, hyperplasia of crypts, decrease in absorption capacity and certain loss of carbohydrate activity may, in combination with changes in the number and type of enterobacteria, induce various degree of post-weaning diarrhoea (Pluske et al., 1997). By now, the prevention and therapy of diseases of sucklings and weanlings was implemented by means of synthetic substances, which enormously burden not only the organisms, but also the living environment as a whole. The extensive use of antibiotics has increased the risk of development of resistance in human and animal pathogens and chemical residues in meat of animals. In progress is the research and development of new methods of biotechnological and natural character that with their complex influence will maximally make efficient the prevention of diseases of animals by the stabilisation of physiological function of biological barriers of the gastrointestinal tract ecosystem. Biological barriers of digestive tract represent the prime and basal protection of organism from negative impacts of external and internal environment, and therefore it is possible to decrease a health risk by its sophisticated modulation. The indigenous microbiota suppresses colonization of incoming bacteria by a process named colonization resistance that is a first line of defence against invasion by exogenous, potential pathogenic organisms or indigenous opportunists. Beneficial microbiota prevent bacterial colonization by competing for epithelium receptors and enteric nutrients, producing antimicrobial compounds such as bacteriocins and metabolizing nutrients to create a restrictive environment which is generally unfavourable for the growth of many enteric pathogens (Bomba et al., 2002; Marinho et al., 2007). Probiotics as natural bioregulators assist the maintenance of the homeostasis of the gastrointestinal tract ecosystem and, during the critical periods of animal life, can play an important role in prevention of diarrhoeic diseases of dietetic and bacterial origin (Bomba et al., 2002; Marinho et al., 2007). Gastrointestinal microflora may be affected by adding probiotic micro-organisms of genera *Lactobacillus*, *Bifidobacterium* (Bomba et al., 2002), *Bacillus*, *Enterococcus* and *Streptococcus* (Scharek et al., 2005) to feed or by their combinations (Bomba et al., 2002; Mathew et al., 1998). Enterococci belong to those lactic-acid bacteria which inhabit human and animal intestines (Devriese et al., 1991). It was observed that *Enterococcus faecium* prevents adherence of enterotoxigenic *Escherichia coli* K 88+ to the surface of intestinal mucosa of piglets (Scharek et al., 2005). In terms of exactitude and interpretability of results, gnotobiotic piglets are an ideal experimental model for the study of digestive processes and their development. The presence of normal microflora influences the structure of the host intestinal mucous membrane, its function and short-chain fatty acids (SCFAs) production. By means of gnotobiotic conditions, we excluded the influence of the normal microflora and sow's milk. The changes in the small intestine, observed under the specific controlled conditions, were compared to the development of the gut in conventionally bred piglets.

The aim of the study was to evaluate the effects of piglet´s age and diet (natural feeding, artificial feeding and gnotobiotic conditions) on the development of microflora, production of short-chain fatty acids (SCFAs), postnatal morphological development and disaccharidase enzymes activity in the small intestine in piglets reared under the sow, piglets fed on milk replacement, as well as in gnotobiotic piglets.
