**2.1.1 Gnotobiotic piglets - 1st experiment**

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- 70 kPa and air flow 8-30 m3.

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;

Differences in the Development of the Small Intestine

**2.2.1 Microbiological analysis** 

forming units (cfu)g-1 of sample.

**2.2.2 Biochemical analysis** 

Czech Republic).

Between Gnotobiotic and Conventionally Bred Piglets 379

of conventional experiments, three piglets from each group were sacrificed at 3 hours post partum and at 2, 7, 14, 21, and 28 days of age. In the second experiment the piglets were slaughtered also on days 35 and 42 of age. The gastrointestinal tract was immediately removed and divided into six segments as follows: stomach, three equal segments of the small intestine, caecum and colon. The total content of each segment was weighed, pH was immediately measured. Intestinal tissue (1 cm2) were taken from the duodenum (5 cm distal to the orifice of the pancreatic duct) and the medial part of both the jejunum and ileum. The samples were fixed in 4% formalin solution for microscopic assessment of mucosal morphology. Sections of jejunum, ileum and caecum (1 g) were collected and processed for microbial counting and short-chain fatty acids (SCFAs) determinations were carried out in the contents from jejunum, ileum and colon. The intestinal segments (duodenum, jejunum and ileum) were rinsed thoroughly with ice-cold saline solution, opened lengthwise and blotted dry. The mucosa was scraped using a glass slide and immediately frozen in liquid nitrogen. Samples of mucosa

For microbial analysis, about 1 g of samples (jejunum, ileum, caecum) was placed in a sterile polyethylene stomacher Lab Blender bag (Seward Medical Limited, London, UK) with 9 ml of sterile anaerobic diluent (0.4 g Na HCO3, 0.05g L-cysteine HCl, 1 ml resazurine 0.1%), 7.5 ml mineral solution I (0.6% K2HPO4), 7.5 ml mineral solution II (1.2% NaCl, 1.2% (NH4)2SO4, 0.6% KH2PO4, 0.12% CaCl2, 0.25% MgSO4 and 84 ml distilled water, pH 6.8) and stomached (Stomacher Lab Blender 80, Seward Medical Limited, London, UK) for 5 min under a CO2 atmosphere. A series of 10-fold dilutions (10-2 to 10-8) were made in the same diluents. From appropriate dilutions, 0.1 ml aliquots were spread onto one non-selective agar plate: trypticase soy blood agar with 10% sheep blood (BBL, Microbiology systems, Cockeysville, USA) for aerobes. Aliquots (0.1 ml) were also spread on 5 selective agar media as follows: Beerens medium (Beerens, 1990) for *Bifidobacterium,* Rogosa agar (Imuna, Šarišské Michaľany, Slovak Republic) for *Lactobacillus,* Enterococcosel agar (BBL) for *Enterococcus*, MacConkey agar (Imuna) for *Coliforms* and Endo agar (Imuna) for *Enterobacteriaceae.* Plates for the enumeration of *aerobic* bacteria were incubated for 2 days at 37oC. Colonies were counted and bacteria were Gram stained and visualized under a microscope for morphological characterization. The viable counts are expressed as the log 10 of colony

After the collection, 1 g of digesta (jejunum, ileum, colon) was diluted in 50 ml of deionized H2O and applied at a volume of 30 µl for analysis of SCFAs. The concentration of formic, acetoacetic, lactic, succinic, acetic, propionic, butyric and valeric acids in the intestinal content was determined by capillary isotachophoresis (ITP). The measurements were done on an "Isotachophoretic analyser ZKI 01" (SR). In the pre-separation capillary, a leading electrolyte of the following composition was used: 10-2 M HCl + 2.2. 10-2 M ε-aminocaproic acid + 0.1% methylhydroxyethylcellulosic acid, pH 4.3. As finishing electrolyte, a solution of 5.10-3 M caproic acid + histidine was used. This electrolytic system worked at 250µA in preseparation and 50 µA in the analytic capillary. pH was measured by a pH meter (LP Prague,

were then stored at -70oC until the analysis of digestive enzyme activities.

1 ml contained 1 × 104 cfu (data analysed in the Laboratory of Gnotobiology). From the 5th day of life, autoclaved water was available to piglets *ad libitum* and they were fed irradiation-sterilized rations intended for early weaning of piglets. At the age of 28 days, the suckling piglets were weaned and fed irradiation-sterilized starter feedstuff *ad libitum* (OŠ-02®, Tajba Čaňa, Slovak Republic, in 1 kg dry matter: crude protein 180 g, fibre 45 g, lysine 11.5 g, methionine and cysteine 6.3 g, threonine 7.5 g, Ca 7 g, P 5.8 g, Na 1.5 g, Cu 10 mg, Zn 100 mg, Mn 30 mg, ME 13 MJ, vitamin A 8 000 IU, vitamin D3 1 000 IU, vitamin E 20 mg, Fe 125 mg, vitamin B2 3 mg, vitamin B12 20 μg, choline 600 mg). A routine microbiological control of gnotobiotic isolators was performed throughout the experiment. Microbiological swabs were taken from isolator walls, surface of animals and from their rectum. The samples were cultivated in PYG medium (Imuna, Slovak Republic). The microbiological control was verified every day on TSA agar with 5% ram's blood (BBL, Microbiology systems, Cockeysville, USA).

#### **2.1.2 Conventional suckled piglets - 2nd experiment**

In the experiment, 24 piglets of both sexes (Large white breed x Landrace) from two litters were included. The pigs were housed in two pens, 12 piglets in each, equipped with automatic heating, forced ventilation and completely slatted floors. The suckling piglets had access to sows 6 times daily (2, 6, 10, 14, 18, 22 h.) and from day 5 onwards the animals were provided commercial mixed feed OŠ-01® (Tajba Čaňa, Slovak Republic, in 1 kg dry matter: crude protein 200 g, fibre 40 g, lysine 14 g, methionine and cysteine 6.3 g, threonine 9.1 g, Ca 8 g, P 6.7 g, Na 2 g, Cu 10 mg, Zn 100 mg, Mn 30 mg, ME 13.3 MJ, vitamin A 8 000 IU, vitamin D3 1 000 IU, vitamin E 20 mg, Fe 125 mg, vitamin B2 3 mg, vitamin B12 20 μg, choline 300 mg) *ad libitum*. The piglets were weaned at 28 days of age, fed starter feedstuff *ad libitum* (OŠ-02®, Tajba Čaňa, Slovak Republic) and moved to 2 pens (375 x 165 cm) where three piglets were housed per pen. The temperature in the nursery was maintained at 32oC during the first week, and was gradually reduced to 25oC between weeks two and six. The animals had free access to water throughout the experiment (42 days).

#### **2.1.3 Conventional replacer-fed piglets - 3rd experiment**

The experiment included 26 piglets of both sexes (Large white breed x Landrace) from two litters. The experiment was carried out in two blocks, 13 piglets in each. The piglets were separated from the sow immediately after birth and had no contact with sow faeces. They were born naturally, were non-colostral, and were fed a commercial milk replacer diluted with distilled water 1:5 (Sanolac Ferkel, Germany), enriched by *Enterococcus faecium* 0.1 × 104 cfu/g of feed*.* Milk was given to piglets individually from a glass bottle 6 times daily (2, 6, 10, 14, 18, 22 h.), *ad libitum.* Starting from day 5, the suckling piglets were offered the same commercial mixed feed OŠ-01® (Tajba Čaňa, Slovak Republic) and were housed under the same hygiene conditions as those in the second experiment.

#### **2.2 Experimental procedure**

All pigs were sacrificed by intracardial euthanasia with 1 ml/kg BW T61® (Intervet International B.V. Boxmeer, The Netherlands). In the first experiment, three hours after birth and at the age of two and seven days, two piglets of each indicated age were sacrificed. Three piglets of each indicated age were sacrificed at the age of 14, 21, 28 and 35 days. In the course

1 ml contained 1 × 104 cfu (data analysed in the Laboratory of Gnotobiology). From the 5th day of life, autoclaved water was available to piglets *ad libitum* and they were fed irradiation-sterilized rations intended for early weaning of piglets. At the age of 28 days, the suckling piglets were weaned and fed irradiation-sterilized starter feedstuff *ad libitum* (OŠ-02®, Tajba Čaňa, Slovak Republic, in 1 kg dry matter: crude protein 180 g, fibre 45 g, lysine 11.5 g, methionine and cysteine 6.3 g, threonine 7.5 g, Ca 7 g, P 5.8 g, Na 1.5 g, Cu 10 mg, Zn 100 mg, Mn 30 mg, ME 13 MJ, vitamin A 8 000 IU, vitamin D3 1 000 IU, vitamin E 20 mg, Fe 125 mg, vitamin B2 3 mg, vitamin B12 20 μg, choline 600 mg). A routine microbiological control of gnotobiotic isolators was performed throughout the experiment. Microbiological swabs were taken from isolator walls, surface of animals and from their rectum. The samples were cultivated in PYG medium (Imuna, Slovak Republic). The microbiological control was verified every day on TSA agar with 5% ram's blood (BBL, Microbiology

In the experiment, 24 piglets of both sexes (Large white breed x Landrace) from two litters were included. The pigs were housed in two pens, 12 piglets in each, equipped with automatic heating, forced ventilation and completely slatted floors. The suckling piglets had access to sows 6 times daily (2, 6, 10, 14, 18, 22 h.) and from day 5 onwards the animals were provided commercial mixed feed OŠ-01® (Tajba Čaňa, Slovak Republic, in 1 kg dry matter: crude protein 200 g, fibre 40 g, lysine 14 g, methionine and cysteine 6.3 g, threonine 9.1 g, Ca 8 g, P 6.7 g, Na 2 g, Cu 10 mg, Zn 100 mg, Mn 30 mg, ME 13.3 MJ, vitamin A 8 000 IU, vitamin D3 1 000 IU, vitamin E 20 mg, Fe 125 mg, vitamin B2 3 mg, vitamin B12 20 μg, choline 300 mg) *ad libitum*. The piglets were weaned at 28 days of age, fed starter feedstuff *ad libitum* (OŠ-02®, Tajba Čaňa, Slovak Republic) and moved to 2 pens (375 x 165 cm) where three piglets were housed per pen. The temperature in the nursery was maintained at 32oC during the first week, and was gradually reduced to 25oC between weeks two and six. The animals

The experiment included 26 piglets of both sexes (Large white breed x Landrace) from two litters. The experiment was carried out in two blocks, 13 piglets in each. The piglets were separated from the sow immediately after birth and had no contact with sow faeces. They were born naturally, were non-colostral, and were fed a commercial milk replacer diluted with distilled water 1:5 (Sanolac Ferkel, Germany), enriched by *Enterococcus faecium* 0.1 × 104 cfu/g of feed*.* Milk was given to piglets individually from a glass bottle 6 times daily (2, 6, 10, 14, 18, 22 h.), *ad libitum.* Starting from day 5, the suckling piglets were offered the same commercial mixed feed OŠ-01® (Tajba Čaňa, Slovak Republic) and were housed under the

All pigs were sacrificed by intracardial euthanasia with 1 ml/kg BW T61® (Intervet International B.V. Boxmeer, The Netherlands). In the first experiment, three hours after birth and at the age of two and seven days, two piglets of each indicated age were sacrificed. Three piglets of each indicated age were sacrificed at the age of 14, 21, 28 and 35 days. In the course

systems, Cockeysville, USA).

**2.1.2 Conventional suckled piglets - 2nd experiment** 

had free access to water throughout the experiment (42 days).

**2.1.3 Conventional replacer-fed piglets - 3rd experiment** 

same hygiene conditions as those in the second experiment.

**2.2 Experimental procedure** 

of conventional experiments, three piglets from each group were sacrificed at 3 hours post partum and at 2, 7, 14, 21, and 28 days of age. In the second experiment the piglets were slaughtered also on days 35 and 42 of age. The gastrointestinal tract was immediately removed and divided into six segments as follows: stomach, three equal segments of the small intestine, caecum and colon. The total content of each segment was weighed, pH was immediately measured. Intestinal tissue (1 cm2) were taken from the duodenum (5 cm distal to the orifice of the pancreatic duct) and the medial part of both the jejunum and ileum. The samples were fixed in 4% formalin solution for microscopic assessment of mucosal morphology. Sections of jejunum, ileum and caecum (1 g) were collected and processed for microbial counting and short-chain fatty acids (SCFAs) determinations were carried out in the contents from jejunum, ileum and colon. The intestinal segments (duodenum, jejunum and ileum) were rinsed thoroughly with ice-cold saline solution, opened lengthwise and blotted dry. The mucosa was scraped using a glass slide and immediately frozen in liquid nitrogen. Samples of mucosa were then stored at -70oC until the analysis of digestive enzyme activities.
