**4. Discussion**

398 New Advances in the Basic and Clinical Gastroenterology

a

 c \*\*\*

\*\* \*

\*\*\* a

*Saccharase*

Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01), c (p < 0.001), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 Fig. 8. Effect of age, weaning and diets on specific activity of saccharase at various locations

duodenum jejunum ileum

age (day)

GP SP GP SP GP SP GP SP GP SP GP SP GP SP

\*\*\*

 b \*\*\*

0 2 7 14 21 28 35

and 2-day old conventional piglets were very low and did not exceed 0.6 µmol/mg protein/hour. After one week of age of piglets, activities of both enzymes increased. The maximum specific activity of maltase peaked on days 14 and 21 (p < 0.05, p < 0.001) in the duodenum and significant difference was observed (p < 0.05) in the jejunal segment. Similarly, activity of saccharase reached highest values on days 14 and 21 of age and differed significantly (p < 0.001) in both the duodenal and jejunal segment. On day 28 of age we observed a decrease in the specific activity of both enzymes with significant difference in saccharase (p < 0.01) in the jejunum. Observation of distribution of maltase in the small intestine of conventional piglets showed that in the direction of terminal ileum, up to the age of 2 weeks the highest activity was detected in the jejunum and the lowest in the ileum. In the following period activity of this enzyme was higher in proximal to medial segments of the intestine than in the distal ones. Throughout the experiment saccharase reached the

The postnatal development of specific activity of enzymes maltase and saccharase showed a similar trend with higher activities of both enzymes in suckled piglets in all digestive tract segments throughout the observation. The highest activity of the enzymes in comparison with gnotobiotic piglets was recorded on days 14 and 21 of age with significant differences in the level of maltase (p < 0.05, p < 0.001) in the duodenum and significantly different activity of the enzyme (p < 0.05) in jejunum. Significantly different activities of saccharase compared to the other group of piglets were observed in the same period (days 14 and 21) in

SP- suckled piglets, GP- gnotobiotic piglets.

0

2

4

6

µmol/mg protein/hour

along the intestinal tract of suckling and gnotobiotic piglets

highest activity in the jejunum and the lowest in the ileum.

duodenum (p < 0.05) and jejunum (p < 0.01, p < 0.001).

#### **4.1 Effect of age and diets on development of the small intestine – Intestinal microflora**

At birth, a young pig which is axenic during its uterine life is suddenly confronted with a complex bacterial environment. Beginning with birth, the young are in contact with several microbial ecosystems - i.e. faeces, contaminated vagina and perineum, but also from the skin and teats of the sow which are usually contaminated as well. It could be assumed that each of these ecosystems contributes to constituting the gut flora of the newborn (Siggers et al., 2007). However, in the following days, simplified microbiota profiles have been characterized, which will become more complex with time, increasing its diversity as the animal grows (Inoue at al., 2005). The population level of the microbiota in various parts of the gastrointestinal tract of monogastric animals depends on the attachment ability, replication period of the microorganism under the physicochemical tract conditions and the emptying rhythm in the part of the gastrointestinal tract under investigation. Swords et al. (1993) studied pig faecal microbiota evolution within the first four months of life, and concluded that the establishment of the adult faecal flora is a large and complex process with three different marked phases in the bacterial succession. The first phase corresponds with the first week of life, the second one, from the end of the first week to conclusion of suckling, and the third phase from weaning to final adaptation to dry food. In this first phase, aerobes and facultative anaerobes from the sow and the environment become the predominant bacterial groups, comprising 80% of the total flora by three hours after birth. The gut colonization is extremely fast, only twelve hours after birth, total bacteria in distal colon reaches counts of 109 cfu/g colonic content (Jensen et al., 1998; Swords et al., 1993). First colonizers modify the gastrointestinal environment (by consumption of molecular oxygen and reduction of the redox potential), making it more favourable for the following colonization by anaerobes. As a result, aerotolerant bacteria are gradually supplanted by strict anaerobes, and 48h after birth, piglets already show 90% of anaerobic bacteria (Swords et al., 1993). Of these bacterial groups, lactobacilli and streptococci become the dominant bacteria at the end of the first week of life and will be maintained for the whole suckling period with counts of around 107-109 cfu/g digesta (Swords et al., 1993). Microbiota remains fairly stable in terms of species composition during the second phase when the piglets receive milk from their mother (Mathew et al., 1996). The diversity of anaerobic bacteria increases in this period (Inoue et al., 2005) and supplantation of aerobic and facultative anaerobic bacteria by anaerobic bacteria become almost completed in this phase. As has been mentioned before, lactobacilli and streptococci continue being dominant bacteria, which are well adapted to utilize substrate from the milk diet. *Clostridium*, *Bacteroides*, *bifidobacteria*, and low densities *Eubacterium*, *Fusobacterium*, *Propionibacterium* and *Streptococcus* spp. are also usually found in this second phase (Swords et al., 1993). In our experiment with piglets fed maternal milk, the gut flora developed very quickly post partum. As early as within 48 hrs post partum, *Escherichia coli*, enterococci and lactobacilli were detected in the content of digesta in suckled piglets, the populations of which represented 105 - 109 bacteria/g per sample. The gut flora of piglets in the proximal part (jejunum) consisted of facultatively anaerobic bacteria (enterococci*, E. coli*) and aerotolerant anaerobes (lactobacilli) counting 104 - 106 bacteria/g per sample. These numbers increased progressively in the ileum and the dominant flora in the posterior portions of the digestive tract (caecum) was facultatively anaerobic bacteria (*E. coli*, enterococci, *Enterobacteriaceae* and lactobacilli) counting 108 - 109 bacteria/g per sample. As

Differences in the Development of the Small Intestine

Between Gnotobiotic and Conventionally Bred Piglets 401

well known that organic acids exhibit antibacterial activity (Piva et al., 2002), increase intestinal absorption of minerals and improve ileal digestion of proteins and amino acids. Their relative levels vary depending on location (stomach, small intestine, large intestine) and diet composition (dietary lactose level, fibre level, etc). Lactic acid is in the greatest concentration in the stomach and small intestine while other organic acids, acetic along with propionic and butyric, are predominant in the large intestine. In our study, in suckled piglets we recorded higher level of acetic acid in the ileal contents in comparison with the replacer-fed piglets throughout the observation, ranging from about 7 to 21.5 mmol/l. Higher concentrations of this acid were observed in the same segment also in gnotobiotic piglets compared to replacer-fed piglets, ranging from 2.05 to 13.69 mmol/l. A similar tendency was recorded in lactic acid, in the ileal content, with higher concentrations in suckled piglets compared to the replacer-fed piglets in which the values later ranged from about 8.2 to 21.4 mmol/l. Higher production of lactic acid in gnotobiotic piglets in comparison with replacer-fed piglets was recorded throughout the observation and ranged from 0.6 to 10.14 mmol/l. Increasing concentrations of lactate in ileal digesta should therefore reflect an increased population and activity of lactic acid bacteria (Pluske et al., 2002). These findings are of importance relative to the management of growing pigs, because lactate has been shown to have antibacterial effects on *E. coli* and *Salmonella* species, and lactobacilli have been shown to inhibit adhesion of enterotoxigenic *E. coli* to the ileal epithelium (Pluske et al., 2002). The ability to generate organic acids, particularly lactic and acetic acid, present one of the mechanisms by which lactobacilli perform their inhibitory effect upon pathogens. With decreasing pH values, the inhibitory activity of the above acids increases, their molecular form being toxic for bacteria. The increased toxicity of acetic acid is attributed to its higher pKa in comparison to lactic acid. Increased lactic acid levels intensify the toxicity of acetic acid. Comparison of lactic acid levels in the jejunal and ileal contents of one week old gnotobiotic piglets (Bomba et al., 1998) and conventional suckling piglets (Zitnan et al., 2001) revealed that the highest levels were found in conventional animals (29.30 and 27.90 mmol/l, resp.) and in *Lactobacillus plantarum* inoculated gnotobiotic piglets (26.60 and 14.20 mmol/l, resp.). High levels of lactic acid were recorded in our study in jejunal and ileal contents of conventional suckling piglets (27.52 and 26.91 mmol/l, resp.) and piglets inoculated with *Enterococus faecium* at the age of 1 week (23.59 and 24.56 mmol/l, resp.). Lower concentrations of this acid were found in replacer-fed piglets (24.92 and 14.42) and the lowest levels of lactic acid in the jejunal and ileal contents (Bomba et al., 1998) were seen in germ-free piglets (4.40 and 6.45 mmol/l, resp.). At the age of 3 weeks, the level of lactic acid in the jejunum of piglets inoculated with *Enterococcus faecium* was lower in comparison with that in the jejunum of piglets inoculated with *Lactobacillus plantarum* (21.94 and 33.15 mmol/l, resp.) but in the ileum of *Enterococcus faecium* inoculated piglets we found the highest level of this acid (24.40 mmol/l) in comparison with all other groups of piglets. The mentioned authors presented different results also with regard to acetic acid, as the highest concentrations of acetic acid in the jejunum and ileum (30.05 and 23.61 mmol/l, resp.) were observed in conventional piglets by Zitnan et al. (2001) and in conventional suckling piglets investigated in our study (33.05 and 21.81 mmol/l, resp.). Lower levels were observed in replacer-fed piglets (21.43 and 10.47 mmol/l, resp.) and in gnotobiotic piglets (Bomba et al. 1998) inoculated with lactobacilli (11.80 and 11.85 mmol/l, resp.) and in those inoculated with *Enterococcus faecium* (8.18 and 24.16 mmol/l, resp.). Similarly low levels were detected also in germ-free piglets (13.15 and 3.9 mmol/l, resp.). On the contrary, at the age of 3 weeks, we recorded higher levels of acetic acid in the jejunal and ileal content of

the piglets grew, the flora progressively changed. The replacement of maternal milk by a milk replacement diet in our third experiment resulted at 2 and 14 days of age in increased numbers of bacterial flora, of the family *Enterobacteriaceae* in the proximal small intestine (jejunum) by 3 log compared to suckled piglets. At the same time a gradual increase in the numbers of coliform bacteria (p < 0.001) in the proximal (jejunum) and terminal (caecum) part of the intestine occurred reaching numbers as much as by 4 log higher in the jejunum at 14 d of age compared to colostral piglets. Similar findings were obtained by (Franklin et al. 2002; Jensen et al., 1998; Pluske et al., 1997) which observed, in addition to higher populations of anaerobic and coliform bacteria, a significant decline in lactobacilli populations in piglets fed replacement milk. The mechanism of the selection has not yet been determined, although feeding may be a primary factor. According to different authors it is almost impossible to prevent neonatal *E. coli* diarrhoea in piglets which do not receive maternal colostrum (Pluske et al., 1997; Xu et al., 1996). Colostrum and also maternal milk contain highly digestible nutrients and components such as immunoglobulins and lysozymes and have both bacteriostatic and antiadhesive properties towards the pathogen *E. coli* (Xu et al., 1996). This protective effect, however, does not seem to be accompanied by an elimination of *E. coli* from the digestive tract. In addition, in both the jejunal and ileal part (p < 0.001) of the small intestine of replacer-fed piglets, high numbers of enterococci were recorded, being by 1-3.5 log higher compared to the colostrum-fed piglets throughout the period of observation. It is likely, however, that this result was influenced by the milk replacement having been enriched by *Enterococcus faecium* counting 104 cfu/g of feed. The human intestinal microbiota is a complex ecosystem, consisting of several hundred (more than 800) different bacterial species. This microbiota plays an important role in human health and nutrition by producing nutrients, preventing colonization of the gut by potential pathogenic microorganisms (Guarner & Malagelada, 2003), and preserving the health of the host through interactions with the developing immune system. The microbiota in early life has been linked to allergy risk (Penders et al., 2007). Major changes in the intestinal microbial composition occur in early life. Sterile *in utero*, the gastrointestinal tract of the newborn infant is rapidly colonized at birth by a myriad of maternal vaginal and faecal bacteria and other sources from its environment. The first few weeks after birth correspond to critical stages of gut colonization. Bacterial colonization of the gastrointestinal tract is influenced by numerous factors including diet, environment, antibiotic treatment, mucosal maturation, and age. Naturally delivered babies experienced a period of 2-3 days in which, as a consequence of the low selective potential of their stomach and small bowel, bacteria invading and reproducing within the gut belong to aerobic species as *Enterobacteriaceae*, streptococci, and staphylococci. These bacteria, arriving from the external environment, belong to species with a pathogenic potential, and therefore, it might seem that they would not be the best choice for the health of neonates. However, the metabolisms of these bacteria are believed to be positive factors in preparing the path to a beneficial enteric flora. In the study of Fallani et al. (2010) they confirmed previously published work (Hopkins et al., 2005; Penders et al., 2007) that bifidobacteria are the predominant group detected in the faeces of pre-weaned infants, followed by *Bacteroides* and enterobacteria.

#### **4.2 Production of SCFAs and pH**

Organic acids are the main metabolites of intestinal fermentation. The degree of their concentration in the digesta reflects the level of intestinal fermentation (Piva et al., 2002). It is

the piglets grew, the flora progressively changed. The replacement of maternal milk by a milk replacement diet in our third experiment resulted at 2 and 14 days of age in increased numbers of bacterial flora, of the family *Enterobacteriaceae* in the proximal small intestine (jejunum) by 3 log compared to suckled piglets. At the same time a gradual increase in the numbers of coliform bacteria (p < 0.001) in the proximal (jejunum) and terminal (caecum) part of the intestine occurred reaching numbers as much as by 4 log higher in the jejunum at 14 d of age compared to colostral piglets. Similar findings were obtained by (Franklin et al. 2002; Jensen et al., 1998; Pluske et al., 1997) which observed, in addition to higher populations of anaerobic and coliform bacteria, a significant decline in lactobacilli populations in piglets fed replacement milk. The mechanism of the selection has not yet been determined, although feeding may be a primary factor. According to different authors it is almost impossible to prevent neonatal *E. coli* diarrhoea in piglets which do not receive maternal colostrum (Pluske et al., 1997; Xu et al., 1996). Colostrum and also maternal milk contain highly digestible nutrients and components such as immunoglobulins and lysozymes and have both bacteriostatic and antiadhesive properties towards the pathogen *E. coli* (Xu et al., 1996). This protective effect, however, does not seem to be accompanied by an elimination of *E. coli* from the digestive tract. In addition, in both the jejunal and ileal part (p < 0.001) of the small intestine of replacer-fed piglets, high numbers of enterococci were recorded, being by 1-3.5 log higher compared to the colostrum-fed piglets throughout the period of observation. It is likely, however, that this result was influenced by the milk replacement having been enriched by *Enterococcus faecium* counting 104 cfu/g of feed. The human intestinal microbiota is a complex ecosystem, consisting of several hundred (more than 800) different bacterial species. This microbiota plays an important role in human health and nutrition by producing nutrients, preventing colonization of the gut by potential pathogenic microorganisms (Guarner & Malagelada, 2003), and preserving the health of the host through interactions with the developing immune system. The microbiota in early life has been linked to allergy risk (Penders et al., 2007). Major changes in the intestinal microbial composition occur in early life. Sterile *in utero*, the gastrointestinal tract of the newborn infant is rapidly colonized at birth by a myriad of maternal vaginal and faecal bacteria and other sources from its environment. The first few weeks after birth correspond to critical stages of gut colonization. Bacterial colonization of the gastrointestinal tract is influenced by numerous factors including diet, environment, antibiotic treatment, mucosal maturation, and age. Naturally delivered babies experienced a period of 2-3 days in which, as a consequence of the low selective potential of their stomach and small bowel, bacteria invading and reproducing within the gut belong to aerobic species as *Enterobacteriaceae*, streptococci, and staphylococci. These bacteria, arriving from the external environment, belong to species with a pathogenic potential, and therefore, it might seem that they would not be the best choice for the health of neonates. However, the metabolisms of these bacteria are believed to be positive factors in preparing the path to a beneficial enteric flora. In the study of Fallani et al. (2010) they confirmed previously published work (Hopkins et al., 2005; Penders et al., 2007) that bifidobacteria are the predominant group detected in the faeces of pre-weaned infants, followed by *Bacteroides*

Organic acids are the main metabolites of intestinal fermentation. The degree of their concentration in the digesta reflects the level of intestinal fermentation (Piva et al., 2002). It is

and enterobacteria.

**4.2 Production of SCFAs and pH** 

well known that organic acids exhibit antibacterial activity (Piva et al., 2002), increase intestinal absorption of minerals and improve ileal digestion of proteins and amino acids. Their relative levels vary depending on location (stomach, small intestine, large intestine) and diet composition (dietary lactose level, fibre level, etc). Lactic acid is in the greatest concentration in the stomach and small intestine while other organic acids, acetic along with propionic and butyric, are predominant in the large intestine. In our study, in suckled piglets we recorded higher level of acetic acid in the ileal contents in comparison with the replacer-fed piglets throughout the observation, ranging from about 7 to 21.5 mmol/l. Higher concentrations of this acid were observed in the same segment also in gnotobiotic piglets compared to replacer-fed piglets, ranging from 2.05 to 13.69 mmol/l. A similar tendency was recorded in lactic acid, in the ileal content, with higher concentrations in suckled piglets compared to the replacer-fed piglets in which the values later ranged from about 8.2 to 21.4 mmol/l. Higher production of lactic acid in gnotobiotic piglets in comparison with replacer-fed piglets was recorded throughout the observation and ranged from 0.6 to 10.14 mmol/l. Increasing concentrations of lactate in ileal digesta should therefore reflect an increased population and activity of lactic acid bacteria (Pluske et al., 2002). These findings are of importance relative to the management of growing pigs, because lactate has been shown to have antibacterial effects on *E. coli* and *Salmonella* species, and lactobacilli have been shown to inhibit adhesion of enterotoxigenic *E. coli* to the ileal epithelium (Pluske et al., 2002). The ability to generate organic acids, particularly lactic and acetic acid, present one of the mechanisms by which lactobacilli perform their inhibitory effect upon pathogens. With decreasing pH values, the inhibitory activity of the above acids increases, their molecular form being toxic for bacteria. The increased toxicity of acetic acid is attributed to its higher pKa in comparison to lactic acid. Increased lactic acid levels intensify the toxicity of acetic acid. Comparison of lactic acid levels in the jejunal and ileal contents of one week old gnotobiotic piglets (Bomba et al., 1998) and conventional suckling piglets (Zitnan et al., 2001) revealed that the highest levels were found in conventional animals (29.30 and 27.90 mmol/l, resp.) and in *Lactobacillus plantarum* inoculated gnotobiotic piglets (26.60 and 14.20 mmol/l, resp.). High levels of lactic acid were recorded in our study in jejunal and ileal contents of conventional suckling piglets (27.52 and 26.91 mmol/l, resp.) and piglets inoculated with *Enterococus faecium* at the age of 1 week (23.59 and 24.56 mmol/l, resp.). Lower concentrations of this acid were found in replacer-fed piglets (24.92 and 14.42) and the lowest levels of lactic acid in the jejunal and ileal contents (Bomba et al., 1998) were seen in germ-free piglets (4.40 and 6.45 mmol/l, resp.). At the age of 3 weeks, the level of lactic acid in the jejunum of piglets inoculated with *Enterococcus faecium* was lower in comparison with that in the jejunum of piglets inoculated with *Lactobacillus plantarum* (21.94 and 33.15 mmol/l, resp.) but in the ileum of *Enterococcus faecium* inoculated piglets we found the highest level of this acid (24.40 mmol/l) in comparison with all other groups of piglets. The mentioned authors presented different results also with regard to acetic acid, as the highest concentrations of acetic acid in the jejunum and ileum (30.05 and 23.61 mmol/l, resp.) were observed in conventional piglets by Zitnan et al. (2001) and in conventional suckling piglets investigated in our study (33.05 and 21.81 mmol/l, resp.). Lower levels were observed in replacer-fed piglets (21.43 and 10.47 mmol/l, resp.) and in gnotobiotic piglets (Bomba et al. 1998) inoculated with lactobacilli (11.80 and 11.85 mmol/l, resp.) and in those inoculated with *Enterococcus faecium* (8.18 and 24.16 mmol/l, resp.). Similarly low levels were detected also in germ-free piglets (13.15 and 3.9 mmol/l, resp.). On the contrary, at the age of 3 weeks, we recorded higher levels of acetic acid in the jejunal and ileal content of

Differences in the Development of the Small Intestine

diagnosed, and *E. coli* K88 was isolated from rectal swabs.

**4.3 Intestinal morphology and disaccharidase activity** 

Between Gnotobiotic and Conventionally Bred Piglets 403

was low and resulted in their low concentrations which did not decrease pH so effectively as it was observed in gnotobiotic piglets inoculated with *Enterococcus faecium.* Deficit of colostral nutrition in these piglets resulted in worsened health in 8 out of total 26 piglets. The disease was peracute and proceeded with physiological temperature. Even though antibiotics were administered, the piglets died within 8 hrs of appearing of the first symptoms. In the piglets, lymphocytic leukocytosis as well as hypochromic anemia were

Due to numerous similarities of the physiology and anatomy of the gastrointestinal tract of man and pigs, the pig model is a very attractive model for human nutritional studies (Miller & Ullrey, 1987). Investigations performed in humans and pigs showed that the portions of total life required to reach chemical maturity for both these species are nearly identical, 4.4% and 4.6, respectively. Even though there are species-specific differences of the placenta and immunological system of pigs and human, the piglets are optimum experimental model for investigations concerning physiology and pathology of the gastrointestinal tract of human newborn (Miller & Ullrey, 1987). The postnatal development of the gastrointestinal (GI) system is a very dynamic process. In the neonatal pig with the mean birth body weight of 1.45kg, the small intestine and pancreas weight contribute to 3.1% and 0.14% of the total body weight, respectively (Zabielski et al., 2008). Within the first four postnatal weeks weight of the piglet is increased >5-fold, with the GI organs growing faster than many other organs of the body (Zabielski et al., 2008). Can we suspect the same changes in human neonates? Presumably yes, but the intensity of the remodelling is not as dramatic. The development in humans is slower, and the growth rate is slower in comparison to pigs. In humans the birth weight is doubled within ca. 170 days. Nevertheless, a number of similarities pig and human in the process of the development can be seen. In the study of Len et al. (2009), similar to our investigations of conventional piglets, the absolute weight of visceral organs and GI tract increased with piglet age. However, when expressed as g/kg empty body weight, the weight of visceral organs decreased with age (Len et al., 2009), which is in agreement with Pluske et al. (2002), who found that the relative weight of the visceral organs of piglets had a tendency to decrease between 14 and 28 days of age. In our study we observed a gradual decrease in the weight of small and large intestine between days 2 and 21 of age in suckled piglets while in gnotobiotic piglets the relative weight of intestines increased gradually throughout the period of observation. In germ-free and monoassociated pigs (Shirkey et al., 2006), the relative small intestine length was reduced compared with conventional pigs. The mechanisms affecting intestinal length are unknown, however, it can be hypothesized that increased small intestine length in conventionalized pigs is a compensatory response to the decreased absorptive capacity associated with decreased surface area (decreased villi length) and/or to direct competition with the microbiota for dietary nutrients. Shirkey et al. (2006) observed that in the proximal region of the small intestine, the relative weights for segments from conventional pigs tended to be higher than those from germ-free and monoassociated pigs. This is consistent with our study, as well as with the previous reports indicating that compared with germ-free animals, conventionally reared animals experience intestinal "thickening" associated primarily with increased *lamina propria* cellularity (Miniats & Valli, 1973) as well as thickening of the submucosa and muscular layers (Furuse & Okumura, 1994). On the

gnotobiotic piglets inoculated with *Enterococcus faecium* (12.86 and 32.08 mmol/l, resp.) in comparison with all other groups of piglets (10.7 and 25.9 mmol/l, resp.) of conventional suckling piglets (Zitnan, 2001), (5.36 and 25.00 mmol/l, resp.) of conventional suckling piglets in our study, (6.87 and 19.86 mmol/l, resp.) of replacer-fed piglets and gnotobiotic piglets inoculated with *Lactobacillus plantarum* (11.85 and 14.2 mmol/l, resp.). Under the influence of more diverse populations of microorganisms the conditions in the colonic content changed with gradual occurrences of propionic, butyric, and valeric acids. Organic acids and ammonia concentrating in the colon were according to Kiare et al. (2007) 5-10-fold bigger than those in the ileum and their increased concentrations resulted in additional fermentation activity of short-chain organic acids. This indicates higher microbial activity and considerable N-metabolism in the caudal segment of the intestine. The most significant increase in production of organic acids in the colonic segment observed in our study involved acetoacetic, acetic, propionic and butyric acids in the group of suckled piglets compared to replacer-fed piglets, at important concentrations of acetoacetic acid from 14 to 28 days of age (p < 0.01). Significant increase was observed also in production of lactic acid in gnotobiotic piglets compared to the replacer-fed animals throughout the observation period with the highest concentrations reached on days 2 (p < 0.05), 14, 21 and 28 of age (p < 0.01). Decreased pH of the gut content and increased production of lactic and acetic acids affects positively optimisation of digestive processes. Bomba et al. (1998) investigated intestinal metabolism of gnotobiotic piglets and recorded significantly lower pH (p<0.05) in the jejunal content in piglets inoculated with *Lactobacillus plantarum* in the 1st week of life in comparison with germ-free piglets. Zitnan et al. (2001) observed pH in the jejunal and ileal content of conventional piglets of the same age. When comparing the actual acidity in individual segments of the small intestine, pH of the jejunum content of germ-free piglets was higher in the first week of life (7.49) in comparison with pH of conventional piglets (6.23) of the same age. Contrary to that, pH of the jejunal content of gnotobiotic piglets inoculated with *Lactobacillus plantarum* was considerably lower (5.63). Similar low pH was recorded in our study in gnotobiotic piglets of the same age inoculated with *Enterococcus faecium* (6.02). Bomba et al. (1998) conducted two experiments to investigate the influence of short-term and continuous preventive administration of *Lactobacillus casei* subs.casei against *E.coli* on actual acidity, production of organic acids and colonisation of jejunum with *E.coli*  O8:K88 in gnotobiotic piglets. After the short-term administration of *L.casei* they recorded lower pH in the jejunal content of experimental piglets (L-E) while pH of the ileal content of these piglets increased significantly (7.63) in comparison with the control (7.03). After continuous administration, the authors recorded lower pH in the experimental group L-E (6.1) in comparison with the control group (6.28). In our experiment we observed a positive influence of *Enterococcus faecium* on intestinal metabolism in replacer-fed piglets in terms of increased production of organic acids (formic acid, acetoacetic, propionic and butyric acid). However, production of lactic acid responsible for decrease in pH was lower in our observations and their concentrations ranged between 6.27 and 20.30 mmol/l in the ileal segment of replacer-fed piglets in comparison with suckled piglets (15.52 to 60.11mmol/l) and failed to induce the corresponding pH reduction. pH in the ileum of non-colostral piglets ranged from 6.9 to 7.6. Although acetic acid reached higher levels in the colon segment, it could become toxic only at low pH of the environment dependent on sufficient concentration of lactic acid in the gut. According to Mufandaedza et al. (2006), decreased active acidity, pH < 5.0, limits even stops growth and multiplication of *E. coli*. Similar conclusions were drawn from our study. Production of organic acids by replacer-fed piglets

gnotobiotic piglets inoculated with *Enterococcus faecium* (12.86 and 32.08 mmol/l, resp.) in comparison with all other groups of piglets (10.7 and 25.9 mmol/l, resp.) of conventional suckling piglets (Zitnan, 2001), (5.36 and 25.00 mmol/l, resp.) of conventional suckling piglets in our study, (6.87 and 19.86 mmol/l, resp.) of replacer-fed piglets and gnotobiotic piglets inoculated with *Lactobacillus plantarum* (11.85 and 14.2 mmol/l, resp.). Under the influence of more diverse populations of microorganisms the conditions in the colonic content changed with gradual occurrences of propionic, butyric, and valeric acids. Organic acids and ammonia concentrating in the colon were according to Kiare et al. (2007) 5-10-fold bigger than those in the ileum and their increased concentrations resulted in additional fermentation activity of short-chain organic acids. This indicates higher microbial activity and considerable N-metabolism in the caudal segment of the intestine. The most significant increase in production of organic acids in the colonic segment observed in our study involved acetoacetic, acetic, propionic and butyric acids in the group of suckled piglets compared to replacer-fed piglets, at important concentrations of acetoacetic acid from 14 to 28 days of age (p < 0.01). Significant increase was observed also in production of lactic acid in gnotobiotic piglets compared to the replacer-fed animals throughout the observation period with the highest concentrations reached on days 2 (p < 0.05), 14, 21 and 28 of age (p < 0.01). Decreased pH of the gut content and increased production of lactic and acetic acids affects positively optimisation of digestive processes. Bomba et al. (1998) investigated intestinal metabolism of gnotobiotic piglets and recorded significantly lower pH (p<0.05) in the jejunal content in piglets inoculated with *Lactobacillus plantarum* in the 1st week of life in comparison with germ-free piglets. Zitnan et al. (2001) observed pH in the jejunal and ileal content of conventional piglets of the same age. When comparing the actual acidity in individual segments of the small intestine, pH of the jejunum content of germ-free piglets was higher in the first week of life (7.49) in comparison with pH of conventional piglets (6.23) of the same age. Contrary to that, pH of the jejunal content of gnotobiotic piglets inoculated with *Lactobacillus plantarum* was considerably lower (5.63). Similar low pH was recorded in our study in gnotobiotic piglets of the same age inoculated with *Enterococcus faecium* (6.02). Bomba et al. (1998) conducted two experiments to investigate the influence of short-term and continuous preventive administration of *Lactobacillus casei* subs.casei against *E.coli* on actual acidity, production of organic acids and colonisation of jejunum with *E.coli*  O8:K88 in gnotobiotic piglets. After the short-term administration of *L.casei* they recorded lower pH in the jejunal content of experimental piglets (L-E) while pH of the ileal content of these piglets increased significantly (7.63) in comparison with the control (7.03). After continuous administration, the authors recorded lower pH in the experimental group L-E (6.1) in comparison with the control group (6.28). In our experiment we observed a positive influence of *Enterococcus faecium* on intestinal metabolism in replacer-fed piglets in terms of increased production of organic acids (formic acid, acetoacetic, propionic and butyric acid). However, production of lactic acid responsible for decrease in pH was lower in our observations and their concentrations ranged between 6.27 and 20.30 mmol/l in the ileal segment of replacer-fed piglets in comparison with suckled piglets (15.52 to 60.11mmol/l) and failed to induce the corresponding pH reduction. pH in the ileum of non-colostral piglets ranged from 6.9 to 7.6. Although acetic acid reached higher levels in the colon segment, it could become toxic only at low pH of the environment dependent on sufficient concentration of lactic acid in the gut. According to Mufandaedza et al. (2006), decreased active acidity, pH < 5.0, limits even stops growth and multiplication of *E. coli*. Similar conclusions were drawn from our study. Production of organic acids by replacer-fed piglets was low and resulted in their low concentrations which did not decrease pH so effectively as it was observed in gnotobiotic piglets inoculated with *Enterococcus faecium.* Deficit of colostral nutrition in these piglets resulted in worsened health in 8 out of total 26 piglets. The disease was peracute and proceeded with physiological temperature. Even though antibiotics were administered, the piglets died within 8 hrs of appearing of the first symptoms. In the piglets, lymphocytic leukocytosis as well as hypochromic anemia were diagnosed, and *E. coli* K88 was isolated from rectal swabs.

#### **4.3 Intestinal morphology and disaccharidase activity**

Due to numerous similarities of the physiology and anatomy of the gastrointestinal tract of man and pigs, the pig model is a very attractive model for human nutritional studies (Miller & Ullrey, 1987). Investigations performed in humans and pigs showed that the portions of total life required to reach chemical maturity for both these species are nearly identical, 4.4% and 4.6, respectively. Even though there are species-specific differences of the placenta and immunological system of pigs and human, the piglets are optimum experimental model for investigations concerning physiology and pathology of the gastrointestinal tract of human newborn (Miller & Ullrey, 1987). The postnatal development of the gastrointestinal (GI) system is a very dynamic process. In the neonatal pig with the mean birth body weight of 1.45kg, the small intestine and pancreas weight contribute to 3.1% and 0.14% of the total body weight, respectively (Zabielski et al., 2008). Within the first four postnatal weeks weight of the piglet is increased >5-fold, with the GI organs growing faster than many other organs of the body (Zabielski et al., 2008). Can we suspect the same changes in human neonates? Presumably yes, but the intensity of the remodelling is not as dramatic. The development in humans is slower, and the growth rate is slower in comparison to pigs. In humans the birth weight is doubled within ca. 170 days. Nevertheless, a number of similarities pig and human in the process of the development can be seen. In the study of Len et al. (2009), similar to our investigations of conventional piglets, the absolute weight of visceral organs and GI tract increased with piglet age. However, when expressed as g/kg empty body weight, the weight of visceral organs decreased with age (Len et al., 2009), which is in agreement with Pluske et al. (2002), who found that the relative weight of the visceral organs of piglets had a tendency to decrease between 14 and 28 days of age. In our study we observed a gradual decrease in the weight of small and large intestine between days 2 and 21 of age in suckled piglets while in gnotobiotic piglets the relative weight of intestines increased gradually throughout the period of observation. In germ-free and monoassociated pigs (Shirkey et al., 2006), the relative small intestine length was reduced compared with conventional pigs. The mechanisms affecting intestinal length are unknown, however, it can be hypothesized that increased small intestine length in conventionalized pigs is a compensatory response to the decreased absorptive capacity associated with decreased surface area (decreased villi length) and/or to direct competition with the microbiota for dietary nutrients. Shirkey et al. (2006) observed that in the proximal region of the small intestine, the relative weights for segments from conventional pigs tended to be higher than those from germ-free and monoassociated pigs. This is consistent with our study, as well as with the previous reports indicating that compared with germ-free animals, conventionally reared animals experience intestinal "thickening" associated primarily with increased *lamina propria* cellularity (Miniats & Valli, 1973) as well as thickening of the submucosa and muscular layers (Furuse & Okumura, 1994). On the

Differences in the Development of the Small Intestine

Between Gnotobiotic and Conventionally Bred Piglets 405

The gastrointestinal tract goes through substantial structural and functional changes in the early postnatal period (Walthall et al., 2005). As the piglets grow, functional changes occur in the expression and kinetics (Fan et al., 2002) of brush border digestive enzymes. Each brush border enzyme shows a specific developmental pattern as the animal ages, which have been associated with the maturation of enterocytes (Walthall et al., 2005). Specifically, changing disaccharidasae activities have been used as an indicator of intestinal maturation. Measurable lactase levels were detected in bush border homogenates and membrane vesicles of the small intestine in the 7th week of pregnancy (Buddington & Malo, 1996). For comparison, activity of lactase in human foetuses was confirmed only later and only in the 34th week of gravidity (Menard & Basque, 2001). Aumaitre & Corring (1978) measured lactase in small intestine homogenates from pig foetuses at 105th day of gravidity and observed that the total activity of lactase in the intestine amounted to only 10% of the activity determined at birth. It was stated that specific activities of lactase in homogenates or membrane vesicles of the small intestine brush border were high at birth and stayed at this level during the first 7-10 days of postnatal life (Torp et al., 1993). In suckling pigs, lactase activity was observed to undergo an initial marked decrease sometime during the second to fifth week of age which was followed by a period when it remained relatively constant or continued to decrease gradually up to 8 weeks of age (Kelly et al., 1991). In terms of enzyme distribution throughout the intestine, neonatal and 1-day old piglets show the highest specific lactase activity in the proximal part of the small intestine and the lowest in the distal part, but at the age of 6-10 days its distribution throughout the intestine was more regular (Buddington & Malo, 1996). The intestinal microbiota has been shown to affect brush border enzyme expression, as the intestine of a germ-free mouse has a different pattern of brush border enzymes than a conventional mouse (Kozakova et al., 2001). The mechanism by which bacteria induce changes in brush border enzyme activities or which bacteria are responsible has not been elucidated. According to (Willing & Kessel, 2009), conventionalization in pigs reduced enterocyte brush border enzyme activity compared with germ-free without a concomitant reduction in gene expression in the case of lactase phlorizin hydrolase. Because of the reduced villus height and increased enterocyte replacement rate observed in conventional as compared with germ-free animals (Furuse & Okumura, 1994), it has been postulated that the higher disaccharidase activity in the small intestine of germ-free as compared with conventional rats is because of an increased number of mature enterocytes (Willing & Kessel, 2009). These reports were confirmed by Reddy & Wostmann (1966) who observed that disaccharidase activity was higer in the small intestine of the germ-free as compared with conventional rats. However, in our study, contrary to previous studies, specific activities of lactase along the entire intestinal tract were higher in conventional piglets throughout the experiment. Kozakova et al. (2001) concluded that individual bacteria can stimulate a similar response, as monoassociation of gnotobiotic mice with *Bifidobacteria bifidum* induces a shift in enzyme activity to a pattern similar to that of a conventional mouse. Aumaitre & Corring (1978) reported that the intestinal tract of foetal (105th day of gravidity) and newborn piglets contained maltase but saccharase was present in one week old piglets. Similar observations for saccharase were presented by Buddington & Malo (1996). However, the studies by James et al. (1987) and Sangild et al. (1991) revealed low activities of saccharase and maltase in the small intestine of newborn piglets. Starting from 1 week of age specific activities of maltase and saccharase abruptly increased reaching maximum at the age of 10 - 16 days and sustained values at the age of approximately 3 weeks (James et al., 1987; Sangild et al., 1991). Similar tendencies of specific activity of

contrary, higher relative weight of the distal part of the intestine was reported in germ-free piglets (Shirkey et al., 2006). Similar results were obtained in our study which showed higher relative weight of the large intestine in gnotobiotic piglets inoculated with *Enterococcus faecium* in comparison with conventional piglets starting from the second week of age up to the weaning (day 28 of age). In addition to an intensive growth of the GI system, during the first month of life an intense rebuilding of the tissues takes place. The most intensive processes are observed in the epithelium of the small intestine (Zabielski et al., 2008). The weight of small intestinal mucosa doubles during the first postnatal day due to a complex of processes involving, accumulation of colostrum proteins in the enterocytes as a result of an open "gut barrier", increase of local blood flow concurrently with a reduction in basal vascular resistance (Nankervis et al., 2001), and finally changes in epithelial cell turnover, namely, increased mitosis accompanied by the inhibition of apoptosis which result in a 2-fold increase in the mitosis/apoptosis ratio within the first 2 postnatal days (Zabielski et al., 2008). The regulation of small intestine development (especially the tissue growth) is in a positive feed-back to colostrum and milk intake (Marion et al., 2003). Currently none artificial feeding system (milk, artificial milk formula, nor feeding with any other compositions like lactose, glucose solutions) could reproduce the developmental characteristics obtained with maternal colostrum feeding (Zabielski et al., 2008). Furthermore, high specificity of colostrum, especially concerning the composition of hormones and bioactive compounds prevents utilization of colostrum of other species as the replacement. In the study of Meslin et al. (1973), the overall mass of the small intestine in germ-free species was decreased, and its surface area was smaller, whereas the villi of the small intestine were unusually uniform in shape and appear slender, with crypts, which were shorter and less populated than in the respective conventional control animals. Our study showed that the jejunal part of the intestinal tract in gnotobiotic pigs was characterized up to 14 days of life by relatively short crypts, extremely long villi and narrow *lamina propria* containing few cells. Reduced crypt depth and increased villus length agree with the previous observations in germ-free pigs (Shirkey et al., 2006; Shurson et al., 1990). In the present study in gnotobiotic piglets villi were the longest in the jejunum and shortest in the duodenum and ileum, whereas crypt depth was shortest in the jejunum and deepest in the duodenum throughout the observation period. These morphological characteristics suggested that the rates of enterocyte proliferation and exfoliation were the highest in the proximal small intestine, as indicated by deep crypts and shorter villi, respectively, with rates decreasing distally along the small intestine (Hampson & Kidder, 1986). In agreement with our morphological findings, Miniats & Valli (1973) reported longer jejunal villi in germ-free pigs but did not measure villi in other regions. Shurson et al. (1990) reported that germ-free pigs had longer ileal and duodenal villi but shorter jejunal villi compared to their conventional counterparts. Similar results were obtained also in our study as the villi in the duodenum and ileum of gnotobiotic piglets were higher in comparison with conventional piglets throughout the experiment. The difference was significant at 3 hours after birth, on days 2 and 7 of age (p < 0.01) and day 14 of age (p < 0.05) in the duodenum and on days 14 and 21 in the ileum (p < 0.05). Shirkey et al. (2006) suggested that regional variation in morphology, especially in the proximal small intestine, is not entirely dependent on microbial colonization but is also influenced by such non-microbial factors as bile salts, pancreatic secretions, and compounds of dietary origin which would be expected to be in higher concentration and have more contact with mucosal surface in the duodenum.

contrary, higher relative weight of the distal part of the intestine was reported in germ-free piglets (Shirkey et al., 2006). Similar results were obtained in our study which showed higher relative weight of the large intestine in gnotobiotic piglets inoculated with *Enterococcus faecium* in comparison with conventional piglets starting from the second week of age up to the weaning (day 28 of age). In addition to an intensive growth of the GI system, during the first month of life an intense rebuilding of the tissues takes place. The most intensive processes are observed in the epithelium of the small intestine (Zabielski et al., 2008). The weight of small intestinal mucosa doubles during the first postnatal day due to a complex of processes involving, accumulation of colostrum proteins in the enterocytes as a result of an open "gut barrier", increase of local blood flow concurrently with a reduction in basal vascular resistance (Nankervis et al., 2001), and finally changes in epithelial cell turnover, namely, increased mitosis accompanied by the inhibition of apoptosis which result in a 2-fold increase in the mitosis/apoptosis ratio within the first 2 postnatal days (Zabielski et al., 2008). The regulation of small intestine development (especially the tissue growth) is in a positive feed-back to colostrum and milk intake (Marion et al., 2003). Currently none artificial feeding system (milk, artificial milk formula, nor feeding with any other compositions like lactose, glucose solutions) could reproduce the developmental characteristics obtained with maternal colostrum feeding (Zabielski et al., 2008). Furthermore, high specificity of colostrum, especially concerning the composition of hormones and bioactive compounds prevents utilization of colostrum of other species as the replacement. In the study of Meslin et al. (1973), the overall mass of the small intestine in germ-free species was decreased, and its surface area was smaller, whereas the villi of the small intestine were unusually uniform in shape and appear slender, with crypts, which were shorter and less populated than in the respective conventional control animals. Our study showed that the jejunal part of the intestinal tract in gnotobiotic pigs was characterized up to 14 days of life by relatively short crypts, extremely long villi and narrow *lamina propria* containing few cells. Reduced crypt depth and increased villus length agree with the previous observations in germ-free pigs (Shirkey et al., 2006; Shurson et al., 1990). In the present study in gnotobiotic piglets villi were the longest in the jejunum and shortest in the duodenum and ileum, whereas crypt depth was shortest in the jejunum and deepest in the duodenum throughout the observation period. These morphological characteristics suggested that the rates of enterocyte proliferation and exfoliation were the highest in the proximal small intestine, as indicated by deep crypts and shorter villi, respectively, with rates decreasing distally along the small intestine (Hampson & Kidder, 1986). In agreement with our morphological findings, Miniats & Valli (1973) reported longer jejunal villi in germ-free pigs but did not measure villi in other regions. Shurson et al. (1990) reported that germ-free pigs had longer ileal and duodenal villi but shorter jejunal villi compared to their conventional counterparts. Similar results were obtained also in our study as the villi in the duodenum and ileum of gnotobiotic piglets were higher in comparison with conventional piglets throughout the experiment. The difference was significant at 3 hours after birth, on days 2 and 7 of age (p < 0.01) and day 14 of age (p < 0.05) in the duodenum and on days 14 and 21 in the ileum (p < 0.05). Shirkey et al. (2006) suggested that regional variation in morphology, especially in the proximal small intestine, is not entirely dependent on microbial colonization but is also influenced by such non-microbial factors as bile salts, pancreatic secretions, and compounds of dietary origin which would be expected to be in higher concentration and

have more contact with mucosal surface in the duodenum.

The gastrointestinal tract goes through substantial structural and functional changes in the early postnatal period (Walthall et al., 2005). As the piglets grow, functional changes occur in the expression and kinetics (Fan et al., 2002) of brush border digestive enzymes. Each brush border enzyme shows a specific developmental pattern as the animal ages, which have been associated with the maturation of enterocytes (Walthall et al., 2005). Specifically, changing disaccharidasae activities have been used as an indicator of intestinal maturation. Measurable lactase levels were detected in bush border homogenates and membrane vesicles of the small intestine in the 7th week of pregnancy (Buddington & Malo, 1996). For comparison, activity of lactase in human foetuses was confirmed only later and only in the 34th week of gravidity (Menard & Basque, 2001). Aumaitre & Corring (1978) measured lactase in small intestine homogenates from pig foetuses at 105th day of gravidity and observed that the total activity of lactase in the intestine amounted to only 10% of the activity determined at birth. It was stated that specific activities of lactase in homogenates or membrane vesicles of the small intestine brush border were high at birth and stayed at this level during the first 7-10 days of postnatal life (Torp et al., 1993). In suckling pigs, lactase activity was observed to undergo an initial marked decrease sometime during the second to fifth week of age which was followed by a period when it remained relatively constant or continued to decrease gradually up to 8 weeks of age (Kelly et al., 1991). In terms of enzyme distribution throughout the intestine, neonatal and 1-day old piglets show the highest specific lactase activity in the proximal part of the small intestine and the lowest in the distal part, but at the age of 6-10 days its distribution throughout the intestine was more regular (Buddington & Malo, 1996). The intestinal microbiota has been shown to affect brush border enzyme expression, as the intestine of a germ-free mouse has a different pattern of brush border enzymes than a conventional mouse (Kozakova et al., 2001). The mechanism by which bacteria induce changes in brush border enzyme activities or which bacteria are responsible has not been elucidated. According to (Willing & Kessel, 2009), conventionalization in pigs reduced enterocyte brush border enzyme activity compared with germ-free without a concomitant reduction in gene expression in the case of lactase phlorizin hydrolase. Because of the reduced villus height and increased enterocyte replacement rate observed in conventional as compared with germ-free animals (Furuse & Okumura, 1994), it has been postulated that the higher disaccharidase activity in the small intestine of germ-free as compared with conventional rats is because of an increased number of mature enterocytes (Willing & Kessel, 2009). These reports were confirmed by Reddy & Wostmann (1966) who observed that disaccharidase activity was higer in the small intestine of the germ-free as compared with conventional rats. However, in our study, contrary to previous studies, specific activities of lactase along the entire intestinal tract were higher in conventional piglets throughout the experiment. Kozakova et al. (2001) concluded that individual bacteria can stimulate a similar response, as monoassociation of gnotobiotic mice with *Bifidobacteria bifidum* induces a shift in enzyme activity to a pattern similar to that of a conventional mouse. Aumaitre & Corring (1978) reported that the intestinal tract of foetal (105th day of gravidity) and newborn piglets contained maltase but saccharase was present in one week old piglets. Similar observations for saccharase were presented by Buddington & Malo (1996). However, the studies by James et al. (1987) and Sangild et al. (1991) revealed low activities of saccharase and maltase in the small intestine of newborn piglets. Starting from 1 week of age specific activities of maltase and saccharase abruptly increased reaching maximum at the age of 10 - 16 days and sustained values at the age of approximately 3 weeks (James et al., 1987; Sangild et al., 1991). Similar tendencies of specific activity of

Differences in the Development of the Small Intestine

Between Gnotobiotic and Conventionally Bred Piglets 407

piglets, it is associated with a variable period of anorexia during the first days after weaning, the deterioration of the digestive function and accumulation of undigested feed as a result of inefficient digestion. During this period, piglets are more susceptible to suffer from postweaning diarrhoea with the proliferation and attachment to the intestinal mucosa of haemolytic strains of *E. coli* (Fairbrother et al., 2005). Nabuurs (1998) and Pluske et al. (1997) stated that predisposition to infections with eneterotoxigenic bacteria depends on a number of factors. Miller et al. (1986) concluded that the problems induced by weaning were caused rather by the changes in the structure of the intestines and specific loss of digestive enzymes than by any great changes in absorption function despite the fact that the data of Nabuurs et al. (1998) were contradictory. Nabuurs (1998) concluded that piglets suffering from postweaning diarrhoea excreted enterotoxigenic *E. coli* strains and *rotavirus,* and that these piglets developed a hyperregenerative villus atrophy, and subsequently a severe loss of net absorption of fluid and electrolytes in the small intestine. A simulated halving of the absorption in the large intestine of weaned piglets aggravates the adverse effects of an enterotoxigenic *E. coli* in the small intestine (Nabuurs, 1998). Franklin et al. (2002) recorded no post-weaning increase in *E.coli* in pigs weaned at 17 days of age, in agreement with the studies of Etheridge et al. (1984) and Mathew et al. (1998), but in contrast with others (Mathew et al., 1996) who reported increase in *E.coli* populations after weaning. We have observed changes during the first week post-weaning in the jejunal part of the digestive tract of colostral piglets that pointed to a decrease in all observed groups of bacteria with the highest decrease by 1-1.8 log for enterococci*, E. coli* and *Enterobacteriaceae.* Mathew et al. (1998) postulated the absence of an *E.coli* increase may be due to weaning pigs into a highly sanitized, environmentally controlled room with limited contact among pigs. Franklin et al. (2002) also observed *E.coli* populations to be lower in pigs remaining on the sow, as have other investigators (Etheridge et al., 1984; Mathew et al., 1996). Jensen (1998) reported that lactobacilli are inversely proportionate to coliform bacteria during 1 week post-weaning. This is also confirmed by the results of Risley et al. (1992), but however, has not been confirmed in our experiments. In the study by Franklin et al. (2002) faecal populations of lactobacilli and *E.coli* followed patterns typical of those observed in the more anterior portions of the gastrointestinal tract. However, faecal bifidobacteria populations increased post-weaning, possibly due to the decrease in lactobacilli and *E.coli* in the posterior gastrointestinal tract. The loss of direct competition may benefit other bacterial populations, including bifidobacteria. The infant´s microbiota initially shows low diversity and instability, but evolves into a more stable adult-type microbiota over the first 24 months of life (Zoetendal et al., 1998). *Bifidobacterium* populations are dominant in the first months of life, especially in breast-fed infants due to the bifinogenic effect of breast milk, while a more diverse microbiota is found in formula-fed infants, weaning children and adults (Gueimonde et al., 2006). In adults and weaned children the major constituents of the colonic microbiota are *Bacteroides*, followed by several genera belonging to the division *Firmicutes*, such as *Eubacterium*, *Ruminococcus* and *Clostridium*, and the genus *Bifidobacterium*. By contrast, in infants the genus *Bifidobacterium* is predominant and also a few genera from the family *Enterobacteriaceae*, as a *Escherichia*, *Raoultella*, and *Klebsiella* (Kurokawa et al., 2007). It is well know that weaning has a dramatic negative impact on the intestinal mucosal morphology of piglets. Significant post-weaning reduction in villus height has been observed by study (Berkeveld et al., 2007). In study of Hedemann et al. (2003) villus height decreased to a minimum during the first 3 days post-weaning and this is in accordance with the other studies showing that villous height is minimal 2-5 days post-weaning (Hampson & Kidder, 1986; Kelly et al., 1991). In the jejunum and ileum of conventional piglets, investigated in our

maltase and saccharase were observed also in our study. When comparing the postnatal development of specific activities of enzymes maltase and saccharase in gnotobiotic and conventional piglets we observed a similar trend but higher activities of both enzymes in all segments of digestive tract of conventional piglets throughout the observation period. In 6 - 7 days old piglets, the distribution of activities of saccharase and maltase was similar along the small intestine but the activities of both enzymes were higher in the proximal to medial parts of the jejunum compared to distal part of the small intestine (Aumaitre & Corring, 1978; Buddington & Malo, 1996). In our study, saccharase activity distribution throughout the small intestine of gnotobiotic and conventional piglets was higher in the jejunum during the entire period of observation. Distribution of maltase along the small intestine changed depending on age, from predominant concentration of the activity in the proximal half of the small intestine at the age of 1-2 weeks (Aumaitre & Corring, 1978) through uniform distribution along the small intestine at the age of 2-3 weeks (Kelly et al., 1991) up to higher activity in the range of 10-15% along 80-90% of the intestine length at the age of 5 - 8 weeks (Hampson & Kidder, 1986). Similar distribution of maltase along the small intestine was observed also in our study in both gnotobiotic and conventional piglets. Transition from milk nutrition to definitive nutrition in children is accompanied with induction of maltase and decreasing activity of lactase as an adaptation of GIT to changes in nutrition with age (Menard & Basque, 2001). Other factors besides age which affect development of disaccharidases activities in the small intestine include: feed offered to piglets in the period of suckling (Hampson & Kidder, 1986), weaning to dry or liquid feed, growth factors, for example epidermal growth factor (James et al., 1987) and hormones, for example insulin (Shulman, 1990), corticosteroids (Kreikemeier et al., 1990), ACTH - adrenocorticotropin (Sangild et al.,1991). Willing & Kessel (2009) concluded that enterocyte upregulation of brush border enzyme expression occurs as either a direct response to microbial colonization or as a feedback mechanisms in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

#### **4.4 Effect of weaning on development of the small intestine of conventionally and gnotobiotic bred piglets**

Another critical phase in the gastrointestinal tract development of young animals is the weaning period. The weaning of piglets usually takes place between 3 and 4 week of life, when the majority of nutrients are ingested with milk. Weaning for farm animals occurs in an early age, when the gastrointestinal system motility, digestive and absorptive functions are not yet matured and prepared for food other than milk. In a wild boar, domestic pig ancestor, the offspring is weaned in much older age and change of the diet is gradual, therefore weaning disorders are nearly nonexistent. In intensive livestock production shorter suckling period benefits in increased number of piglets born per year, but at the negative side is an increased number of weaning disorders (Zabielski et al., 2008). Weaning is associated with mixing of piglets from different litters and sometimes also with the transport of animals from the place of birth to specialized nursery units. This results in profound social and environmental stress which is a caused also by the changes in the diet. The gastrointestinal tract has to adapt to the new type of feed, which leads to changes in myenteron motility, enzymes secretion and activity, and the composition of bacterial flora (Barszcz & Skomial, 2011). According to Lalles et al. (2007) weaning is a critical phase for

maltase and saccharase were observed also in our study. When comparing the postnatal development of specific activities of enzymes maltase and saccharase in gnotobiotic and conventional piglets we observed a similar trend but higher activities of both enzymes in all segments of digestive tract of conventional piglets throughout the observation period. In 6 - 7 days old piglets, the distribution of activities of saccharase and maltase was similar along the small intestine but the activities of both enzymes were higher in the proximal to medial parts of the jejunum compared to distal part of the small intestine (Aumaitre & Corring, 1978; Buddington & Malo, 1996). In our study, saccharase activity distribution throughout the small intestine of gnotobiotic and conventional piglets was higher in the jejunum during the entire period of observation. Distribution of maltase along the small intestine changed depending on age, from predominant concentration of the activity in the proximal half of the small intestine at the age of 1-2 weeks (Aumaitre & Corring, 1978) through uniform distribution along the small intestine at the age of 2-3 weeks (Kelly et al., 1991) up to higher activity in the range of 10-15% along 80-90% of the intestine length at the age of 5 - 8 weeks (Hampson & Kidder, 1986). Similar distribution of maltase along the small intestine was observed also in our study in both gnotobiotic and conventional piglets. Transition from milk nutrition to definitive nutrition in children is accompanied with induction of maltase and decreasing activity of lactase as an adaptation of GIT to changes in nutrition with age (Menard & Basque, 2001). Other factors besides age which affect development of disaccharidases activities in the small intestine include: feed offered to piglets in the period of suckling (Hampson & Kidder, 1986), weaning to dry or liquid feed, growth factors, for example epidermal growth factor (James et al., 1987) and hormones, for example insulin (Shulman, 1990), corticosteroids (Kreikemeier et al., 1990), ACTH - adrenocorticotropin (Sangild et al.,1991). Willing & Kessel (2009) concluded that enterocyte upregulation of brush border enzyme expression occurs as either a direct response to microbial colonization or as a feedback mechanisms in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host

with the intestinal microbiota for available nutrients.

**gnotobiotic bred piglets** 

**4.4 Effect of weaning on development of the small intestine of conventionally and** 

Another critical phase in the gastrointestinal tract development of young animals is the weaning period. The weaning of piglets usually takes place between 3 and 4 week of life, when the majority of nutrients are ingested with milk. Weaning for farm animals occurs in an early age, when the gastrointestinal system motility, digestive and absorptive functions are not yet matured and prepared for food other than milk. In a wild boar, domestic pig ancestor, the offspring is weaned in much older age and change of the diet is gradual, therefore weaning disorders are nearly nonexistent. In intensive livestock production shorter suckling period benefits in increased number of piglets born per year, but at the negative side is an increased number of weaning disorders (Zabielski et al., 2008). Weaning is associated with mixing of piglets from different litters and sometimes also with the transport of animals from the place of birth to specialized nursery units. This results in profound social and environmental stress which is a caused also by the changes in the diet. The gastrointestinal tract has to adapt to the new type of feed, which leads to changes in myenteron motility, enzymes secretion and activity, and the composition of bacterial flora (Barszcz & Skomial, 2011). According to Lalles et al. (2007) weaning is a critical phase for piglets, it is associated with a variable period of anorexia during the first days after weaning, the deterioration of the digestive function and accumulation of undigested feed as a result of inefficient digestion. During this period, piglets are more susceptible to suffer from postweaning diarrhoea with the proliferation and attachment to the intestinal mucosa of haemolytic strains of *E. coli* (Fairbrother et al., 2005). Nabuurs (1998) and Pluske et al. (1997) stated that predisposition to infections with eneterotoxigenic bacteria depends on a number of factors. Miller et al. (1986) concluded that the problems induced by weaning were caused rather by the changes in the structure of the intestines and specific loss of digestive enzymes than by any great changes in absorption function despite the fact that the data of Nabuurs et al. (1998) were contradictory. Nabuurs (1998) concluded that piglets suffering from postweaning diarrhoea excreted enterotoxigenic *E. coli* strains and *rotavirus,* and that these piglets developed a hyperregenerative villus atrophy, and subsequently a severe loss of net absorption of fluid and electrolytes in the small intestine. A simulated halving of the absorption in the large intestine of weaned piglets aggravates the adverse effects of an enterotoxigenic *E. coli* in the small intestine (Nabuurs, 1998). Franklin et al. (2002) recorded no post-weaning increase in *E.coli* in pigs weaned at 17 days of age, in agreement with the studies of Etheridge et al. (1984) and Mathew et al. (1998), but in contrast with others (Mathew et al., 1996) who reported increase in *E.coli* populations after weaning. We have observed changes during the first week post-weaning in the jejunal part of the digestive tract of colostral piglets that pointed to a decrease in all observed groups of bacteria with the highest decrease by 1-1.8 log for enterococci*, E. coli* and *Enterobacteriaceae.* Mathew et al. (1998) postulated the absence of an *E.coli* increase may be due to weaning pigs into a highly sanitized, environmentally controlled room with limited contact among pigs. Franklin et al. (2002) also observed *E.coli* populations to be lower in pigs remaining on the sow, as have other investigators (Etheridge et al., 1984; Mathew et al., 1996). Jensen (1998) reported that lactobacilli are inversely proportionate to coliform bacteria during 1 week post-weaning. This is also confirmed by the results of Risley et al. (1992), but however, has not been confirmed in our experiments. In the study by Franklin et al. (2002) faecal populations of lactobacilli and *E.coli* followed patterns typical of those observed in the more anterior portions of the gastrointestinal tract. However, faecal bifidobacteria populations increased post-weaning, possibly due to the decrease in lactobacilli and *E.coli* in the posterior gastrointestinal tract. The loss of direct competition may benefit other bacterial populations, including bifidobacteria. The infant´s microbiota initially shows low diversity and instability, but evolves into a more stable adult-type microbiota over the first 24 months of life (Zoetendal et al., 1998). *Bifidobacterium* populations are dominant in the first months of life, especially in breast-fed infants due to the bifinogenic effect of breast milk, while a more diverse microbiota is found in formula-fed infants, weaning children and adults (Gueimonde et al., 2006). In adults and weaned children the major constituents of the colonic microbiota are *Bacteroides*, followed by several genera belonging to the division *Firmicutes*, such as *Eubacterium*, *Ruminococcus* and *Clostridium*, and the genus *Bifidobacterium*. By contrast, in infants the genus *Bifidobacterium* is predominant and also a few genera from the family *Enterobacteriaceae*, as a *Escherichia*, *Raoultella*, and *Klebsiella* (Kurokawa et al., 2007). It is well know that weaning has a dramatic negative impact on the intestinal mucosal morphology of piglets. Significant post-weaning reduction in villus height has been observed by study (Berkeveld et al., 2007). In study of Hedemann et al. (2003) villus height decreased to a minimum during the first 3 days post-weaning and this is in accordance with the other studies showing that villous height is minimal 2-5 days post-weaning (Hampson & Kidder, 1986; Kelly et al., 1991). In the jejunum and ileum of conventional piglets, investigated in our

Differences in the Development of the Small Intestine

062505.

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populations and volatile fatty acid concentrations in the jejunum, ileum and cecum,

study, we observed a post-weaning decrease in the height of villi significant on day 35 of age in the jejunum (p < 0.01). Elongation of the crypts post-weaning has been observed in several studies (Hampson & Kidder, 1986; Hedemann et al., 2003) and was confirmed in the present experiment. In our study, in the first week after weaning, conventional piglets showed significant deepening of crypts in duodenal (p < 0.001), jejunal (p < 0.01) and ileal (p < 0.05) segments. Villous atrophy may result both from increased rate of cell loss leading to higher rate of mitosis in crypts and their hyperplasia and from slower rate of cell renewal resulting from the reduction of cell division, i.e. in case of underfeeding. During the time of weaning villous shape also undergoes modifications. The marked and abrupt morphological response to weaning in the small intestine, characterized by transformation from a dense finger-like villi population to a smooth, compact, tongue-shaped luminal villi was observed in previous study (Skrzypek et al., 2005) and in the present study. The morphological changes observed in the small intestine around weaning are closely related to changes in the mucosal enzyme activity observed at the same time. When shortening of the villi is associated with cell loss, loss of mature enterocytes where digestive enzymes are located also occurs. The disaccharidases have been the most commonly investigated mucosal enzymes in relation to weaning of piglets (Kelly et al., 1991). Morphological changes in the small intestine of piglets after weaning are accompanied by smaller activity of brush border enzymes, lactase and sucrase (Pacha, 2000). In our study we registered that of lactase activity of gnotobiotic piglets decreased in the weeks 4 (p < 0.001) and 5 (p < 0.001) to levels similar to those noticed at birth. Similarly, we recorded a post-weaning decrease in lactase specific activity also in conventional piglets. The results of these studies have been used to interpret the digestive and absorptive capacity of the small intestine as well as the maturity of the enterocytes.
