**3.2 Acidity**

380 New Advances in the Basic and Clinical Gastroenterology

The lactase (EC 3.2.1.23), maltase (EC 3.2.1.20) and saccharase (EC 3.2.1.26) activities were measured according to Mir et al. (1997). Mucosa samples (200 mg) were homogenized for 3 min with 1 mL saline solution at 0oC. The homogenate was transferred to a test tube together with 2.5 mL (2 × aliquot) of saline solution. Three reaction tubes were filled with 100 µL of the homogenate and placed in a 37oC water bath, and then 400 µL of 56 mM lactose, maltose, saccharose in citrate buffer (pH 6.6, 0.01 mM) were added, respectively. After shaking and incubation for 30 min enzyme activity was stopped in boiling water. The reaction tubes were centrifuged at 2000 × g (30 min, 5oC). The individual enzymes were determined using enzymatic UV method (Boehringer Mannheim, Germany). Protein content in homogenate was started according to Bradford et al. (1976) and the results were

Fixed intestinal segments were rinsed with water, the samples were dehydrated in a graded series of absolute ethanol (30%, 50%, 70%, 90%), cleared with benzene, saturated with and embedded in paraffin. Sections of 7 μm thickness (10 slices of each sample) were stained with haematoxylin/eosin and observed under a light microscope. The length of 10 villi and depth of 10 crypts was determined by a computer operated Image C picture analysis system (Intronic GmbH, Berlin, Germany) and the IMES analysis software, using a colour video

Statistical analysis was performed using Statistic software PRIZMA (version 3.0). All the data were presented as means ± SEM. To estimate the effect of age and weaning on the concentration of SCFAs, bacterial count, disaccharidase activity and intestinal morphology, the data were evaluated statistically by one-way analysis of variance (ANOVA) followed by a multiple comparison Tukey´s test. Significant differences between the two groups of piglets were tested using analysis of variance and Student´s t-test. Probability values less

The deficit of colostral feeding on day 4 of life caused clinical symptoms of disease in 8 replacer-fed piglets (i.e. 30.8 % of the total number of 26 piglets). The other piglets from this group were healthy. The sick piglets were apathic and did not show any interest in feeding. The disease was peracute and proceeded with physiological temperature. Even though antibiotics were administered, the piglets died within 8 hrs of the first appearance of symptoms. Rectal smears and blood for hematological examination were taken from the piglets and both pathological and anatomical dissections were carried out. In the piglets, lymphocytic leukocytosis as well as hypochromic anaemia were diagnosed, and *E. coli* K88 was isolated from rectal swabs. The colonies from the final dilutions were verified by slide agglutination with K88ab antiserum (Imuna Šarišské Michaľany, Slovakia). The pathological and anatomical dissection for peracuteness of the course of the disease revealed only

camera (Sony 3 CCD) and a light microscope (Axiolab, Carl Zeiss Jena, Germany).

than 0.05 were used as the criterion for statistical significance.

**2.2.3 Disaccharidase activity** 

expressed as µmol/ mg protein/ hour.

**2.2.4 Small intestinal morphology** 

**2.2.5 Statistical analysis** 

**3.1 Health status of animals** 

**3. Results** 

The actual acidity of stomach digesta in replacer-fed piglets ranged more widely - i.e. from pH 1.7 to 3.8. During the period of observation, only on day 2 of age the pH of stomach contents of these piglets was significantly lower (p<0.01, p<0.05) than in suckled piglets with pH ranging from 2.9 to 3.7 and gnotobiotic piglets with 2.7 - 4.1 pH range (Table 1). In the proximal segment of GIT (content of duodenum and jejunum) of gnotobiotic piglets we recorded between days 7 and 28 days of age the lowest levels of pH which differed significantly on day 14 of age (p<0.001) in duodenum and on day 21 of age (p<0.01) in jejunum in comparison with replacer-fed piglets. The pH level in the caudal segment of GIT (ileal content) of suckled piglets was lower in comparison with replacer-fed piglets and significantly lower on days 2 (p<0.05) and 21 (p<0.01) of age. The ileal and colonic pH were on average lower by 0.08 to 0.5 in the group of suckled piglets and the pH values ranged from 6.27 to 7.17 and from 6.50 to 7.21 compared to replacer-fed piglets in which the pH of the ileal content ranged from 6.93 to 7.64 and the pH of the colonic content ranged from 6.24 to 7.53.

#### **3.3 Effect of age and weaning on production of SCFAs in the intestinal tract of gnotobiotic and conventionally bred piglets**

### **3.3.1 Conventional suckled piglets (natural feeding)**

Concentration of both acetoacetic and acetic acid in the jejunal content of suckled piglets (Table 2) within the period of milk nutrition was the highest at 7 days of age (p < 0.001 and p < 0.01, respectively). Subsequently the values declined at 2 weeks of age to 14.76 mmol/l of acetoacetic and to 28.02 mmol/l of acetic acid. This decline continued in acetoacetic acid by day 35 of age to 6.10 mmol/l and in acetic acid by week 4 of life to 7.71 mmol/l (p < 0.01). The concentration of lactic acid in jejunal contents was comparable to that of both acetoacetic and acetic acid, with a mean decline of 11.85 mmol lactic acid/l between day 2 and day 21. But a pronounced increase in the concentration of lactic acid was recorded at 1st week post-weaning - i.e. 53.91 mmol/l. The course of the concentration of both acetoacetic and lactic acid in the ileal content (Table 3) was largely similar to that recorded in the jejunal content. Under the influence of more diverse populations of microorganisms, the conditions in the colonic content changed. Propionic acid concentration (Table 4) increased gradually up to weaning (28 days) and then markedly after weaning (day 35: p < 0.01 and on day 42: p < 0.05). The most pronounced production of acids in the colonic content was observed in acetoacetic acid with highest concentrations at day 14 and 28 of age (p < 0.001 and p < 0.001) and a sudden 4-fold decline at 1st week post-weaning (p < 0.01). In acetic acid, a gradual increase in values was recorded from 7 days of age (11.91 mmol/l), with its highest concentration at 2 weeks post-weaning (p < 0.001).

#### **3.3.2 Conventional replacer-fed piglets (artificial feeding)**

In both acetoacetic and lactic acid, the highest levels in the jejunal content in replacer-fed piglets (Table 2) were recorded at 7 days of age (19.89 mmol/l and 24.92 mmol/l, p < 0.01,

Differences in the Development of the Small Intestine

Between Gnotobiotic and Conventionally Bred Piglets 383

Table 2. Effect of age, weaning and diets on production of SCFAs in the jejunum (mmol/l) of gnotobiotic and conventionally bred piglets. ND- not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different

(SC,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)


ND- not detectable, NS- not significant, SP- suckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets, St- stomach, D- duodenum, J- jejunum, Ile- ileum, C- colon, GIT- gastrointestinal tract Significantly different (SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Table 1. The pH along the gastrointestinal tract of gnotobiotic and conventionally bred piglets

respectively), with a slight decline in the concentration up to the end of observation. Concentrations of acetoacetic acid in the colonic content (Table 4) were similar to those in the jejunal content except on day 7 of age when a value of 24.09 mmol/l was recorded (p < 0.05). With lactic acid, the highest concentration was seen on day 7 of age (31.17 mmol/l), with a sudden 10-fold decline from day 14 of age (3.15 mmol/l) up to 28 days of life. Acetic acid concentration was relatively stable from 2 to 21 days of life. Thereafter, a marked increase in the concentration was observed at 4 weeks of life (p < 0.01).

#### **3.3.3 Gnotobiotic piglets**

The concentration of acetoacetic acid in the jejunal content of gnotobiotic piglets (Table 2) reached the highest level at the age of 7 days, in the period of milk nutrition (p < 0.05), in comparison with the concentration recorded three hours after birth (3.35 mmol/l). A more


Table 2. Effect of age, weaning and diets on production of SCFAs in the jejunum (mmol/l) of gnotobiotic and conventionally bred piglets. ND- not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different (SC,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

St D J Ile C SEM SC

× RFP

GP × RFP

Segments of GIT p - value

Suckled piglets 3.7 5.5 6.1 6.6a 7.2 0.27 p < 0.05 - Replacer-fed piglets 1.7ba 5.7 6.8 7.4 7.4 0.12 p < 0.01 p < 0.05 Gnotobiotic piglets 4.1 ND 7.2 8.0 7.7 0.39 - -

Suckled piglets 2.9 5.6 6.2 6.3 6.5 0.09 NS - Replacer-fed piglets 3.8 5.8 6.0 6.9 6.3b 0.22 NS p < 0.01 Gnotobiotic piglets 4.1 4.5 5.9 7.4 7.0 0.56 - -

Suckled piglets 3.4 5.4 6.1 7.2 6.7 0.09 NS - Replacer-fed piglets 2.5 5.5 5.9 7.1 7.5 0.18 NS - Gnotobiotic piglets 3.5 4.2c 5.4 7.0 6.4 0.23 - p < 0.001

Suckled piglets 3.3 5.2 6.2a 6.8b 7.4 0.15 p < 0.01 - Replacer-fed piglets 3.4 5.4 6.7 7.5 7.5 0.23 - - Gnotobiotic piglets 3.1 4.2 5.9b 6.9 6.7 0.34 - p < 0.01

Suckled piglets 3.2 5.8 5.9 6.6 6.5 0.18 NS - Replacer-fed piglets 2.9 6.0 6.7 7.6 6.2 0.25 NS NS Gnotobiotic piglets 2.7 4.4 5.8 6.7 6.6 0.34 - NS ND- not detectable, NS- not significant, SP- suckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets, St- stomach, D- duodenum, J- jejunum, Ile- ileum, C- colon, GIT- gastrointestinal tract

Table 1. The pH along the gastrointestinal tract of gnotobiotic and conventionally bred

respectively), with a slight decline in the concentration up to the end of observation. Concentrations of acetoacetic acid in the colonic content (Table 4) were similar to those in the jejunal content except on day 7 of age when a value of 24.09 mmol/l was recorded (p < 0.05). With lactic acid, the highest concentration was seen on day 7 of age (31.17 mmol/l), with a sudden 10-fold decline from day 14 of age (3.15 mmol/l) up to 28 days of life. Acetic acid concentration was relatively stable from 2 to 21 days of life. Thereafter, a marked

The concentration of acetoacetic acid in the jejunal content of gnotobiotic piglets (Table 2) reached the highest level at the age of 7 days, in the period of milk nutrition (p < 0.05), in comparison with the concentration recorded three hours after birth (3.35 mmol/l). A more

*Day 21* p < 0.05

Significantly different (SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

increase in the concentration was observed at 4 weeks of life (p < 0.01).

*Day 2*

*Day 7*

*Day 14*

*Day 28*

piglets

**3.3.3 Gnotobiotic piglets** 

Differences in the Development of the Small Intestine

Replacer-fed

Gnotobiotic

*Acetoacetic acid* 

Replacer-fed

Gnotobiotic

Replacer-fed

Gnotobiotic

*Succinic acid* 

Replacer-fed

Gnotobiotic

Replacer-fed

Gnotobiotic

*Propionic acid* 

Replacer-fed

Gnotobiotic

Replacer-fed

Gnotobiotic

*Butyric acid* 

*Acetic acid* 

*Lactic acid* 

Between Gnotobiotic and Conventionally Bred Piglets 385

Suckled piglets ND 6.37 6.70 4.79 9.23 14.65 12.19 13.08 1.50 NS NS -

piglets ND 9.66 8.38 14.35a 8.55 7.41 ND ND 1.20 - p < 0.05 NS

piglets 3.28 9.02 26.17 22.82 20.84a 12.68 21.47 ND 3.57 NS - p <

Suckled piglets ND 9.06 26.17 13.58 17.11 14.16 4.18\* 5.14 3.48 p < 0.05 NS -

piglets ND 5.39 22.48 10.12a 8.78 9.13 ND ND 2.75 - NS p <

piglets 6.37 6.84 13.42 6.14 13.08 7.68 6.17 ND 2.00 NS - NS

Suckled piglets ND 25.63 26.91c 21.64 15.52 37.58 60.11 55.90 12.61 NS p < 0.001 -

piglets ND 6.27 14.42 13.41 20.30 16.17 ND ND 1.79 - NS NS

piglets 8.72 6.87 24.56b 12.25 24.40 20.67 23.73 ND 7.34 NS - p <

Suckled piglets ND 7.24 23.62 11.07c 11.07 9.72 4.85 5.47 1.97 NS p < 0.001 -

piglets ND 4.72 2.91 3.40 8.38 7.51 ND ND 0.85 - NS NS

piglets 1.87 2.91 3.69 4.56 5.02 5.03 2.50 ND 0.79 NS - NS

piglets ND 15.10 10.47 20.13 19.86 13.43 ND ND 2.03 - NS NS

piglets 7.71 11.14 24.16 22.18 32.08 21.58 25.41 ND 5.48 NS - NS

Suckled piglets ND ND 3.18 6.71 8.38 ND ND ND 1.06 - NS -

piglets ND ND 5.30 4.57 5.03 7.24 ND ND 1.36 - NS NS

piglets ND 2.21 6.17 ND 1.84 3.52 1.17 ND 0.48 NS - NS

Suckled piglets ND 2.68 ND ND 1.67 ND 2.25 3.48 0.68 - NS -

piglets ND 2.51 ND ND ND 3.02 ND ND 0.84 - NS NS

piglets ND 2.01 ND 3.52 3.38 7.24 1.71 ND 1.03 NS - NS

Table 3. Effect of age, weaning and diets on production of SCFAs in the ileum (mmol/l) of gnotobiotic and conventionally bred piglets. ND - not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different

(SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Suckled piglets ND 35.23c 21.81 27.18a 25.00 34.00 11.34 15.83 5.74 NS p < 0.05,

0 2 7 14 21 28 35 42 SEM 28 × 35 SC × RFP GP

× RFP

0.05

0.05

0.01

p < 0.001 -

Age (days) p - value

*Formic acid*

pronounced post-weaning decrease in the level of acetoacetic acid was recorded one week after weaning (p < 0.05). The proportion of lactic acid was relatively stable from day 7 to 28 of life and ranged between 19.96 and 23.59 mmol/l. Afterwards, in the 5th week of life, the lactic acid level increased to 31.25 mmol/l. Similarly, acetic acid level was relatively stable during the first two weeks of life of piglets, then increased significantly (p < 0.01) at the age of 28 days compared to the level at 3 hours after birth (6.57 mmol/l). The most important production of acids in the colon of gnotobiotic piglets (Table 4) was the production of acetic acid which remained relatively stable between days 7 and 35 of life and the difference between its concentration recorded at 3 hours after the birth (6.94 mmol/l) and that determined on day 7 of life (41.98 mmol/l) was significant (p < 0.05). A significant difference in concentration of lactic acid was recorded on day 28 of age (p < 0.001, p < 0.01 ) compared to the level on the second day (8.18 mmol/l) and 21st day (14.49 mmol/l). Subsequently, we recorded a non-significant, 3-fold increase in the level of this acid at 5 weeks of age (to 76.30 mmol/l). A similar tendency was recorded for acetoacetic and propionic acid in the colonal content with the exception of the level recorded at 3 hours after the birth. The level of acetoacetic acid was significantly higher on day 21 of age (16.22 mmol/l, p < 0.01) compared to the second day of life (6.71 mmol/l).

#### **3.4 Effect of diets on production of SCFAs in the intestinal tract of gnotobiotic and conventionally bred piglets**

The concentration of acetoacetic acid in the jejunal content (Table 2) was higher in suckled piglets at 7 days of life (28.69 mmol/l) compared to the replacer-fed animals in which the level of the above acid represented 19.89 mmol/l. In both groups of piglets observed, well-balanced levels of the above acid were recorded thereafter. The course of the concentration of lactic acid up to day 7 of age was the same in 3 groups of piglets with a subsequent increase in replacer-fed piglets at 21 days of age (p < 0.01) compared to suckled animals. In the acetic acid of suckled piglets, significantly higher levels were recorded at day 2 and 14 of age compared to the replacer-fed piglets (p < 0.05, p < 0.001). The proportion of acetic acid in gnotobiotic piglets was up to two weeks of life considerably lower in comparison with both investigated groups and the decrease on day 14 of life was significant (p < 0.05) in comparison with replacer-fed piglets. The course of the concentration of acetoacetic acid in the ileal content (Table 3) was the same in all 3 observed groups except at 3 weeks of age of replacer-fed piglets. While the content of acetoacetic acid represented 17.11 mmol/l in suckled piglets at 3 weeks of life, in the noncolostral group a decline to 8.78 mmol/l was observed. The level of acetic acid in the ileal contents of suckled piglets was higher throughout the period of investigation ranging from about 7 to 21.5 mmol/l compared with the replacer-fed piglets which showed the highest concentration on day 2 (p < 0.001) and 14 of life (p < 0.05). Insignificantly higher concentrations of acetic acid in the ileal content were observed also in gnotobiotic piglets (2.05 - 13.69 mmol/l) compared to replacer-fed piglets. A similar tendency was recorded in lactic acid, in the ileal content up to 14 days of age, with higher concentrations in suckled piglets as compared to the replacer-fed piglets in which the values later ranged about 8.2 till 21.4 mmol/l and a significantly higher level of the acid was recorded at 7 days of age (p < 0.001). Higher production of lactic acid by gnotobiotic piglets (0.6 - 10.14 mmol/l) was observed throughout the observation period in comparison with replacerfed piglets with a significant increase on day 7 of age (p < 0.01).

pronounced post-weaning decrease in the level of acetoacetic acid was recorded one week after weaning (p < 0.05). The proportion of lactic acid was relatively stable from day 7 to 28 of life and ranged between 19.96 and 23.59 mmol/l. Afterwards, in the 5th week of life, the lactic acid level increased to 31.25 mmol/l. Similarly, acetic acid level was relatively stable during the first two weeks of life of piglets, then increased significantly (p < 0.01) at the age of 28 days compared to the level at 3 hours after birth (6.57 mmol/l). The most important production of acids in the colon of gnotobiotic piglets (Table 4) was the production of acetic acid which remained relatively stable between days 7 and 35 of life and the difference between its concentration recorded at 3 hours after the birth (6.94 mmol/l) and that determined on day 7 of life (41.98 mmol/l) was significant (p < 0.05). A significant difference in concentration of lactic acid was recorded on day 28 of age (p < 0.001, p < 0.01 ) compared to the level on the second day (8.18 mmol/l) and 21st day (14.49 mmol/l). Subsequently, we recorded a non-significant, 3-fold increase in the level of this acid at 5 weeks of age (to 76.30 mmol/l). A similar tendency was recorded for acetoacetic and propionic acid in the colonal content with the exception of the level recorded at 3 hours after the birth. The level of acetoacetic acid was significantly higher on day 21 of age (16.22 mmol/l, p < 0.01) compared

**3.4 Effect of diets on production of SCFAs in the intestinal tract of gnotobiotic and** 

fed piglets with a significant increase on day 7 of age (p < 0.01).

The concentration of acetoacetic acid in the jejunal content (Table 2) was higher in suckled piglets at 7 days of life (28.69 mmol/l) compared to the replacer-fed animals in which the level of the above acid represented 19.89 mmol/l. In both groups of piglets observed, well-balanced levels of the above acid were recorded thereafter. The course of the concentration of lactic acid up to day 7 of age was the same in 3 groups of piglets with a subsequent increase in replacer-fed piglets at 21 days of age (p < 0.01) compared to suckled animals. In the acetic acid of suckled piglets, significantly higher levels were recorded at day 2 and 14 of age compared to the replacer-fed piglets (p < 0.05, p < 0.001). The proportion of acetic acid in gnotobiotic piglets was up to two weeks of life considerably lower in comparison with both investigated groups and the decrease on day 14 of life was significant (p < 0.05) in comparison with replacer-fed piglets. The course of the concentration of acetoacetic acid in the ileal content (Table 3) was the same in all 3 observed groups except at 3 weeks of age of replacer-fed piglets. While the content of acetoacetic acid represented 17.11 mmol/l in suckled piglets at 3 weeks of life, in the noncolostral group a decline to 8.78 mmol/l was observed. The level of acetic acid in the ileal contents of suckled piglets was higher throughout the period of investigation ranging from about 7 to 21.5 mmol/l compared with the replacer-fed piglets which showed the highest concentration on day 2 (p < 0.001) and 14 of life (p < 0.05). Insignificantly higher concentrations of acetic acid in the ileal content were observed also in gnotobiotic piglets (2.05 - 13.69 mmol/l) compared to replacer-fed piglets. A similar tendency was recorded in lactic acid, in the ileal content up to 14 days of age, with higher concentrations in suckled piglets as compared to the replacer-fed piglets in which the values later ranged about 8.2 till 21.4 mmol/l and a significantly higher level of the acid was recorded at 7 days of age (p < 0.001). Higher production of lactic acid by gnotobiotic piglets (0.6 - 10.14 mmol/l) was observed throughout the observation period in comparison with replacer-

to the second day of life (6.71 mmol/l).

**conventionally bred piglets** 


Table 3. Effect of age, weaning and diets on production of SCFAs in the ileum (mmol/l) of gnotobiotic and conventionally bred piglets. ND - not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different (SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)


The value of acetoacetic acid in the colonic content (Table 4) was significantly higher in the group of suckled piglets from day 14 to 28 of age (p < 0.01), compared to replacer-fed

Table 4. Effect of age, weaning and diets on production of SCFAs in the colon (mmol/l) of gnotobiotic and conventionally bred piglets. ND- not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different (SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Differences in the Development of the Small Intestine

compared to suckled piglets.

**piglets and replacer-fed piglets** 

observation in all parts of the intestine.

Between Gnotobiotic and Conventionally Bred Piglets 387

piglets. In the period between weeks 2 to 4 of life, production of this acid by gnotobiotic piglets was also higher in comparison with replacer-fed piglets with significant increase at 3 weeks of life (p < 0.001). While the dynamics of lactic acid in the colon of suckled and replacer-fed piglets was similar with the exception of week 1, and its levels were low and did not exceed 7 mmol/l, gnotobiotic piglets produced higher level of this acid with peaks at 2 days (p < 0.05) and 14, 21 and 28 days of age (p < 0.01) compared to the replacer-fed animals. Dynamics of acetic acid in colonal content of suckled piglets resembled that in jejunal and ileal contents and reached higher levels with the exception of days 2 and 7 of age. Significant difference (p < 0.01) was observed at 3 hours after the birth in comparison with replacer-fed piglets. Acetic acid in replacer-fed piglets showed an opposite trend in the proximal section of the intestinal tract where we recorded a gradual increase in concentrations up to the end of observation with highest levels at 2 days (p < 0.01) and 28 days of age (p < 0.05) compared to the gnotobiotic piglets. In the colon of replacer-fed piglets we recorded also higher production of propionic acid on days 21 and 28 of age (p < 0.01) compared to the gnotobiotic piglets and butyric acid at 2 days of age (p < 0.05)

**3.5 Effect of diets on development of microflora in the digestive tract of suckling** 

however, total aerobes populations maintained at a constant level of 9 log cfu/g.

In suckled piglets, an increase in the followed microflora population was recorded towards the caudal part of the intestine. Bacterial populations ranged from 4 to 8 log cfu/g in the jejunum, from 4 to 9 log cfu/g in the ileum, and from 6 to 9 log cfu/g in the caecum. The lactobacilli in the ileum slightly increased within the period of observation ranging from 1 to 2 log. The cfu of *E. coli* and *Enterobacteriaceae* in the ileum remained more or less stable over time, while they declined in the caecum. The course of development of total aerobes was similar in the jejunum and ileum throughout the period of observation, in the colon,

Likewise, in conventional piglets fed on milk replacement, an increase in the microflora population was observed towards the caudal part of the intestine. Bacterial populations ranged from 4 to 8 log cfu/g in the jejunum, from 4 to 9 log cfu/g in the ileum and from 6 to 9 log cfu/g in the caecum. In all parts of the intestine, *E. coli* and *Enterobacteriaceae* increased by 1 to 3 log units between days 2 and 14, and decreased thereafter until day 28. In the jejunum and ileum lactobacilli and enterococci slightly increased throughout the period of observation, in the colon, however, lactobacilli population*s* persisted at a constant level of 9 log cfu/g. *Enterococcus* spp*.* in colon contents declined by about 2 log units until 28 days of age. The course of the development of total aerobes was the same throughout the period of

Total lactobacilli populations in the jejunal content were significantly higher in the group of conventional replacer-fed piglets with highest counts on days 21 and 28 of age (p < 0.001 and p < 0.001) compared to the group of suckled piglets (Figure 1). The course of *E. coli*  development in the jejunal content was the same in both observed groups except at 2 weeks of age. Whereas total *E. coli* populations were lower in the group of suckled piglets at 2 weeks of life, i.e. 3.78 log cfu/g, a significant increase was observed in group of replacer-fed piglets (p < 0.001). In conventional piglets fed on milk replacement, an increase in enterococci populations (Table 5) in the jejunal content was seen throughout the period of

The value of acetoacetic acid in the colonic content (Table 4) was significantly higher in the group of suckled piglets from day 14 to 28 of age (p < 0.01), compared to replacer-fed

Table 4. Effect of age, weaning and diets on production of SCFAs in the colon (mmol/l) of gnotobiotic and conventionally bred piglets. ND- not detectable, NS- not significant, SPsuckled piglets, GP- gnotobiotic piglets, RFP- replacer-fed piglets. Significantly different

(SP,GP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

piglets. In the period between weeks 2 to 4 of life, production of this acid by gnotobiotic piglets was also higher in comparison with replacer-fed piglets with significant increase at 3 weeks of life (p < 0.001). While the dynamics of lactic acid in the colon of suckled and replacer-fed piglets was similar with the exception of week 1, and its levels were low and did not exceed 7 mmol/l, gnotobiotic piglets produced higher level of this acid with peaks at 2 days (p < 0.05) and 14, 21 and 28 days of age (p < 0.01) compared to the replacer-fed animals. Dynamics of acetic acid in colonal content of suckled piglets resembled that in jejunal and ileal contents and reached higher levels with the exception of days 2 and 7 of age. Significant difference (p < 0.01) was observed at 3 hours after the birth in comparison with replacer-fed piglets. Acetic acid in replacer-fed piglets showed an opposite trend in the proximal section of the intestinal tract where we recorded a gradual increase in concentrations up to the end of observation with highest levels at 2 days (p < 0.01) and 28 days of age (p < 0.05) compared to the gnotobiotic piglets. In the colon of replacer-fed piglets we recorded also higher production of propionic acid on days 21 and 28 of age (p < 0.01) compared to the gnotobiotic piglets and butyric acid at 2 days of age (p < 0.05) compared to suckled piglets.

#### **3.5 Effect of diets on development of microflora in the digestive tract of suckling piglets and replacer-fed piglets**

In suckled piglets, an increase in the followed microflora population was recorded towards the caudal part of the intestine. Bacterial populations ranged from 4 to 8 log cfu/g in the jejunum, from 4 to 9 log cfu/g in the ileum, and from 6 to 9 log cfu/g in the caecum. The lactobacilli in the ileum slightly increased within the period of observation ranging from 1 to 2 log. The cfu of *E. coli* and *Enterobacteriaceae* in the ileum remained more or less stable over time, while they declined in the caecum. The course of development of total aerobes was similar in the jejunum and ileum throughout the period of observation, in the colon, however, total aerobes populations maintained at a constant level of 9 log cfu/g.

Likewise, in conventional piglets fed on milk replacement, an increase in the microflora population was observed towards the caudal part of the intestine. Bacterial populations ranged from 4 to 8 log cfu/g in the jejunum, from 4 to 9 log cfu/g in the ileum and from 6 to 9 log cfu/g in the caecum. In all parts of the intestine, *E. coli* and *Enterobacteriaceae* increased by 1 to 3 log units between days 2 and 14, and decreased thereafter until day 28. In the jejunum and ileum lactobacilli and enterococci slightly increased throughout the period of observation, in the colon, however, lactobacilli population*s* persisted at a constant level of 9 log cfu/g. *Enterococcus* spp*.* in colon contents declined by about 2 log units until 28 days of age. The course of the development of total aerobes was the same throughout the period of observation in all parts of the intestine.

Total lactobacilli populations in the jejunal content were significantly higher in the group of conventional replacer-fed piglets with highest counts on days 21 and 28 of age (p < 0.001 and p < 0.001) compared to the group of suckled piglets (Figure 1). The course of *E. coli*  development in the jejunal content was the same in both observed groups except at 2 weeks of age. Whereas total *E. coli* populations were lower in the group of suckled piglets at 2 weeks of life, i.e. 3.78 log cfu/g, a significant increase was observed in group of replacer-fed piglets (p < 0.001). In conventional piglets fed on milk replacement, an increase in enterococci populations (Table 5) in the jejunal content was seen throughout the period of

Differences in the Development of the Small Intestine

*Day 2*

*Day 7*

*Day 14*

*Day 21*

*Day 28*

*Day 35*

*Day 42*

\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Between Gnotobiotic and Conventionally Bred Piglets 389

Content

Enterococci 6.36 6.83 6.10 7.70\*\*\* 8.08 8.69\* 0.31 *Enterobacteiaceae* 4.00 6.95 6.89\*\*\* 4.55 8.95 7.92 0.25 Total aerobes 7.35 7.97 9.04 8.31 9.33 9.35 0.19

Enterococci 6.19 7.22 3.96 7.42\*\*\* 7.49 8.73\* 0.29 *Enterobacteiaceae* 4.43 4.02 7.24\*\*\* 5.27 8.51 8.35 0.19 Total aerobes 6.56 8.15\*\*\* 7.76 8.45\*\* 9.24 9.57 0.14

Enterococci 5.12 6.80 5.06 7.49\*\*\* 6.82 7.41 0.31 *Enterobacteiaceae* 4.11 7.72\*\*\* 7.16\*\*\* 8.17 7.37 9.42 0.26 Total aerobes 7.51 8.13\* 7.46 8.83\*\* 9.15 9.43 0.19

Enterococci 4.93 7.42\*\*\* 6.01 6.93\*\*\* 6.91 6.57 0.14 *Enterobacteiaceae* 5.38 4.69 6.69 6.57 7.10 8.13 0.19 Total aerobes 6.93 8.22\*\*\* 8.77 9.35 9.13 9.12 0.29

Enterococci 5.52 7.46\*\*\* 6.38 8.95\*\*\* 7.17 6.94 0.16 *Enterobacteiaceae* 4.81 4.74 6.71\*\* 5.96 6.07 6.53 0.28 Total aerobes 7.95 7.65 8.89 9.20 9.05 8.79 0.21

Enterococci 4.45 ND 5.15 ND 7.17 ND 0.39 *Enterobacteiaceae* 3.00 ND 5.79 ND 5.98 ND 0.19 Total aerobes 6.61 ND 9.26 ND 9.38 ND 0.14

Enterococci 6.75 ND 7.94 ND 8.91 ND 0.19 *Enterobacteiaceae* 5.38 ND 5.30 ND 5.36 ND 0.14 Total aerobes 7.80 ND 8.07 ND 9.13 ND 0.51

Table 5. Effect of diets on bacterial population (log10 cfu/g of digesta) at various locations

fed piglets than those in suckled piglets throughout the period of observation with significant difference between days 7 and 28 of age (p < 0.001). In suckled piglets, total *Enterobacteriaceae* populations were by 2 log higher by 1 week of life compared to the

SP- suckled piglets (n=3), RFP- replacer-fed piglets (n=3), ND- not detectable

along the intestinal tract of suckling piglets and replacer-fed piglets

Jejunum Ileum Caecum

SP RFP SP RFP SP RFP SEM

Each bar represents the mean ± SE of 3 piglets. Significantly different (SP vs RFP): c (p < 0.001)

Fig. 1. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the jejunum of suckling piglets and replacer-fed piglets

observation. In suckled piglets, on the other hand, total enterococci populations were by 0.5 - 2.5 log lower. The highest enterococci populations in the jejunal content of replacer-fed piglets were recorded at 21 and 28 d of age (p < 0.001 and p < 0.001, respectively). Total counts of *Enterobacteriaceae* in the jejunal content were the highest in replacer-fed piglets at 2 (6.95 log cfu/g) and 14 days of age (p < 0.001) compared to the group of suckled piglets in which lower numbers (by 3 logs) were seen at 2 days of life (4.00 log cfu/g) and at 14 days of age the populations were by 3.6 log lower (4.11 log cfu/g). The course of the development of total aerobes in the jejunal content was the same in both groups of piglets, however, with the difference that in replacer-fed piglets, the total numbers of observed bacteria were by 0.5 to 1.2 log higher with significantly higher numbers at 7 (p < 0.001), 14 (p < 0.05), and 21 days of age (p < 0.001).

In the ileal content of replacer-fed piglets we detected higher lactobacilli populations (Figure 2) compared to the suckled animals throughout the period of observation except at 14 days of age with significantly higher numbers at 7 and 21 days of age (p < 0.01 and p < 0.05). The course of development of *E. coli* was the same in both groups, however, with the difference that in the group of suckled piglets the total numbers were higher by 0.5 log except at 14 days of age when an increase in the total *E. coli* populations was recorded (p < 0.001). Total enterococci populations in the ileal content (Table 5) were higher by 1 to 3.5 log in replacer-

*Lactobacilli*

2 7 14 21 28 35 42 age (day)

c c

*E. coli*

2 7 14 21 28 35 42 age (day)

replacer-fed piglets suckled piglets

Fig. 1. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the jejunum of

c

observation. In suckled piglets, on the other hand, total enterococci populations were by 0.5 - 2.5 log lower. The highest enterococci populations in the jejunal content of replacer-fed piglets were recorded at 21 and 28 d of age (p < 0.001 and p < 0.001, respectively). Total counts of *Enterobacteriaceae* in the jejunal content were the highest in replacer-fed piglets at 2 (6.95 log cfu/g) and 14 days of age (p < 0.001) compared to the group of suckled piglets in which lower numbers (by 3 logs) were seen at 2 days of life (4.00 log cfu/g) and at 14 days of age the populations were by 3.6 log lower (4.11 log cfu/g). The course of the development of total aerobes in the jejunal content was the same in both groups of piglets, however, with the difference that in replacer-fed piglets, the total numbers of observed bacteria were by 0.5 to 1.2 log higher with significantly higher numbers at 7 (p < 0.001), 14 (p < 0.05), and 21 days

In the ileal content of replacer-fed piglets we detected higher lactobacilli populations (Figure 2) compared to the suckled animals throughout the period of observation except at 14 days of age with significantly higher numbers at 7 and 21 days of age (p < 0.01 and p < 0.05). The course of development of *E. coli* was the same in both groups, however, with the difference that in the group of suckled piglets the total numbers were higher by 0.5 log except at 14 days of age when an increase in the total *E. coli* populations was recorded (p < 0.001). Total enterococci populations in the ileal content (Table 5) were higher by 1 to 3.5 log in replacer-

Each bar represents the mean ± SE of 3 piglets. Significantly different (SP vs RFP): c (p < 0.001)

log 10/g

log 10/g

suckling piglets and replacer-fed piglets

of age (p < 0.001).


SP- suckled piglets (n=3), RFP- replacer-fed piglets (n=3), ND- not detectable

\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Table 5. Effect of diets on bacterial population (log10 cfu/g of digesta) at various locations along the intestinal tract of suckling piglets and replacer-fed piglets

fed piglets than those in suckled piglets throughout the period of observation with significant difference between days 7 and 28 of age (p < 0.001). In suckled piglets, total *Enterobacteriaceae* populations were by 2 log higher by 1 week of life compared to the

Differences in the Development of the Small Intestine

log 10/g

Each bar represents the mean ± SE of 3 piglets.

5

6

7

8

9

10

log 10/g

**gnotobiotic and conventionally bred piglets** 

piglets and replacer-fed piglets

of weaning (Table 6).

Significantly different (SP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Fig. 3. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the colon of suckling

2 7 14 21 28 35 42

replacer-fed piglets suckled piglets

From day 2 of age we recorded a gradual increase in body weight of gnotobiotic piglets from 0.75 kg up to 1.80 kg (p < 0.05) on day 14 of age and 3.90 kg (p < 0.01) on day 21 of age. On day 28 (day of weaning) and one week after weaning the body weight of piglets was decreased insignificantly (Table 6). The increase in relative weight of the small intestine resembled that of the large intestine throughout the period of investigation with the exception that the relative weight of the large intestine in comparison with the weight on day 2 of age was increased significantly on day 21 (p < 0.05), 28 (p < 0.05) and 35 of age (p < 0.001). Similar trend of body weight increase as that recorded in gnotobiotic piglets was observed also in conventional piglets, increasing from 1.41 kg at 2 days of age to 3.50 kg at 14 days of age (p < 0.05). The body weight of piglets increased gradually up to the end of observation with significant increase on day 35 of life (p < 0.01) in comparison with the day

The development of relative weight of the small and large intestine of suckled piglets was similar and the relative weights decreased gradually between days 2 and 21 of age. On day

**3.6 Effect of age, weaning and diets on development of intestinal morphology in** 

Between Gnotobiotic and Conventionally Bred Piglets 391

*Lactobacilli*

a

2 7 14 21 28 35 42 age (day)

*E. coli*

c

b

Each bar represents the mean ± SE of 3 piglets Significantly different (SP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Fig. 2. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the ileum of suckling piglets and replacer-fed piglets

replacer-fed piglets. Thereafter, the populations of observed bacteria slightly declined up to the end of the period of observation. A significant increase in the *Enterobacteriaceae*  populations in replacer-fed piglets was recorded at 14 days of age (p < 0.001). Numbers of total aerobes in the ileal content were by 0.5 - 1.5 log higher in replacer-fed piglets compared to colostral animals with significant difference on day 7 (p < 0.01) and 14 of age (p < 0.01).

In the caecum of suckled piglets, *E. coli* populations gradually declined throughout the period of observation (Figure 3) compared with replacer-fed piglets in which a significant increase in numbers was recorded at 14 and 21 days of age (p < 0.001 and p < 0.01, respectively). The course of the development of lactobacilli and total aerobes in the content of the caecum was the same. Total enterococci populations in replacer-fed piglets (Table 5) were by 0.6 - 1.2 log higher by day 14 of age, with significant enterococci populations at 2 and 7 days of age (p < 0.05 and p< 0.05). After day 14, the cfu of *Enterococcus* spp. in the caecum contents of replacer-fed piglets were about 0.3 log units lower than those of suckled piglets. A significant increase in the *Enterobacteriaceae*  populations in replacer-fed piglets compared to the second group was recorded from day 14 of age (p < 0.001) to the end of the period of observation (p < 0.01) at 21 days of age and (p < 0.05) at 28 days of age.

*Lactobacilli*

c

a

a

2 7 14 21 28 35 42 age (day)

*E. coli*

c

2 7 14 21 28 35 42

replacer-fed piglets suckled piglets

Each bar represents the mean ± SE of 3 piglets

log 10/g

log 10/g

piglets and replacer-fed piglets

(p < 0.05) at 28 days of age.

(p < 0.01).

Significantly different (SP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

b

Fig. 2. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the ileum of suckling

replacer-fed piglets. Thereafter, the populations of observed bacteria slightly declined up to the end of the period of observation. A significant increase in the *Enterobacteriaceae*  populations in replacer-fed piglets was recorded at 14 days of age (p < 0.001). Numbers of total aerobes in the ileal content were by 0.5 - 1.5 log higher in replacer-fed piglets compared to colostral animals with significant difference on day 7 (p < 0.01) and 14 of age

In the caecum of suckled piglets, *E. coli* populations gradually declined throughout the period of observation (Figure 3) compared with replacer-fed piglets in which a significant increase in numbers was recorded at 14 and 21 days of age (p < 0.001 and p < 0.01, respectively). The course of the development of lactobacilli and total aerobes in the content of the caecum was the same. Total enterococci populations in replacer-fed piglets (Table 5) were by 0.6 - 1.2 log higher by day 14 of age, with significant enterococci populations at 2 and 7 days of age (p < 0.05 and p< 0.05). After day 14, the cfu of *Enterococcus* spp. in the caecum contents of replacer-fed piglets were about 0.3 log units lower than those of suckled piglets. A significant increase in the *Enterobacteriaceae*  populations in replacer-fed piglets compared to the second group was recorded from day 14 of age (p < 0.001) to the end of the period of observation (p < 0.01) at 21 days of age and

Each bar represents the mean ± SE of 3 piglets. Significantly different (SP vs RFP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

Fig. 3. Effect of diets on bacterial population (lactobacilli and *E.coli*) in the colon of suckling piglets and replacer-fed piglets

#### **3.6 Effect of age, weaning and diets on development of intestinal morphology in gnotobiotic and conventionally bred piglets**

From day 2 of age we recorded a gradual increase in body weight of gnotobiotic piglets from 0.75 kg up to 1.80 kg (p < 0.05) on day 14 of age and 3.90 kg (p < 0.01) on day 21 of age. On day 28 (day of weaning) and one week after weaning the body weight of piglets was decreased insignificantly (Table 6). The increase in relative weight of the small intestine resembled that of the large intestine throughout the period of investigation with the exception that the relative weight of the large intestine in comparison with the weight on day 2 of age was increased significantly on day 21 (p < 0.05), 28 (p < 0.05) and 35 of age (p < 0.001). Similar trend of body weight increase as that recorded in gnotobiotic piglets was observed also in conventional piglets, increasing from 1.41 kg at 2 days of age to 3.50 kg at 14 days of age (p < 0.05). The body weight of piglets increased gradually up to the end of observation with significant increase on day 35 of life (p < 0.01) in comparison with the day of weaning (Table 6).

The development of relative weight of the small and large intestine of suckled piglets was similar and the relative weights decreased gradually between days 2 and 21 of age. On day

Differences in the Development of the Small Intestine

Content

*Day 0*

*Day 2*

*Day 7*

*Day 14*

*Day 21*

*Day 28*

*Day 35*

*Day 42*

\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

SP- suckled piglets, GP- gnotobiotic piglets, ND- not detectable

< 0.05) in gnotobiotic piglets on day 35 (Figure 4).

the intestinal tract of suckling piglets and gnotobiotic piglets

Between Gnotobiotic and Conventionally Bred Piglets 393

Villus height, µm 253.67 371.29 689.68 679.43 574.80 587.37 47.11 Crypt depth, µm 129.13 81.63 53.67 67.12 48.14 79.43 1.96

Villus height, µm 308.42\*\*\* 424.81 701.57 725.18 588.29 455.05 45.66 Crypt depth, µm 140.23 104.15 69.77 72.66 77.77 82.54 2.73

Villus height, µm 374.98\*\*\* 502.63 718.21 691.74 401.54\*\*\* 393.00 28.60 Crypt depth, µm 188.00 124.10 83.82 90.97 71.18 111.35 5.03

Villus height, µm 547.83\*\*\* 605.14 661.59 581.23\* 369.52 424.16 29.03 Crypt depth, µm 151.79 119.27 92.53 103.47 178.96 118.22 3.96

Villus height, µm 454.48\*\* 478.43\*\* 492.42\*\*\* 442.86\*\*\* 351.01 423.29 34.21 Crypt depth, µm 168.54 162.33\* 129.44 136.17 134.26 176.15\* 3.60

Villus height, µm 351.81\*\* 349.21 447.67 401.93 361.69 358.57 31.64 Crypt depth, µm 220.94 197.82 164.80 145.50 165.94 157.69 3.70

Villus height, µm 364.13 396.07 324.23\*\* 387.44 310.00 336.54 25.33 Crypt depth, µm 343.56\*\*\* 205.10 230.11\*\* 164.23 206.80\* 166.91 5.83

Villus height, µm 379.44 ND 440.08 ND 358.65 ND 18.32 Crypt depth, µm 322.66 ND 223.39 ND 174.81 ND 4.22

Table 7. Effect of age and weaning on small intestinal morphology at various locations along

age (p < 0.01). Comparison of both animal groups showed that villi in the jejunum of conventional piglets were higher between days 14 and 28 of age, the difference being significant on day 14 (p < 0.05). In the post-weaning period, villi were significantly higher (p

When comparing both mentioned groups, higher villi were observed in ileum of gnotobiotic piglets, resembling the situation in duodenum, with significant difference (p < 0.05) on days

Duodenum Jejunum Ileum

SP GP SP GP SP GB SEM


Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01)

\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Table 6. Effect of age, weaning and diets on weight, gut weight of suckling piglets and gnotobiotic piglets

28 of age the relative weights showed an insignificant increase but in the first week postweaning the relative weights of both small and large intestine increased significantly (p < 0.001) in comparison with all periods of investigation. The weight of conventional piglets was greater throughout the experiment and significant differences (p < 0.05) were recorded on days 14 and 35. Completely opposite trend was recorded for relative weights of small and large intestines of these piglets (Table 6). While relative weights of intestines of gnotobiotic piglets gradually increased throughout the experiment with the exception of day 28 of life, the weight of small and large intestine in suckled piglets decreased gradually between days 2 and 21 of life. Between days 2 and 28 of life the relative weight of the intestines of gnotobiotic piglets was higher compared to conventional piglets with significant difference on day 21 of life (p < 0.01). In the post-weaning period (one week postweaning), the weight of small and large intestine of suckled piglets was significantly higher (p < 0.05) in comparison with gnotobiotic piglets in the same period of observation.

The development of the height of villi in individual segments of the small intestine of gnotobiotic piglets is shown in Table 7. On day 21, the height of villi in the duodenum was decreased significantly (p < 0.01). A decrease in their length was also recorded on day 28 but the difference was insignificant. In the duodenum of suckled piglets, the height of villi (Table 7) increased significantly (p < 0.001) in the period between birth and day 14 of age. Subsequently, their length decreased gradually up to the weaning (p < 0.01). Throughout the experiment, the willi in the duodenum of gnotobiotic piglets were higher and their height differed significantly from that of conventional piglets at 3 hours after birth, days 2 and 7 of age (p < 0.01) and day 14 of age (p < 0.05) (Figure 4).

The length of jejunal villi of gnotobiotic piglets (Table 7) increased from day 0 up to day 2 of age reaching maximum of 725.18 m. In the subsequent 5 weeks, the length of villi decreased gradually down to 387.44 m in the week after weaning and the decrease was significant on days 14 (p < 0.05) and 21 of age (p < 0.001). In the jejunum of suckled piglets we observed a gradual but insignificant increase in villi height in the period from birth up to day 7 of life. In the following period, from day 14 of age till one week post-weaning we recorded gradual decrease in the height of villi, significant on days 21 (p < 0.001) and 35 of

Weight (kg) 1.41 2.05 3.50\*a 4.30 5.35 8.55\*\*a 0.39 Small intestinal weight (g/kg) 67.88 64.59 43.59 41.88 44.27 156.6\*\*\*a 7.08 Large intestinal weight (g/kg) 25.52 24.65 15.68 15.53 23.38 125.4\*\*\*a 2.38

Weight (kg) 0.75 1.29 1.80\* 3.90\*\* 3.65 3.25 0.46 Small intestinal weight (g/kg) 42.47 52.59 53.02 64.68b 55.41 60.67 6.14 Large intestinal weight (g/kg) 13.89 18.31 20.07 47.41\*b 42.1\* 85.63\*\*\* 4.07

Table 6. Effect of age, weaning and diets on weight, gut weight of suckling piglets and

(p < 0.05) in comparison with gnotobiotic piglets in the same period of observation.

and 7 of age (p < 0.01) and day 14 of age (p < 0.05) (Figure 4).

The development of the height of villi in individual segments of the small intestine of gnotobiotic piglets is shown in Table 7. On day 21, the height of villi in the duodenum was decreased significantly (p < 0.01). A decrease in their length was also recorded on day 28 but the difference was insignificant. In the duodenum of suckled piglets, the height of villi (Table 7) increased significantly (p < 0.001) in the period between birth and day 14 of age. Subsequently, their length decreased gradually up to the weaning (p < 0.01). Throughout the experiment, the willi in the duodenum of gnotobiotic piglets were higher and their height differed significantly from that of conventional piglets at 3 hours after birth, days 2

The length of jejunal villi of gnotobiotic piglets (Table 7) increased from day 0 up to day 2 of age reaching maximum of 725.18 m. In the subsequent 5 weeks, the length of villi decreased gradually down to 387.44 m in the week after weaning and the decrease was significant on days 14 (p < 0.05) and 21 of age (p < 0.001). In the jejunum of suckled piglets we observed a gradual but insignificant increase in villi height in the period from birth up to day 7 of life. In the following period, from day 14 of age till one week post-weaning we recorded gradual decrease in the height of villi, significant on days 21 (p < 0.001) and 35 of

28 of age the relative weights showed an insignificant increase but in the first week postweaning the relative weights of both small and large intestine increased significantly (p < 0.001) in comparison with all periods of investigation. The weight of conventional piglets was greater throughout the experiment and significant differences (p < 0.05) were recorded on days 14 and 35. Completely opposite trend was recorded for relative weights of small and large intestines of these piglets (Table 6). While relative weights of intestines of gnotobiotic piglets gradually increased throughout the experiment with the exception of day 28 of life, the weight of small and large intestine in suckled piglets decreased gradually between days 2 and 21 of life. Between days 2 and 28 of life the relative weight of the intestines of gnotobiotic piglets was higher compared to conventional piglets with significant difference on day 21 of life (p < 0.01). In the post-weaning period (one week postweaning), the weight of small and large intestine of suckled piglets was significantly higher

*Suckled piglets* 

*Gnotobiotic piglets* 

gnotobiotic piglets

\*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01)

Age (day) 2 7 14 21 28 35 SEM


SP- suckled piglets, GP- gnotobiotic piglets, ND- not detectable \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Table 7. Effect of age and weaning on small intestinal morphology at various locations along the intestinal tract of suckling piglets and gnotobiotic piglets

age (p < 0.01). Comparison of both animal groups showed that villi in the jejunum of conventional piglets were higher between days 14 and 28 of age, the difference being significant on day 14 (p < 0.05). In the post-weaning period, villi were significantly higher (p < 0.05) in gnotobiotic piglets on day 35 (Figure 4).

When comparing both mentioned groups, higher villi were observed in ileum of gnotobiotic piglets, resembling the situation in duodenum, with significant difference (p < 0.05) on days

Differences in the Development of the Small Intestine

suckling piglets

Between Gnotobiotic and Conventionally Bred Piglets 395

*Gnotobiotic piglets* 

*Suckled piglets*  (a, b) 3 hours after birth, (c, d) 2 days of age, (e, f) 7 days of age, (g, h) 28 days of age, (ch) 35days of age of GP, (i) 42 days of age of SP, SP- suckled piglets, GP- gnotobiotic piglets, Magnification x 125.

Fig. 5. Light micrograph of hematoxylin and eosin-stained jejunal mucosa of gnotobiotic and

time of weaning (day 28 of age) the differentiated basis of intestinal villi in both observed groups of piglets consisted of thin fibrous tissue containing fascicles of smooth muscle cells. On the surface of the villi we were able to observe goblet cells interspersed among enterocytes (Figure 5/g, h). The enterocyte nuclei were located in the medial part of the cytoplasm. The villi stroma was infiltrated with small number of lymphocytes and plasmatic cells. By day 35 of life, the jejunal villi acquired tongue-like shape (Figure 5/ch). On day 42 of age intestinal crypts of suckled piglets (Figure 5/i) almost completely filled up *lamina propria* of the small intestine and their bases almost reached *lamina muscularis mucosae.* 

a c e

g ch

b d f

h i

duodenum jejunum ileum

Fig. 4. Effect of diets on small intestinal morphology at various locations along the intestinal tract of suckling and gnotobiotic piglets

14 and 21 of age with the exception of day 2 of life when ileal villi were significantly higher (p < 0.001) in conventional piglets (Figure 4).

The postnatal changes in the depth of crypts (Table 7) were the same in all small intestinal segments of gnotobiotic piglets. From the birth up to day 35 of life the crypts in the jejunal segment gradually deepened and reached 164.23 m on day 35 of life. Similar development was observed also in the duodenal segment of the small intestine, where, with the exception of slight decrease on day 14 of age, the depth of crypts gradually increased throughout the observation period with significant difference on day 21 of age (p < 0.05). One week after weaning the depth of crypts in the duodenum reached 205.1 m. A significant increase in the depth of crypts (p < 0.05) was recorded on day 21 of age also in the ileal segment of gnotobiotic piglets. Also dynamics of development of the depth of crypts was similar in all small intestine segments of colostral piglets. In the first post-weaning week we recorded a significant deepening of crypts in duodenal (p < 0.001), jejunal (p < 0.01) and ileal (p < 0.05) segments.

Staining with haematoxylin/eosin showed that the small intestinal mucosa of gnotobiotic piglets and suckled piglets at 3 h after birth (Figure 5/a,b) and on day 2 of age (Figure 5/c,d) was covered by population of dense, finger-like villi of the same height. While on the surface of villi of gnotobiotic piglets we were able to observe enterocytes with apically located nucleus, in suckled piglets the enterocytes had apically to medially located nucleus. The fibrous base of intestinal villi was poorly differentiated and the intestinal crypts were small. By day 7 of age of gnotobiotic piglets (Figure 5/e) the height of villi decreased but their diameter increased. In the same period the villi in suckled piglets preserved their finger-like shape with medially to basally located nucleus in enterocytes (Figure 5/f). At the

SP- suckled piglets, GP- gnotobiotic piglets Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

*Villus height*

<sup>a</sup><sup>a</sup>

Fig. 4. Effect of diets on small intestinal morphology at various locations along the intestinal

duodenum jejunum ileum

age (day)

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

a a

a

0 2 7 14 21 28 35

14 and 21 of age with the exception of day 2 of life when ileal villi were significantly higher

The postnatal changes in the depth of crypts (Table 7) were the same in all small intestinal segments of gnotobiotic piglets. From the birth up to day 35 of life the crypts in the jejunal segment gradually deepened and reached 164.23 m on day 35 of life. Similar development was observed also in the duodenal segment of the small intestine, where, with the exception of slight decrease on day 14 of age, the depth of crypts gradually increased throughout the observation period with significant difference on day 21 of age (p < 0.05). One week after weaning the depth of crypts in the duodenum reached 205.1 m. A significant increase in the depth of crypts (p < 0.05) was recorded on day 21 of age also in the ileal segment of gnotobiotic piglets. Also dynamics of development of the depth of crypts was similar in all small intestine segments of colostral piglets. In the first post-weaning week we recorded a significant deepening of crypts in duodenal (p < 0.001), jejunal (p < 0.01) and ileal (p < 0.05)

Staining with haematoxylin/eosin showed that the small intestinal mucosa of gnotobiotic piglets and suckled piglets at 3 h after birth (Figure 5/a,b) and on day 2 of age (Figure 5/c,d) was covered by population of dense, finger-like villi of the same height. While on the surface of villi of gnotobiotic piglets we were able to observe enterocytes with apically located nucleus, in suckled piglets the enterocytes had apically to medially located nucleus. The fibrous base of intestinal villi was poorly differentiated and the intestinal crypts were small. By day 7 of age of gnotobiotic piglets (Figure 5/e) the height of villi decreased but their diameter increased. In the same period the villi in suckled piglets preserved their finger-like shape with medially to basally located nucleus in enterocytes (Figure 5/f). At the

SP- suckled piglets, GP- gnotobiotic piglets

b

µm

tract of suckling and gnotobiotic piglets

segments.

(p < 0.001) in conventional piglets (Figure 4).

Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01), c (p < 0.001)

c

b

b

*Suckled piglets* 

(a, b) 3 hours after birth, (c, d) 2 days of age, (e, f) 7 days of age, (g, h) 28 days of age, (ch) 35days of age of GP, (i) 42 days of age of SP, SP- suckled piglets, GP- gnotobiotic piglets, Magnification x 125.

Fig. 5. Light micrograph of hematoxylin and eosin-stained jejunal mucosa of gnotobiotic and suckling piglets

time of weaning (day 28 of age) the differentiated basis of intestinal villi in both observed groups of piglets consisted of thin fibrous tissue containing fascicles of smooth muscle cells. On the surface of the villi we were able to observe goblet cells interspersed among enterocytes (Figure 5/g, h). The enterocyte nuclei were located in the medial part of the cytoplasm. The villi stroma was infiltrated with small number of lymphocytes and plasmatic cells. By day 35 of life, the jejunal villi acquired tongue-like shape (Figure 5/ch). On day 42 of age intestinal crypts of suckled piglets (Figure 5/i) almost completely filled up *lamina propria* of the small intestine and their bases almost reached *lamina muscularis mucosae.* 

Differences in the Development of the Small Intestine

Between Gnotobiotic and Conventionally Bred Piglets 397

was recorded in duodenum and the lowest in ileum where lactase levels were 4-fold lower compared to proximal small intestine segments. Significant increase in the activity of enzyme was observed in jejunum (p < 0.001) and ileum (p < 0.01) of conventional piglets on day 7 of age. Comparison of both animal groups (Figure 6) showed that specific activity of lactase in the intestinal tract was higher in conventional piglets throughout the experiment with significantly higher lactase activity in duodenum from day 7 to 35 of age (p < 0.05, p <

The post-natal dynamics of maltase (Figure 7) and saccharase (Figure 8) in gnotobiotic piglets took a completely different course. Very low levels of maltase in the jejunum detected at birth (0.3 µmol/mg protein/hour) and during the 1st week of life slightly increased in the following period and on the 14th day reached 1.2 µmol/mg protein/hour. The slightly increased specific activity of maltase persisted also in the following period. Maltase distribution (Figure 7) throughout the small intestine changed depending on the age with a predominant concentration of maltase activity in the jejunum at the age of 1-2 weeks, through a higher activity in the duodenal part at the age of 21 days, until a balanced distribution in the proximal and medial part of the small intestine was achieved at the age of 4-5 weeks. Similarly also very low specific saccharase activity in the jejunum (Figure 8) recorded until the 1st week of life (1.12 µmol/mg protein/hour) progressively increased with a maximum on the 21st day of life (p < 0.001), sustained values were reached in the 4th and 5th week of the life of piglets. Saccharase activity distribution throughout the small intestine of gnotobiotic piglets was higher in the jejunum during the entire period of observation. Specific activities of maltase (Figure 7) and saccharase (Figure 8) in newborn

0.01) and significantly higher level in the jejunum (p < 0.05) on day 7 of age.

Significantly different (SP vs GP): a (p < 0.05), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

gnotobiotic piglets.

0

2

4

6

µmol/mg protein/hour

Fig. 7. Effect of age, weaning and diets on specific activity of maltase at various locations along the intestinal tract of suckling and gnotobiotic piglets, SP- suckled piglets, GP-

age (day)

duodenum jejunum ileum

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

 a \*\* \*

 a \*\*\* \*\* a

0 2 7 14 21 28 35

*Maltase*

Intestinal villi were shorter and acquired tongue-like shape. Such characteristic development was observed in all investigated segments of the small intestine which, however, differed by morphometric parameters of villi height.

#### **3.7 Effect of age, weaning and diets on development of specific activity of disaccharidases in the small intestine of gnotobiotic and conventionally bred piglets**

In our study we registered that development of lactase activity of gnotobiotic piglets was similar in the duodenum and jejunum in the period from day 0 to 35 of age. The specific activity of lactase (Figure 6) in the duodenum reached at birth the level of 3.4 µmol/mg protein/hour. From the 2nd day of life the increasing enzyme activity was noticed with a measured maximum on day 7 of life (p < 0.001). High enzyme levels were observed until the 21th day of life (5.80 µmol/mg protein/hour), after which the enzyme activity decreased in the weeks 4 (p < 0.001) and 5 (p < 0.001) to levels similar to those noticed at birth. In terms of lactase activity distribution throughout the small intestine, higher activity in the duodenum and jejunum were noticed than in distal parts of the small intestine, where the incidence of enzyme was 4 times lower. The specific activity of lactase in the duodenum of conventional piglets (Figure 6) reached at birth 3.6 µmol/mg protein/hour. The course of activity of this enzyme during our observations resembled that of gnotobiotic piglets with maximum recorded on day 7 of age (p < 0.001), gradual decrease from day 14 of life and significant decrease (p < 0.001) on the day of weaning and in the 1st post-weaning weak. As far as the distribution of lactase in the small intestine was concerned, the highest activity of lactase

Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 Fig. 6. Effect of age, weaning and diets on specific activity of lactase at various locations along the intestinal tract of suckling and gnotobiotic piglets, SP- suckled piglets, GPgnotobiotic piglets

Intestinal villi were shorter and acquired tongue-like shape. Such characteristic development was observed in all investigated segments of the small intestine which,

**disaccharidases in the small intestine of gnotobiotic and conventionally bred piglets**  In our study we registered that development of lactase activity of gnotobiotic piglets was similar in the duodenum and jejunum in the period from day 0 to 35 of age. The specific activity of lactase (Figure 6) in the duodenum reached at birth the level of 3.4 µmol/mg protein/hour. From the 2nd day of life the increasing enzyme activity was noticed with a measured maximum on day 7 of life (p < 0.001). High enzyme levels were observed until the 21th day of life (5.80 µmol/mg protein/hour), after which the enzyme activity decreased in the weeks 4 (p < 0.001) and 5 (p < 0.001) to levels similar to those noticed at birth. In terms of lactase activity distribution throughout the small intestine, higher activity in the duodenum and jejunum were noticed than in distal parts of the small intestine, where the incidence of enzyme was 4 times lower. The specific activity of lactase in the duodenum of conventional piglets (Figure 6) reached at birth 3.6 µmol/mg protein/hour. The course of activity of this enzyme during our observations resembled that of gnotobiotic piglets with maximum recorded on day 7 of age (p < 0.001), gradual decrease from day 14 of life and significant decrease (p < 0.001) on the day of weaning and in the 1st post-weaning weak. As far as the distribution of lactase in the small intestine was concerned, the highest activity of lactase

**3.7 Effect of age, weaning and diets on development of specific activity of** 

Significantly different (SP vs GP): a (p < 0.05), b (p < 0.01), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

gnotobiotic piglets

Fig. 6. Effect of age, weaning and diets on specific activity of lactase at various locations along the intestinal tract of suckling and gnotobiotic piglets, SP- suckled piglets, GP-

however, differed by morphometric parameters of villi height.

was recorded in duodenum and the lowest in ileum where lactase levels were 4-fold lower compared to proximal small intestine segments. Significant increase in the activity of enzyme was observed in jejunum (p < 0.001) and ileum (p < 0.01) of conventional piglets on day 7 of age. Comparison of both animal groups (Figure 6) showed that specific activity of lactase in the intestinal tract was higher in conventional piglets throughout the experiment with significantly higher lactase activity in duodenum from day 7 to 35 of age (p < 0.05, p < 0.01) and significantly higher level in the jejunum (p < 0.05) on day 7 of age.

The post-natal dynamics of maltase (Figure 7) and saccharase (Figure 8) in gnotobiotic piglets took a completely different course. Very low levels of maltase in the jejunum detected at birth (0.3 µmol/mg protein/hour) and during the 1st week of life slightly increased in the following period and on the 14th day reached 1.2 µmol/mg protein/hour. The slightly increased specific activity of maltase persisted also in the following period. Maltase distribution (Figure 7) throughout the small intestine changed depending on the age with a predominant concentration of maltase activity in the jejunum at the age of 1-2 weeks, through a higher activity in the duodenal part at the age of 21 days, until a balanced distribution in the proximal and medial part of the small intestine was achieved at the age of 4-5 weeks. Similarly also very low specific saccharase activity in the jejunum (Figure 8) recorded until the 1st week of life (1.12 µmol/mg protein/hour) progressively increased with a maximum on the 21st day of life (p < 0.001), sustained values were reached in the 4th and 5th week of the life of piglets. Saccharase activity distribution throughout the small intestine of gnotobiotic piglets was higher in the jejunum during the entire period of observation. Specific activities of maltase (Figure 7) and saccharase (Figure 8) in newborn

duodenum jejunum ileum

Significantly different (SP vs GP): a (p < 0.05), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001

Fig. 7. Effect of age, weaning and diets on specific activity of maltase at various locations along the intestinal tract of suckling and gnotobiotic piglets, SP- suckled piglets, GPgnotobiotic piglets.

Differences in the Development of the Small Intestine

**4. Discussion** 

**microflora** 

Between Gnotobiotic and Conventionally Bred Piglets 399

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

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

duodenum jejunum ileum

Fig. 8. Effect of age, weaning and diets on specific activity of saccharase at various locations along the intestinal tract of suckling and gnotobiotic piglets

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

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

SP- suckled piglets, GP- gnotobiotic piglets. 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
