Effects of Dietary Fructooligosaccharide on Digestive Enzyme Activities, Intestinal Microflora and Morphology of Growing Pigs

One hundred and twenty-eight growing barrows (Jiaxing Black×Duroc×Landrace) at an average BW of 20.8 kg were allocated to four treatments for 42 days, each of which was replicated four times with eight pigs per replicate and used to investigate the effects of fructooligosaccharide (FOS) on digestive enzyme activities, intestinal microflora and morphology of growing pigs. The pigs received the same basal corn-soybean meal diet and FOS was added to the basal diet at 0, 2, 4, 6 g/kg diet at the expense of corn, respectively. As compared to control, supplementation with 4 and 6 g/kg FOS significantly improved average daily gain and feed efficiency. Addition of FOS enhanced the growth of Bifidobacterium and Lactobacillus, but inhibited Clostridium and Escherichia coli in the small intestinal and proximal colonic contents. Supplementation with 4 and 6 g/kg FOS significantly improved the activities of total protease, trypsin and amylase in the small intestinal contents. However, FOS had no significant effect on the activity of lipase in the small intestinal contents as well as the digestive enzymes in pancreas. Morphological measurement of jejunal mucosa did show response to consumption of FOS. Villus height and the villus height to crypt depth ratio at the jejunal mucosa were significantly higher with 4 and 6 g/kg FOS supplementation as compared to control. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 12 : 1784-1789)


INTRODUCTION
In recent years, numerous investigations have been conducted to study effects of oligosaccharides on commensal bacteria and health of young animals and human infants (Gibson and Wang, 1994). Fructooligosaccharide (FOS) is β-linked fructose unit to the fructose moiety of sucrose. Because the β-linkages between fructose monomers cannot be hydrolyzed by enzymes of endogenous origin, FOS escapes enzymatic digestion in the small intestine and forms a substrate for the gastrointestinal microflora (Tokunaga et al., 1989). The FOS has been shown to enhance the growth of Bifidobacterium and Lactobacillus, but inhibit Escherichia coli in the large intestine (Hidaka et al., 1986b(Hidaka et al., , 1991Bunce et al., 1995;Roberfroid et al., 1998). There are large numbers of microorganisms in the small intestine of pigs, so FOS is fermented to some extent in the small intestine of pigs (Bolduan, et al., 1993). However, there is extremely limited information on the effect of FOS on activity of the microflora in the small intestine. Moreover, data on the effects of FOS on the digestive enzyme activities and intestinal morphology in pigs are lacking.
Therefore, an experiment was carried out to investigate the effects of dietary FOS on intestinal microflora, the digestive enzyme activities of pancreas and small intestinal contents, and intestinal morphology of growing pigs.

Animals and experimental diets
A total of 128 growing barrows (Jiaxing Black×Duroc×Landrace) at an average BW of 20.8 Kg were allocated to 4 treatments for 42 days, each of which was replicated four times with eight pigs per replicate. The pigs received the same basal diet based on corn-soybean meal and FOS was added to the basal diet at 0, 2, 4, 6 g/kg diet at the expense of corn, respectively. The FOS (Meioligo-P) was provided by Meiji Seika kaisha, Ltd. (Tokyo, Japan) and the concentration of oligosaccharides was analyzed by HPLC. The main ingredients of this product are glucose, fructose, sucrose and FOS. The glucose, fructose and sucrose amount to 2.8% and the FOS amount to 97.2%. The FOS consist of three kinds of materials (GF n : G refers to glucosyl moiety, F to the fructosyl moiety and n indicates the number of the fructosyl moieties in the molecules, n=2, 3, 4). GF 2 , GF 3 and GF 4 amount to 46.8, 39.3 and 11.1%, respectively. Diets were formulated to meet or exceed nutrient requirements suggested by the NRC (1998) for 20 to 50 kg pigs. Antibiotic was excluded from all diets (Table 1). All pigs were given ad libitum access to feed and water. Growth performance results such as average daily gain (ADG), average daily feed intake (ADFI), and feed/gain (F/G) were collected.
At the 42th day of the feeding trial, eight pigs from each treatment (two pigs per pen) were slaughtered under general anaesthesia. The pigs were then immediately eviscerated in order to collect intestinal samples.

The digestive enzyme activities in pancreatic tissue and small intestinal contents
Sampling procedure : The contents taken from the small intestine were digesta from the distal end of the duodenum to the ileo-caecal junction. A homogenous intestinal digesta sample was collected by massaging the tract from both ends. The digesta sample were stored immediately at -20°C until used. Enzyme activity analyses of the samples obtained from the small intestine were performed on freeze-dried material, which was extracted with 1 mM HCl (50 mg lyophilized digesta in 1 ml 1 mM HCl ) for 1 h at 4°C followed by centrifugation (3,000×g) according to Jensen et al. (1998). The supernatants were then collected for analysis of protease, trypsin, chymotrypsin, amylase and lipase activities.

Intestinal microbial populations
Samples of the contents from the small intestine (from the distal end of the duodenum to the ileo-caecal junction) and proximal colon were immediately collected into Qorpak glass containers under CO 2 , sealed, and put on ice until they were transported to the lab for enumeration of microbial populations. Ten grams of mixed contents were blended under CO 2 in 90 mL of anaerobic dilution (ADS, Bryant and Allison, 1961). Further serial dilutions were made in ADS for anaerobic bacterial enumeration (Bryant, 1972). The initial dilution in ADS was also used as a source for serial dilutions in PBS for enumeration of aerobic bacterial populations. Triplicate plates were then inoculated with 0.1 ml samples and incubated at 37° aerobically or anaerobically as appropriate. Three dilutions were plated for each medium. Bacteria were enumerated on Wilkins Chalgren Agar (Oxoid; total anaerobes), MRS Agar (Oxoid; Lactobacillus), Reinforced Clostridial Agar plus supplements (Munoa and Pares, 1988; Bifidobacterium), Sulphite-Polymyxin Milk Agar (Mevissen-Cerhage et al., 1987;Clostridium), and MacConkey's No.2 (Oxoid; Escherichia coli). Single colonies were removed from selective media plates and grown in peptone yeast glucose (PYG) broth (Holdeman et al., 1977). Subsequently, the bacteria were characterized to genus level on the basis of colonial appearance, Gram reaction, spore production, cell morphology and fermentation end-product formation (Holdeman et al., 1977).

Histomorphometry
At slaughter, specimens (0.5 cm×0.5 cm) of intestinal tissue from the mid-jejunum were excised, rinsed in physiological saline. Samples were preserved in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Villus height and crypt depth were measured using image processing and analysis system

Statistical analysis
One way analysis of variance was performed using the General Linear Model (GLM) Procedure of SAS (1989). Differences among means were tested using Duncan's multiple range test. A significant level of 0.05 was used.

Growth performance
Growth performance of pigs fed different levels of dietary FOS is presented in Table 2. As compared to control, supplementation with 4 and 6 g/kg FOS significantly improved ADG and feed conversion ratio. However, feed intake was unaffected by dietary treatments.

Intestinal microflora
The total viable counts of anaerobes and the constitutions of microbes in the intestinal contents were shown in Table 3. As compared to control, supplementation with FOS increased the viable counts of Bifidobacterium and Lactobacillus, while reduced those of Clostridium and Escherichia coli in the small intestinal and proximal colonic contents.

Digestive enzymes
The results on the effects of FOS on the digestive enzyme activities in the pancreas and the small intestinal contents of growing pigs are shown in Table 4 and Table 5, respectively. Supplementation with 4 and 6 g/kg FOS significantly improved the activities of total protease, trypsin and amylase in the small intestinal contents. However, FOS had no significant effect on the activities of chymotrypsin and lipase in the small intestinal contents as well as the digestive enzymes in pancreas.

Morphological measurement of jejunal mucosa
Morphological measurement of jejunal mucosa did show response to consumption of FOS (Table 6). Villus height and the villus height to crypt depth ratio at the jejunal mucosa were significantly higher with 4 and 6 g/kg FOS supplementation as compared to control.

Effects of dietary FOS on growth performance and intestinal microflora of growing pigs
Numerous workers have reported increased growth and improved feed conversion ratio as a consequence of FOS inclusion in young pigs' diets (Hidaka et al., 1986a;Fukuyasu et al., 1987). Our study also verified this. Other authors, however, reported no or slightly negative effects of FOS on young pigs' growth performance (Kornegay et al., 1992;Farnworth et al., 1992).
It was reported that the biological effects of FOS on animals were mainly due to its preferential stimulatory effect on number of the health-promoting genus (Bifidobacterium and Lactobacillus), whilst maintaining populations of unprofitable or potential pathogens (Escherichia coli and Clostridium) at relatively low levels. The increases in numbers of Bifidobacterium and Lactobacillus and decreases in numbers of Escherichia coli and Clostridium in the colon of pigs supplemented with FOS in our study were in agreement with the work of most other research groups (Hidaka et al., 1986b(Hidaka et al., , 1991Fukuyasu et al., 1987;Bunce et al., 1995;Xu et al., 2002).
There are large numbers of microorganisms in the small intestine of pigs, so FOS is fermented to some extent in the small intestine of pigs (Bolduan et al., 1993). The results in the present study indicated that bacterial populations in the small intestinal digesta were affected by the supplementation of FOS. In an another study, Bolduan et al. (1993) reported that supplementation with 2 g/kg FOS for weaned pigs did not affect bacterial populations in the small intestinal digesta. To the author's knowledge, this is the only study that has investigated the effects of supplementing diets with FOS on the bacterial populations in the small intestine in the pigs. The reason of the lack of effects of FOS supplementation on the small intestinal bacterial populations in that study may be that the concentration of FOS (2 g/kg) was not adequate to alter microbial populations. In studies with poultry (Bailey et al., 1991), a 3.75 g/kg concentration of FOS was not sufficient to affect colonization of Salmonella typhimurium, whereas a 7.5 g/kg level affected concentrations of that species. Secondy, the overall intestine health of those piglets may have been a factor in the lack of response to the dietary treatments. Oligosaccharides may not selectively enrich for Bifidobacterium when the indigenous population is high before treatment (Hidaka et al., 1986b;Howard et al., 1995). Addition of Bifidobacterium or oligosaccharedes to the diet of humans has been shown to have no effect when the natural level of Bifidobacterium is high (Hidaka et al., 1986b(Hidaka et al., , 1991. For the animals in that study, the number of Bifidobacterium might have been enough that FOS did not increase Bifidobacterium numbers.

Effects of dietary FOS on the digestive enzyme activities in the pancreas and the small intestinal contents
The results in the present study indicate that FOS has no significant effect on the digestive enzymes in pancreas. Ikegami et al. (1990) reported that when alginic acid and calcium alginate, insoluble polysaccharides that did not contribute to viscosity, were given to rats, they had no effect on the digestive enzymes in pancreas. However, administration of sodium alginate, the highly viscous polysaccharides, led to changes in the exocrine pancreaticbiliary function and may have increased the digestive enzymes in pancreas. The FOS used in this study might have little effect on the viscosity, so it did not affect the pancreatic enzyme activity.
The results on the effects of FOS on the digestive enzyme activity in the small intestinal contents indicated that supplementation with 4 and 6 g/kg FOS significantly improved the activities of total protease, trypsin and amylase. The intestinal microflora data showed that FOS exerted a preferential stimulatory effect on Bifidobacterium and Lactobacillus, whilst suppressed Escherichia coli and Clostridium in the small intestine. Such changes in microbial ecosystem in the presence of FOS might contribute to the observed effects on the digestive enzyme activity in the small intestinal contents. The Bifidobacterium and Lactobacillus colonizing the intestine have been reported to deliver enzymes, thus increasing the intestine digestive enzyme activity (Sissons, 1989). However, the Escherichia coli and Clostridium may damage the villus and microvillus of intestinal mucosa and inhibit the secretion of digestive enzymes (Gao, 1998). Moreover, the Clostridium could secrete the proteolytic enzymes, which may take the intestine digestive enzymes as selective nutrients, thus increasing the degradation of digestive enzymes (Conway, 1994).
However, in the present study, FOS has no significant effect on the activities of lipase in the small intestinal contents. Some investigators reported that addition of FOS to diets caused significantly greater fecal lipid excretion in rats (Delzenne, 1993;Kim et al., 1998). Addition of FOS enhanced the growth of Bifidobacterium and Lactobacillus, which had the action of precipitation and assimilation with bile salt (Zhan, 1998), thus increasing fecal bile acid excretion (Delzenne, 1993;Kim et al., 1998) and decreasing its intestinal concentration. Intestinal bile acid has a great impact on the lipid emulsification and the activities of lipase. Therefore, the increases in numbers of Bifidobacterium and Lactobacillus and decreases in numbers of Clostridium in the small intestine of pigs supplemented with FOS resulted in two-sided effects on the activities of lipase: On the one hand, this facilitated lipase secretion and inhibited its degradation; on the other hand, this might lead to the decrease of intestinal bile acid, thus possibly reduced the activity of lipase. It is possible that such two-sided effects of microbial ecosystem might explain why the activity of lipase was not affected in the present study. However, this remains to be further investigated.

Effects of dietary FOS on the change in intestinal morphology
The structure of the intestinal mucosa can reveal some information on gut health. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology such as shorter villus and deeper crypts have been associated with the presence of toxins (Yason et al., 1987;Anonymous, 1999). A shortening of the villus decreases the surface area for nutrient absorption. The crypt can be regarded as the villus factory, and a large crypt indicates fast tissue turnover and a high demand for new tissue. Demand for energy and protein for gut maintenance is higher compared to other organs. A fast-growing broiler devotes about 12% of the newly synthesized protein to the digestive tract (Anonymous, 1999). Any additional tissue turnover will increase nutrient requirement for maintenance and will therefore lower the efficiency of the animal. Changes in intestinal morphology as descried above can lead to poor nutrient absorption, increased secretion in the gastrointestinal tract, diarrhoea, reduced disease resistance and lower overall performance.
In the present study, the increase in villus height and villus height: crypt depth ratio of the jejunal mucosa in FOS-fed pigs was found. It is likely that these changes are due to FOS's ability to improve the intestinal microflora and are not a direct action of FOS on the intestinal tissue. It is suggested that the energy conserved by the reduced turnover rate of the epithelial cells might be utilized for lean tissue mass synthesis and might explain some of the improvements seen in body weight gain and feed conversion with FOS.