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Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto
Comparative Biochemistry and Physiology Part A 133 (2002) 95–104
Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto Mongkol Samanya, Koh-en Yamauchi* Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken 761-0795, Japan Received 5 February 2002; received in revised form 10 April 2002; accepted 15 April 2002
Abstract Two experiments were conducted. In experiment 1, chickens were fed dried Bacillus subtilis var. natto for 3 or 28 days. Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding. In these birds, blood ammonia concentration was decreased (P-0.05). Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups. These results suggest that the B. subtilis natto depressed ammonia concentration. In experiment 2, chickens were fed dietary B. subtilis natto for 28 days. These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls. Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B. subtilis natto. These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken. The present results may suggest that the B. subtilis natto has the potential to be a beneficial microorganism in chickens. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Layer chickens; Bacillus subtilis var. natto; Probiotic; Ammonia; Villus histology; Light microscope; Electron microscope
1. Introduction In the poultry industry, antibiotics are in widespread use to prevent poultry pathogens and disease so as to improve meat and egg production. However, continued use of dietary antibiotics has resulted in common problems, such as the development of drug-resistant bacteria (Sorum and Sunde, 2001), imbalance of normal microflora (Andremont, 2000) and drug residues in the bird body (Burgat, 1991). As a result of these problems, it has become necessary to develop alternatives using beneficial microorganisms. A probiotic is a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989), and is recommended as an effective alternative to antibiotics *Corresponding author. Tel.yfax: q81-87-891-3053. E-mail address: [email protected] (K. Yamauchi).
(Sissons, 1989; Tournut, 1989). After feedings of probiotics, improvements in growth performance and feed efficiency have been reported in turkeys (Jiraphocakul et al., 1990) and in broiler chicks (Santoso et al., 1995; Cavazzoni et al., 1998). In addition, improved egg mass, egg weight and egg size in layers (Nahashon et al., 1994), as well as suppressed cholesterol in cocks (Endo et al., 1999) and in broilers (Santoso et al., 1995), have also been reported. However, the effects of dietary probiotics on histological alterations to intestinal villi are still unclear. Natto is a traditional Japanese health food made by fermenting boiled soybeans in rice straw containing probiotics (Tamura, 1989; Tonouti et al., 2000). Bacillus subtilis var. natto (Ashiuchi et al., 1998) cultured from natto is a Gram-positive spore-forming bacterium. Although there have been a few investigations of the effects of B. subtilis in poultry (Jiraphocakul et al., 1990; Santoso et al., 1995, 2001), little information is
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 1 2 1 - 6
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Table 1 Composition of dried B. subtilis natto Nutrient
Composition (%)
Crude protein Crude fat Crude fiber Ash Water
52 0.5 4.5 6.5 7
available on the effects of B. subtilis natto on nutrient metabolism and histological alterations to intestinal villi in chickens. We performed two experiments: in experiment 1, growth performance, internal organs and nutrient metabolism, such as ammonia and amylase, were examined in adult male chickens fed dietary B. subtilis natto. In experiment 2, growth performance and intestinal villus histological alterations were studied.
lected to observe the gross anatomical changes to internal organs. Intestinal contents (digesta) were collected by massaging the tract from the jejunum to the ileum, and were immediately placed on ice and stored at y20 8C until used. 2.2. Gross anatomical protocol for internal organs After decapitation, the gizzard, pancreas, liver, heart, kidney, separate intestinal parts and ceca were collected. The empty weight of the organs was recorded. 2.3. Ammonia and glucose in blood Venous blood was used for ammonia analysis, and serum, separated from the blood by centrifugation, was used for glucose analysis. Ammonia and glucose were spectrophotometrically analyzed with commercial kits (Wako Pure Chemical Industries, Tokyo, Japan).
2. Materials and methods 2.1. Experiment 1: animals, housing and experimental design Adult male white leghorn chickens (Gallus gallus domesticus) (Julia strain) were fed dried B. subtilis natto (Table 1; Bacillus subtilis Natto Powder-710䉸, Kitamura Co Ltd, Aichi, Japan) at 0, 0.5, 1 and 3% levels to the basal mash diet (Table 2; Nippon Formula Feed Manufacturing Co Ltd, Kanagawa, Japan). The B. subtilis natto culture contained approximately 1=108 –1=1010 microorganismsyg. Birds were placed into individual cages in a controlled environment with a 14-h light photoperiod (06:00–20:00 h) at a mean environmental temperature of 21 8C. Birds were given ad libitum access to water and each diet for 3 or 28 days. Each dietary group for the 3- and 28-day feeding periods had five and seven birds, respectively. Feed intake and body weight gain were measured at the end of the feeding experiment for the 3-day period, and weekly for the 28day period. At the end of each experimental feeding period, blood was collected between 09:00 and 11:00 h from the basilic vein on the wing of five birds from each group for each feeding period. The blood samples were then centrifuged at 2000=g at 4 8C for 15 min. Blood serum was then kept at y20 8C until used. In the case of the 28-day feeding period, internal organs were col-
2.4. Amylase and lipase in the intestinal contents The intestinal digesta samples were diluted 10fold (wyv) in 2 mM sodium phosphate buffer (pH 7.0) containing 6 mM NaCl and homogenized for 1 min, followed by sonication for 1 min. The sample was then centrifuged at 19 000=g for 20 min at 4 8C. To prevent possible enzyme degraTable 2 Composition of the basal diet Ingredients and nutrients Ground cornqmilo Soybean meal Fish meal Rice bran Concentrate mixturea Crude protein Crude fat Crude fiber Ash Calcium Phosphorus ME (kcalykg) a
Composition (%) 64 19 6 3 8 17 3 6 12.5 2.6 0.55 2800
Provided the following per kg of diet: vitamin A, 8000 IU; vitamin D3, 1500 IU; vitamin E, 7.5 mg; vitamin K3, 1 mg; vitamin B1, 1.05 mg; vitamin B2, 5 mg; vitamin B6, 3.75 mg; vitamin B12, 0.0045 mg; biotin, 0.15 mg; folic acid, 0.375 mg; pantothenate, 3 mg; niacin, 20 mg; choline, 3150 mg; iodine, 0.4 mg; manganese, 75 mg; iron, 180 mg; and zinc, 52.5 mg.
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dation, the supernatants from digesta were kept on ice throughout the preparation. Supernatants were stored at y20 8C for enzyme assay. Amylase was analyzed by a spectrophotometric assay with a commercial kit (Wako Pure Chemical Industries, Tokyo, Japan), and lipase with kits from Dainippon Pharmaceutical Co Ltd (Osaka, Japan) and Maruko Seiyaku Co Ltd (Nagoya, Japan). When the amylase activity of samples was too high, 2 mM sodium phosphate buffer (pH 7.0) containing 6 mM sodium chloride was added to dilute the samples. 2.5. Experiment 2: animals, housing and experimental design Adult male white leghorn chickens were randomly divided into four groups of 13 birds each, as follows: dietary addition of dried B. subtilis natto to basal mash diet at was carried out at levels of 0, 0.2, 0.5 and 1%. Birds were placed into individual cages in a controlled environment with a 14-h light photoperiod (06:00–20:00 h) at a mean temperature of 15 8C. Birds were given ad libitum access to water and each diet for 4 weeks. Feed intake and body weight gain were measured every week. This experiment was performed according to the humane care guidelines provided by the Kagawa Medical School. 2.6. Tissue sampling At the end of each experimental period, four birds from each group were randomly killed by decapitation under light anesthesia with diethyl ether. A mixture of 3% glutaraldehyde and 4% paraformaldehyde fixative solution in 0.1 M cacodylate buffer (pH 7.4) was injected into the intestinal lumen of the middle part of each intestinal segment. Whole small intestine was removed and put into the same fixative solution: the intestinal segment from the gizzard to pancreatic and bile duct as duodenum, jejunum from the duct to Meckel’s diverticulum, and ileum from the diverticulum to the ileo-cecal-colonic junction. The tissue samples were taken from the middle part of each intestinal segment. A 2-cm length of duodenum was excised for scanning electron microscopic observations and placed in the same fixative solution described above. Another 2-cm length of each intestinal segment was excised for light microscop-
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ic observations and kept in Bouin’s solution. Light microscopic samples were taken immediately proximal to the section collected for scanning electron microscopic. 2.7. Light microscopy The fixed samples were embedded in paraplast. Transverse sections were cut into 5-mm samples, and every 10th section was collected. After staining with hematoxylin–eosin, the following values were measured using a Nikon Cosmozone 1S image analyzer (Nikon). 2.7.1. Measurement of villus height For villus height measurement, 10 villi having the lamina propria were selected per section. The villus length was measured from the villus tip to the bottom, not including the intestinal crypt. An average of these 10 villi per section was expressed as a mean villus height for each section. A total of eight sections were counted per bird. Then, an average of these eight sections per bird was expressed as a mean villus height for each bird. Finally, these four mean villus heights from four birds were expressed as a mean villus height for one treatment group. 2.7.2. Measurement of absorptive epithelial cell area For measurement of a single cell area on a 5mm transverse section, the epithelial cell layer was randomly measured in the middle part of the villi, then the number of cell nuclei within this measured epithelial cell layer was counted. Finally, the area of the epithelial cell layer was divided by the number of cell nuclei to give an epithelial cell area. Two cell areas for each transverse section were calculated. An average of these two cell areas per section was expressed as a mean for each section. A total of eight sections were counted per bird. Then, an average of these eight sections per bird was expressed as a mean cell area for each bird. Finally, these four mean cell areas from four birds were expressed as a mean cell area for one treatment group. 2.7.3. Measurement of cell mitosis in the crypt For measurement of the number of cell mitoses in the crypt, all cell mitoses observed in one transverse section were measured. A total of cell mitosis numbers were counted from five different
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Table 3 Feed intake, body weight gain and feed efficiency in chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 (ns 5) and 28 days (ns7) (mean"S.E.) Parameter
Level of B. subtilis natto 0%
0.5%
1%
3%
Fed for 3 days Feed intake (g) Body weight gain (g) Feed efficiency
177.5"15.41 23.0"4.63 0.12"0.022
174.3"7.35 23.0"4.35 0.13"0.022
187.3"13.25 23.8"3.14 0.13"0.015
171.4"8.21 20.0"4.47 0.12"0.009
Fed for 28 days Feed intake (g) Body weight gain (g) Feed efficiency
1908.7"36.7 150.0"13.78 0.073"0.007
1944.1"47.6 163.3"18.6 0.083"0.008
1903.4"49 148.6"12.0 0.075"0.005
1929.4"45 164.3"12.5 0.085"0.006
sections for each bird, and these five values were used to calculate the mean cell mitosis for one bird. Finally, four mean cell mitoses from four birds were expressed as the mean cell mitosis for one treatment group. 2.8. Scanning electron microscopy The samples were longitudinally slit at the nonmesenteric side along the entire length, and intestinal contents were washed away with 0.01 M phosphate-buffered saline, pH 7.4. Tissue samples were pinned flat to prevent curling within the same fixative used above at room temperature for 1 h. A block was cut into a 4=7-mm2 squares and fixed for a further 1 h. The pieces were rinsed with 10 mM sodium cacodylate buffer (pH 7.4) and post-fixed with 1% osmium tetroxide in icecold buffer for 2 h. The specimens were dried in a critical-point drying apparatus (Hitachi HCP-1, Japan) using liquid carbon dioxide as the medium. The dried specimens were coated with platinum (RMC-Eiko RE vacuum coater, Japan) and examined with a scanning electron microscope (Hitachi S-800, Japan) at 8 kV. Morphological alterations of the villus tip surface were compared among groups. 2.9. Statistical analysis The average of all parameters for each bird from each treatment group was analyzed across all treatment groups by ANOVA analysis with a Duncan’s multiple range test (STATVIEW program; Abacus Concepts Inc). Differences at P-0.05 were considered as significant.
3. Results 3.1. Experiment 1: growth performance Table 3 shows feed intake and body weight gain of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 or 28 days. Growth performance was not different amongst the groups, except that feed efficiency in all chickens fed dietary B. subtilis natto for 28 days tended to be improved. 3.2. Gross anatomical observations of internal organs Wet weight of gizzard, pancreas, liver, heart, kidney, each intestinal part and ceca was not different among groups of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (Table 4). 3.3. Determination of ammonia and glucose in blood Table 5 shows ammonia and glucose concentrations in blood of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 or 28 days. In the case of the 3-day dietary period, ammonia concentration in the blood tended to be depressed in all groups fed B. subtilis natto compared to concentrations found in the 0% group; even the ammonia level in the 0.5% B. subtilis natto-fed group was lower than the 0% group (P-0.05). The concentration of glucose did not differ from one group to another. In the case of the 28-day dietary period, the ammonia concentration in blood was depressed in all dietary B. subtilis natto groups compared to levels in the 0% group (P-0.05).
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Table 4 Wet weight of internal organs in chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (mean"S.E.; ns 7) Internal organ
Gizzard (g) Pancreas (g) Liver (g) Heart (g) Kidney (g) Duodenum (g) Jejunum (g) Ileum (g) Ceca (g)
Level of B. subtilis natto 0%
0.5%
1%
3%
24.19"1.02 2.54"0.17 27.24"1.30 11.59"0.37 3.16"0.36 7.23"0.39 12.20"0.79 7.36"0.43 4.00"0.14
26.78"0.98 2.67"0.06 26.73"0.92 11.30"0.40 3.37"0.26 7.84"0.45 13.83"0.42 7.87"0.47 3.94"0.27
28.60"1.64 2.84"0.22 29.97"1.82 12.93"1.23 3.82"0.56 7.80"0.64 14.69"1.03 9.09"0.77 4.74"0.49
25.20"0.75 2.51"0.12 27.51"0.65 10.90"0.22 3.48"0.17 7.29"0.31 13.10"0.50 7.51"0.30 3.61"0.17
The concentration of glucose in serum was not altered in any group.
3.6. Intestinal villus height, cell area and cell mitosis
3.4. Determination of amylase and lipase in intestinal digesta
Fig. 1 shows intestinal villus height, cell area and cell mitosis in each intestinal segment in chickens fed 0, 0.2, 0.5 and 1% dietary B. subtilis natto. All parameters in all intestinal parts showed a tendency to be higher in all chickens fed dietary B. subtilis natto than in the 0% group. Compared to the 0% dietary B. subtilis natto group, villus height of the duodenum in the 0.2% group and the ileum in the 1% group were higher (P-0.05). Cell area of the duodenum in the 0.2 and 0.5% groups, the jejunum in all groups, and the ileum in the 0.5 and 1% groups was broader (P-0.05). Cell mitosis of the jejunum in the 0.5% group was increased (P-0.05). Amongst dietary groups, a significant difference was not observed.
Amylase and lipase activity in the intestinal digesta was not significantly different among groups fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (Table 5). 3.5. Experiment 2: growth performance Table 6 shows feed intake, body weight gain and feed efficiency in chickens fed 0, 0.2, 0.5 and 1% dietary B. subtilis natto for 28 days. Growth performance tended to be higher in the 0.2 and 0.5% groups than the 0% group.
Table 5 Ammonia and glucose concentration in the blood (ns5), and amylase and lipase activity in the intestinal content (ns7) of chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 and 28 days (mean"S.E.) Parameter
Level of B. subtilis natto 0%
0.5%
1%
3%
Fed for 3 days Ammonia (mgydl) Glucose (mgydl)
401.7"9.2a 213.2"5.2
345.1"5.4b 217.2"7.2
362.9"21.3ab 221.7"2.8
359.7"12.2ab 212.8"2.7
Fed for 28 days Ammonia (mgydl) Glucose (mgydl) Amylase (IU) Lipase (IU)
424.5"16a 218.9"3.6 353.6"17.6 99.1"21.8
368.1"3b 217.5"7.5 375.4"42 108.4"24.2
341.6"16.1b 218.5"5.6 363.9"29.9 99.1"19.2
347.7"7.3b 228.4"5.1 388.2"18.5 115.4"25.9
a b
Means within each grouping with different letter designations differ (P-0.05).
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Table 6 Feed intake, body weight gain and feed efficiency in chickens fed dietary dried B. subtilis natto at 0, 0.2, 0.5 and 1% levels for 28 days (mean"S.E.; ns13) Parameter
Feed intake (g) Body weight gain (g) Feed efficiency
Level of B. subtilis natto 0%
0.2%
0.5%
1%
2190"73.79 144.3"13.57 0.066"0.006
2208"44.97 164.7"12.94 0.075"0.006
2279"113.9 174.6"18.72 0.075"0.007
2279"113.9 148.3"13.08 0.067"0.005
3.7. Villus tip surface
4. Discussion
Fig. 2 illustrates the duodenal villus tip surface of chickens fed dietary B. subtilis natto at (a) 0, (b) 0.2, (c) 0.5 and (d) 1% levels. The flat cell outline in the 0% group (arrows in a) developed cell protuberances (arrows in b), and large protruded cell clusters (stars in b) were frequently observed in the 0.2% group. In the 0.5% group, cell clusters (stars in c) became smaller, but cell protuberances remained (arrows in c). However, in the 1% group, cell clusters disappeared, and only cell protuberances were found (arrows in d).
The improved growth performance of domestic fowl fed probiotics (Jiraphocakul et al., 1990; Santoso et al., 1995; Cavazzoni et al., 1998) is thought to be induced by the total effects of probiotic action, including the maintenance of normal intestinal microflora, increased digestive enzyme activity and decreased ammonia production (Jin et al., 1997, 2000). The histological changes in chicken intestines reported herein provide new information regarding the potential for using probiotics in chicken feed.
Fig. 1. Intestinal villus height, cell area and cell mitosis number in each intestinal segment of chickens fed dietary dried B. subtilis natto at 0, 0.2, 0.5, and 1% levels (mean"S.E.; ns4). All parameters tended to be activated in all dietary groups. abMeans within each grouping with different letter designations differ (P-0.05).
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Fig. 2. Duodenal villus tip surface of chickens fed dietary dried B. subtilis natto at (a) 0, (b) 0.2, (c) 0.5 and (d) 1%. Arrows indicate cell protuberances; stars indicate protruded cell clusters. It is evident that only flat cell outlines (arrows in a) are present in the 0% group, cell protuberances (arrows in b) and large, protruded cell clusters (stars in b) in the 0.2% group, cell protuberances (arrows in c) and small, protruded cell clusters (stars in c) in the 0.5% group, and only cell protuberances (arrows in d) in the 1% group. Scale bar (23 mm) is common to all pictures (300=).
In experiment 1, blood ammonia concentration was depressed in chickens fed 0.5% dietary B. subtilis natto for 3 days, and in all dietary groups for 28 days. It has been reported that uric acid produced in the chicken liver is partially excreted into the intestine and hydrolyzed into ammonia by microbial urease (Wrong, 1981; Karasawa et al., 1988). In addition, urease-producing bacteria are known to inhibit chicken growth (Lev et al., 1957) and the ammonia produced by these bacteria is toxic to chickens (Visek, 1978). Dietary probiotics have been shown to suppress the growth of urease-
producing bacteria (Yeo and Kim, 1997) and have subsequently been shown to reduce ammonia levels in chicken ceca (Endo and Nakano, 1999; Endo et al., 1999). The present results suggest that B. subtilis natto is also an effective microorganism for suppressing ammonia production. In addition, the results may mean that B. subtilis natto would be beneficial for improving chicken health. In experiment 1, we found depressed ammonia levels in the blood after feeding dietary B. subtilis natto. The effects of dietary B. subtilis natto on the histological stimulation of intestinal villi were
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observed in experiment 2. We think that the depressed blood ammonia concentration may significantly relate to a decrease in ammonia concentration in the intestine after feeding a probiotic. Reduced blood ammonia concentrations indicated increased fixation of ammonia in the intestinal lumen of pigs fed probiotics (Scheuermann, 1993). Yeo and Kim (1997) reported that ammonia produced in the intestine could enter the blood stream. Decreased ammonia concentration in small intestinal content was found in pigs fed Bacillus cereus (Kirchgessner et al., 1993) and in ceca of chicken fed probiotics (Endo and Nakano, 1999; Endo et al., 1999). Ammonia is known to be toxic to villus histology, and reduces gastric mucosal DNA synthesis (Warzecha et al., 2000) and cell proliferation (Rabkin et al., 1993). The activated cell mitosis observed here in chickens fed dietary B. subtilis natto might be induced by depression of ammonia production. In addition, ammonia was reported to cause histological damage, loss of mucus and DNA in the colon (Lin and Visek, 1991), histological damage in the gastric mucosa (Murakami et al., 1990) and acute gastric lesions (Warzecha et al., 2000). Such histological damage was not observed in the present B. subtilis natto-fed birds showing depressed levels of ammonia. As intestinal epithelial cells originating in the crypt migrate along the villus surface upward to the villus tip, and are extruded into the intestinal lumen within 48 to 96 h (Imondi and Bird, 1966; Potten, 1998), the longer villi are induced by activated cell mitosis, and no mucosal damage would result due to the depressed ammonia concentration. This corresponds with the long villi in the ileum of chicks and turkeys treated with Lactobacillus reuteri (Dunham et al., 1993), and probiotics have been shown to induce gut epithelial-cell proliferation in rats (Ichikawa et al., 1999). In addition, long villi were induced in the jejunum and ileum of turkeys fed dietary amylase (Ritz et al., 1995). Amylase is secreted by Bacillus subtilis (Hao et al., 1989; Haddaoui et al., 1999; Duran-Paramo et al., 2000). Amylase genes were also cloned in B. subtilis natto (Emori et al., 1990). It is not clear at present if the high villi observed here were induced by amylase, but it would appear to be related to amylase. Caspary (1992) has described that increased villus height suggests an increased surface area capable of greater absorption of available nutrients. It is understood that greater villus height and numerous cell mitoses in the intestine are
indicators that the function of the intestinal villi is activated (Langhout et al., 1999; Yasar and Forbes, 1999; Shamoto and Yamauchi, 2000). These facts suggest that the villus function might be activated after feeding dietary B. subtilis natto. Activated cell mitosis under the light microscope corresponds with large protruded cell clusters and cell protuberances on the villus tip surface under the depressed ammonia condition of chickens fed dietary B. subtilis natto. Cell clusters and protuberances appear to have occurred due to activation of the cell mitosis rate more so than the cell extrusion rate; this has resulted in an accumulation of cells on the villus tip. Furthermore, although ammonia was closely associated with mucosal histological damage, lesions and loss (Lin and Visek, 1991; Warzecha et al., 2000), cell damage was not found on the villus surface. This also suggests that cell accumulation on the villus tip showing no damage or loss was due to the depression of ammonia production. These cell protuberances have been morphologically demonstrated as displaying activated function in the epithelial cells (Shamoto and Yamauchi, 2000; Tarachai and Yamauchi, 2000; Samanya and Yamauchi, 2001), which suggests that the function of absorptive epithelial cells might be activated in the depressed ammonia condition of chickens fed dietary B. subtilis natto. After being fed dietary B. subtilis, an increase in body weight gain and improved feed efficiency have been reported in heavy meat-type poultry, such as turkeys (Jiraphocakul et al., 1990) and broiler chicks (Santoso et al., 1995, 2001). Yeo and Kim (1997) reported that daily body weight gain was increased only in the early life stage of broiler chicks, but was not different during the second 3-week period after feeding the dietary probiotics. In addition, in laying pullets (Nahashon et al., 1994) and young layer pullets (Nahashon et al., 1996) fed Lactobacillus, these diets improved body weight gain. In contrast, Goodling et al. (1987) described no increase in egg production or feed efficiency. In the layer cocks fed dietary B. subtilis natto in the present work, a significant improvement in growth performance could not be obtained, although feed efficiency tended to be slightly elevated in the 0.2 and 0.5% groups. The reason for this ineffectiveness of B. subtilis natto may be attributed to the fact that adult male layers were used, because these birds do not require as much metabolic energy. However, slightly
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improved feed efficiency in the 0.2 and 0.5% dietary groups suggests that B. subtilis natto may be a useful microorganism in adult layer cocks, and might induce a tendency towards improved growth performance in adult male layers. In conclusion, B. subtilis natto depresses ammonia concentration in blood, resulting in an activation of intestinal function. This suggests that B. subtilis natto has potential to be a useful microorganism in chickens. References Andremont, A., 2000. Consequences of antibiotic therapy to the intestinal ecosystem. Ann. Fr. Anesth. Reanim. 19, 395–402. Ashiuchi, M., Tani, K., Soda, K., Misono, H., 1998. Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate. J. Biochem. 123, 1156–1163. Burgat, V., 1991. Residues of drugs of veterinary use in food. Rev. Prat. 41, 985–990. Caspary, W.F., 1992. Physiology and pathophysiology of intestinal absorption. Am. J. Clin. Nutr. 55, 299S–308S. Cavazzoni, V., Adami, A., Castrovilli, C., 1998. Performance of broiler chickens supplemented with Bacillus coagulans as probiotic. Br. Poult. Sci. 39, 526–529. Dunham, H.J., Williams, C., Edens, F.W., Casas, I.A., Dobrogosz, W.J., 1993. Lactobacillus reuteri immunomodulation of stressor-associated diseases in newly hatched chickens and turkeys. Poult. Sci. 72, 103. Duran-Paramo, E., Garcia-Kirchner, O., Hervagault, J.F., Thomas, D., Barbotin, J.N., 2000. Alpha-amylase production by free and immobilized Bacillus subtilis. Appl. Biochem. Biotechnol. 84-86, 479–485. Emori, M., Takagi, M., Maruo, B., Yano, K., 1990. Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633. J. Bacteriol. 172, 4901–4908. Endo, T., Nakano, M., 1999. Influence of a probiotic on productivity, meat components, lipid metabolism, caecal flora and metabolites, and raising environment in broiler production. Anim. Sci. J. 70, 207–218. Endo, T., Nakano, M., Shimizu, S., Fukushima, M., Miyoshi, S., 1999. Effect of a probiotic on the lipid metabolism of cocks fed on a cholesterol-enriched diet. Biosci. Biotechnol. Biochem. 63, 1569–1575. Fuller, R., 1989. Probiotic in man and animal. J. Appl. Bacteriol. 66, 365–378. Goodling, A.C., Cerniglia, G.J., Hebert, J.A., 1987. Production performance of white leghorn layers fed Lactobacillus fermentation products. Poult. Sci. 66, 480–486. Haddaoui, E., Chambert, R., Petit-Glatron, M.F., Lindy, O., Sarvas, M., 1999. Bacillus subtilis alpha-amylase: the ratelimiting step of secretion is growth phase-independent. FEMS Microbiol. Lett. 173, 127–131.
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Hao, J., Pazlarova, J., Strnadova, M., Chaloupka, J., 1989. Regulation of extracellular proteins and alpha-amylase secretion by temperature in Bacillus subtilis. Foliar Microbiol. 34, 179–184. Ichikawa, H., Kuroiwa, T., Inagaki, A., et al., 1999. Probiotic bacteria stimulate gut epithelial cell proliferation in rat. Digest. Dis. Sci. 44, 2119–2123. Imondi, A.R., Bird, F.H., 1966. The turnover of intestinal epithelium in the chick. Poult. Sci. 45, 142–147. Jin, L.Z., Ho, Y.W., Abdullah, N., Jalaludin, S., 1997. Probiotics in poultry: modes of action. World Poult. Sci. J. 53, 351–368. Jin, L.Z., Ho, H.W., Abdullah, N., Jalaludin, S., 2000. Digestive and bacteria enzyme activities in broilers fed diets supplemented with Lactobacillus cultures. Poult. Sci. 79, 886–891. Jiraphocakul, S., Sullivan, T.W., Shahani, K.M., 1990. Influence of a dried Bacillus subtilis culture and antibiotics on performance and intestinal microflora in turkeys. Poult. Sci. 69, 1966–1973. Karasawa, Y., Kawai, H., Hosono, A., 1988. Ammonia production from amino acids and urea in the caecal contents of the chicken. Comp. Biochem. Physiol. 90B, 205–207. Kirchgessner, M., Roth, F.X., Eidelsburger, U., Gedek, B., 1993. The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets. 1. Effect on the growth parameters and gastrointestinal environment. Arch. Tierernahr. 44, 111–121. Langhout, D.J., Schutte, J.B., Van, L.P., Wiebenga, J., Tamminga, S., 1999. Effect of dietary high and low methylated citrus pectin on the activity of the ileal microflora and morphology of the small intestinal wall of broiler chicks. Br. Poult. Sci. 40, 340–347. Lev, M., Briggs, C.A.E., Coates, M.E., 1957. The gut flora of the chick. 3. Differences in cecal flora between ‘infected’, uninfected and penicillin-fed chicks. J. Anim. Sci. 16, 364–372. Lin, H.C., Visek, W.J., 1991. Colon mucosa cell damage by ammonia in rats. J. Nutr. 121, 887–893. Murakami, M., Yoo, J.K., Teramura, S., et al., 1990. Generation of ammonia and mucosal lesion formation following hydrolysis of urea by urease in the rat stomach. J. Clin. Gastroenterol. 12, S104–109. Nahashon, S.N., Nakaue, H.S., Mirosh, L.W., 1994. Production variable and nutrient retention in single comb white leghorn laying pullets fed diets supplemented with direct-fed microbials. Poult. Sci. 73, 1699–1711. Nahashon, S.N., Nakaue, H.S., Mirosh, L.W., 1996. Performance of single comb white leghorn layers fed a diet with a live microbial during the growth and egg-laying phase. Anim. Feed Sci. Technol. 61, 17–26. Potten, C.S., 1998. Stem cells in the gastrointestinal epithelium: numbers, characteristics and death. Philos. Trans. R. Soc. B 353, 821–830. Rabkin, R., Palathumpat, M., Tsao, T., 1993. Ammonium chloride alters renal tubular cell growth and protein turnover. Lab. Invest. 68, 427–438. Ritz, C.W., Hulet, R.M., Self, B.B., Denbow, D.M., 1995. Growth and intestinal morphology of male turkeys as influenced by dietary supplementation of amylase and xylanase. Poult. Sci. 74, 1329–1334.
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Samanya, M., Yamauchi, K., 2001. Morphological change of the intestinal villi in chicken fed dietary charcoal powder, including wood vinegar compound. J. Poult. Sci. 38, 289–301. Santoso, U., Tanaka, K., Ohtani, S., 1995. Effect of dried Bacillus subtilis culture on growth, body composition and hepatic lipogenic enzyme activity in female broiler chicks. Br. J. Nutr. 74, 523–529. Santoso, U., Tanaka, K., Ohtani, S., Sakaida, M., 2001. Effect of fermented product from Bacillus subtilis on feed conversion efficiency, lipid accumulation and ammonia production in broiler chicks. Asian–Aust. J. Anim. Sci. 14, 333–337. Scheuermann, S.E., 1993. Effect of the probiotic Paciflor (CIP 5832) on energy and protein metabolism in growing pigs. Anim. Feed Sci. Technol. 41, 181–189. Shamoto, K., Yamauchi, K., 2000. Recovery responses of chick intestinal villus morphology to different refeeding procedures. Poult. Sci. 79, 718–723. Sissons, J.W., 1989. Potential of probiotic organisms to prevent diarrhoea and promote digestion in farm animals: a review. J. Sci. Food Agric. 49, 1–13. Sorum, H., Sunde, M., 2001. Resistance to antibiotics in the normal flora of animals. Vet. Res. 32, 227–241. Tamura, M., 1989. Development of low-smelling natto. Lifesci. Biotechnol. 5, 104–108. In Japanese. Tarachai, P., Yamauchi, K., 2000. Effects of luminal nutrient absorption, intraluminal physical stimulation, and intrave-
nous parenteral alimentation on the recovery responses of duodenal villus morphology following feed withdrawal in chickens. Poult. Sci. 79, 1578–1585. Tonouti, A., Oka, H., Kurotaki, K., Takeda, K., 2000. Isolation of Bacillus subtilis (natto) useful for the manufacture of natto. Bull. Facult. Agric. Hirosaki Univ. 3, 14–18. In Japanese. Tournut, J., 1989. Applications of probiotics to animal husbandry. Rev. Sci. Technol. Off. Int. Epiz. 8, 551–566. Visek, W.J., 1978. The mode of growth promotion antibiotics. J. Anim. Sci. 46, 1447–1469. Warzecha, Z., Dembinski, A., Brzozowski, T., et al., 2000. Gastroprotective effect of histamine and acid secretion on ammonia-induced gastric lesions in rats. Scan. J. Gastroenterol. 35, 916–924. Wrong, O.M., 1981. Nitrogen compounds. In: Wrong, O.M., Edmonds, C.J., Chadwick, V.S. (Eds.), The Large Intestine: Its Role in Mammalian Nutrition and Homeostasis. John Wiley and Sons, New York, pp. 133–211. Yasar, S., Forbes, J.M., 1999. Performance and gastro-intestinal response of broiler chicks fed on cereal grain-based foods soaked in water. Br. Poult. Sci. 40, 65–76. Yeo, J., Kim, K.I., 1997. Effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on growth and intestinal urease activity in broiler chicks. Poult. Sci. 76, 381–385.
Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto Mongkol Samanya, Koh-en Yamauchi* Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken 761-0795, Japan Received 5 February 2002; received in revised form 10 April 2002; accepted 15 April 2002
Abstract Two experiments were conducted. In experiment 1, chickens were fed dried Bacillus subtilis var. natto for 3 or 28 days. Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding. In these birds, blood ammonia concentration was decreased (P-0.05). Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups. These results suggest that the B. subtilis natto depressed ammonia concentration. In experiment 2, chickens were fed dietary B. subtilis natto for 28 days. These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls. Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B. subtilis natto. These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken. The present results may suggest that the B. subtilis natto has the potential to be a beneficial microorganism in chickens. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Layer chickens; Bacillus subtilis var. natto; Probiotic; Ammonia; Villus histology; Light microscope; Electron microscope
1. Introduction In the poultry industry, antibiotics are in widespread use to prevent poultry pathogens and disease so as to improve meat and egg production. However, continued use of dietary antibiotics has resulted in common problems, such as the development of drug-resistant bacteria (Sorum and Sunde, 2001), imbalance of normal microflora (Andremont, 2000) and drug residues in the bird body (Burgat, 1991). As a result of these problems, it has become necessary to develop alternatives using beneficial microorganisms. A probiotic is a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989), and is recommended as an effective alternative to antibiotics *Corresponding author. Tel.yfax: q81-87-891-3053. E-mail address: [email protected] (K. Yamauchi).
(Sissons, 1989; Tournut, 1989). After feedings of probiotics, improvements in growth performance and feed efficiency have been reported in turkeys (Jiraphocakul et al., 1990) and in broiler chicks (Santoso et al., 1995; Cavazzoni et al., 1998). In addition, improved egg mass, egg weight and egg size in layers (Nahashon et al., 1994), as well as suppressed cholesterol in cocks (Endo et al., 1999) and in broilers (Santoso et al., 1995), have also been reported. However, the effects of dietary probiotics on histological alterations to intestinal villi are still unclear. Natto is a traditional Japanese health food made by fermenting boiled soybeans in rice straw containing probiotics (Tamura, 1989; Tonouti et al., 2000). Bacillus subtilis var. natto (Ashiuchi et al., 1998) cultured from natto is a Gram-positive spore-forming bacterium. Although there have been a few investigations of the effects of B. subtilis in poultry (Jiraphocakul et al., 1990; Santoso et al., 1995, 2001), little information is
1095-6433/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 1 2 1 - 6
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Table 1 Composition of dried B. subtilis natto Nutrient
Composition (%)
Crude protein Crude fat Crude fiber Ash Water
52 0.5 4.5 6.5 7
available on the effects of B. subtilis natto on nutrient metabolism and histological alterations to intestinal villi in chickens. We performed two experiments: in experiment 1, growth performance, internal organs and nutrient metabolism, such as ammonia and amylase, were examined in adult male chickens fed dietary B. subtilis natto. In experiment 2, growth performance and intestinal villus histological alterations were studied.
lected to observe the gross anatomical changes to internal organs. Intestinal contents (digesta) were collected by massaging the tract from the jejunum to the ileum, and were immediately placed on ice and stored at y20 8C until used. 2.2. Gross anatomical protocol for internal organs After decapitation, the gizzard, pancreas, liver, heart, kidney, separate intestinal parts and ceca were collected. The empty weight of the organs was recorded. 2.3. Ammonia and glucose in blood Venous blood was used for ammonia analysis, and serum, separated from the blood by centrifugation, was used for glucose analysis. Ammonia and glucose were spectrophotometrically analyzed with commercial kits (Wako Pure Chemical Industries, Tokyo, Japan).
2. Materials and methods 2.1. Experiment 1: animals, housing and experimental design Adult male white leghorn chickens (Gallus gallus domesticus) (Julia strain) were fed dried B. subtilis natto (Table 1; Bacillus subtilis Natto Powder-710䉸, Kitamura Co Ltd, Aichi, Japan) at 0, 0.5, 1 and 3% levels to the basal mash diet (Table 2; Nippon Formula Feed Manufacturing Co Ltd, Kanagawa, Japan). The B. subtilis natto culture contained approximately 1=108 –1=1010 microorganismsyg. Birds were placed into individual cages in a controlled environment with a 14-h light photoperiod (06:00–20:00 h) at a mean environmental temperature of 21 8C. Birds were given ad libitum access to water and each diet for 3 or 28 days. Each dietary group for the 3- and 28-day feeding periods had five and seven birds, respectively. Feed intake and body weight gain were measured at the end of the feeding experiment for the 3-day period, and weekly for the 28day period. At the end of each experimental feeding period, blood was collected between 09:00 and 11:00 h from the basilic vein on the wing of five birds from each group for each feeding period. The blood samples were then centrifuged at 2000=g at 4 8C for 15 min. Blood serum was then kept at y20 8C until used. In the case of the 28-day feeding period, internal organs were col-
2.4. Amylase and lipase in the intestinal contents The intestinal digesta samples were diluted 10fold (wyv) in 2 mM sodium phosphate buffer (pH 7.0) containing 6 mM NaCl and homogenized for 1 min, followed by sonication for 1 min. The sample was then centrifuged at 19 000=g for 20 min at 4 8C. To prevent possible enzyme degraTable 2 Composition of the basal diet Ingredients and nutrients Ground cornqmilo Soybean meal Fish meal Rice bran Concentrate mixturea Crude protein Crude fat Crude fiber Ash Calcium Phosphorus ME (kcalykg) a
Composition (%) 64 19 6 3 8 17 3 6 12.5 2.6 0.55 2800
Provided the following per kg of diet: vitamin A, 8000 IU; vitamin D3, 1500 IU; vitamin E, 7.5 mg; vitamin K3, 1 mg; vitamin B1, 1.05 mg; vitamin B2, 5 mg; vitamin B6, 3.75 mg; vitamin B12, 0.0045 mg; biotin, 0.15 mg; folic acid, 0.375 mg; pantothenate, 3 mg; niacin, 20 mg; choline, 3150 mg; iodine, 0.4 mg; manganese, 75 mg; iron, 180 mg; and zinc, 52.5 mg.
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dation, the supernatants from digesta were kept on ice throughout the preparation. Supernatants were stored at y20 8C for enzyme assay. Amylase was analyzed by a spectrophotometric assay with a commercial kit (Wako Pure Chemical Industries, Tokyo, Japan), and lipase with kits from Dainippon Pharmaceutical Co Ltd (Osaka, Japan) and Maruko Seiyaku Co Ltd (Nagoya, Japan). When the amylase activity of samples was too high, 2 mM sodium phosphate buffer (pH 7.0) containing 6 mM sodium chloride was added to dilute the samples. 2.5. Experiment 2: animals, housing and experimental design Adult male white leghorn chickens were randomly divided into four groups of 13 birds each, as follows: dietary addition of dried B. subtilis natto to basal mash diet at was carried out at levels of 0, 0.2, 0.5 and 1%. Birds were placed into individual cages in a controlled environment with a 14-h light photoperiod (06:00–20:00 h) at a mean temperature of 15 8C. Birds were given ad libitum access to water and each diet for 4 weeks. Feed intake and body weight gain were measured every week. This experiment was performed according to the humane care guidelines provided by the Kagawa Medical School. 2.6. Tissue sampling At the end of each experimental period, four birds from each group were randomly killed by decapitation under light anesthesia with diethyl ether. A mixture of 3% glutaraldehyde and 4% paraformaldehyde fixative solution in 0.1 M cacodylate buffer (pH 7.4) was injected into the intestinal lumen of the middle part of each intestinal segment. Whole small intestine was removed and put into the same fixative solution: the intestinal segment from the gizzard to pancreatic and bile duct as duodenum, jejunum from the duct to Meckel’s diverticulum, and ileum from the diverticulum to the ileo-cecal-colonic junction. The tissue samples were taken from the middle part of each intestinal segment. A 2-cm length of duodenum was excised for scanning electron microscopic observations and placed in the same fixative solution described above. Another 2-cm length of each intestinal segment was excised for light microscop-
97
ic observations and kept in Bouin’s solution. Light microscopic samples were taken immediately proximal to the section collected for scanning electron microscopic. 2.7. Light microscopy The fixed samples were embedded in paraplast. Transverse sections were cut into 5-mm samples, and every 10th section was collected. After staining with hematoxylin–eosin, the following values were measured using a Nikon Cosmozone 1S image analyzer (Nikon). 2.7.1. Measurement of villus height For villus height measurement, 10 villi having the lamina propria were selected per section. The villus length was measured from the villus tip to the bottom, not including the intestinal crypt. An average of these 10 villi per section was expressed as a mean villus height for each section. A total of eight sections were counted per bird. Then, an average of these eight sections per bird was expressed as a mean villus height for each bird. Finally, these four mean villus heights from four birds were expressed as a mean villus height for one treatment group. 2.7.2. Measurement of absorptive epithelial cell area For measurement of a single cell area on a 5mm transverse section, the epithelial cell layer was randomly measured in the middle part of the villi, then the number of cell nuclei within this measured epithelial cell layer was counted. Finally, the area of the epithelial cell layer was divided by the number of cell nuclei to give an epithelial cell area. Two cell areas for each transverse section were calculated. An average of these two cell areas per section was expressed as a mean for each section. A total of eight sections were counted per bird. Then, an average of these eight sections per bird was expressed as a mean cell area for each bird. Finally, these four mean cell areas from four birds were expressed as a mean cell area for one treatment group. 2.7.3. Measurement of cell mitosis in the crypt For measurement of the number of cell mitoses in the crypt, all cell mitoses observed in one transverse section were measured. A total of cell mitosis numbers were counted from five different
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Table 3 Feed intake, body weight gain and feed efficiency in chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 (ns 5) and 28 days (ns7) (mean"S.E.) Parameter
Level of B. subtilis natto 0%
0.5%
1%
3%
Fed for 3 days Feed intake (g) Body weight gain (g) Feed efficiency
177.5"15.41 23.0"4.63 0.12"0.022
174.3"7.35 23.0"4.35 0.13"0.022
187.3"13.25 23.8"3.14 0.13"0.015
171.4"8.21 20.0"4.47 0.12"0.009
Fed for 28 days Feed intake (g) Body weight gain (g) Feed efficiency
1908.7"36.7 150.0"13.78 0.073"0.007
1944.1"47.6 163.3"18.6 0.083"0.008
1903.4"49 148.6"12.0 0.075"0.005
1929.4"45 164.3"12.5 0.085"0.006
sections for each bird, and these five values were used to calculate the mean cell mitosis for one bird. Finally, four mean cell mitoses from four birds were expressed as the mean cell mitosis for one treatment group. 2.8. Scanning electron microscopy The samples were longitudinally slit at the nonmesenteric side along the entire length, and intestinal contents were washed away with 0.01 M phosphate-buffered saline, pH 7.4. Tissue samples were pinned flat to prevent curling within the same fixative used above at room temperature for 1 h. A block was cut into a 4=7-mm2 squares and fixed for a further 1 h. The pieces were rinsed with 10 mM sodium cacodylate buffer (pH 7.4) and post-fixed with 1% osmium tetroxide in icecold buffer for 2 h. The specimens were dried in a critical-point drying apparatus (Hitachi HCP-1, Japan) using liquid carbon dioxide as the medium. The dried specimens were coated with platinum (RMC-Eiko RE vacuum coater, Japan) and examined with a scanning electron microscope (Hitachi S-800, Japan) at 8 kV. Morphological alterations of the villus tip surface were compared among groups. 2.9. Statistical analysis The average of all parameters for each bird from each treatment group was analyzed across all treatment groups by ANOVA analysis with a Duncan’s multiple range test (STATVIEW program; Abacus Concepts Inc). Differences at P-0.05 were considered as significant.
3. Results 3.1. Experiment 1: growth performance Table 3 shows feed intake and body weight gain of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 or 28 days. Growth performance was not different amongst the groups, except that feed efficiency in all chickens fed dietary B. subtilis natto for 28 days tended to be improved. 3.2. Gross anatomical observations of internal organs Wet weight of gizzard, pancreas, liver, heart, kidney, each intestinal part and ceca was not different among groups of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (Table 4). 3.3. Determination of ammonia and glucose in blood Table 5 shows ammonia and glucose concentrations in blood of chickens fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 or 28 days. In the case of the 3-day dietary period, ammonia concentration in the blood tended to be depressed in all groups fed B. subtilis natto compared to concentrations found in the 0% group; even the ammonia level in the 0.5% B. subtilis natto-fed group was lower than the 0% group (P-0.05). The concentration of glucose did not differ from one group to another. In the case of the 28-day dietary period, the ammonia concentration in blood was depressed in all dietary B. subtilis natto groups compared to levels in the 0% group (P-0.05).
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Table 4 Wet weight of internal organs in chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (mean"S.E.; ns 7) Internal organ
Gizzard (g) Pancreas (g) Liver (g) Heart (g) Kidney (g) Duodenum (g) Jejunum (g) Ileum (g) Ceca (g)
Level of B. subtilis natto 0%
0.5%
1%
3%
24.19"1.02 2.54"0.17 27.24"1.30 11.59"0.37 3.16"0.36 7.23"0.39 12.20"0.79 7.36"0.43 4.00"0.14
26.78"0.98 2.67"0.06 26.73"0.92 11.30"0.40 3.37"0.26 7.84"0.45 13.83"0.42 7.87"0.47 3.94"0.27
28.60"1.64 2.84"0.22 29.97"1.82 12.93"1.23 3.82"0.56 7.80"0.64 14.69"1.03 9.09"0.77 4.74"0.49
25.20"0.75 2.51"0.12 27.51"0.65 10.90"0.22 3.48"0.17 7.29"0.31 13.10"0.50 7.51"0.30 3.61"0.17
The concentration of glucose in serum was not altered in any group.
3.6. Intestinal villus height, cell area and cell mitosis
3.4. Determination of amylase and lipase in intestinal digesta
Fig. 1 shows intestinal villus height, cell area and cell mitosis in each intestinal segment in chickens fed 0, 0.2, 0.5 and 1% dietary B. subtilis natto. All parameters in all intestinal parts showed a tendency to be higher in all chickens fed dietary B. subtilis natto than in the 0% group. Compared to the 0% dietary B. subtilis natto group, villus height of the duodenum in the 0.2% group and the ileum in the 1% group were higher (P-0.05). Cell area of the duodenum in the 0.2 and 0.5% groups, the jejunum in all groups, and the ileum in the 0.5 and 1% groups was broader (P-0.05). Cell mitosis of the jejunum in the 0.5% group was increased (P-0.05). Amongst dietary groups, a significant difference was not observed.
Amylase and lipase activity in the intestinal digesta was not significantly different among groups fed dietary B. subtilis natto at 0, 0.5, 1 and 3% levels for 28 days (Table 5). 3.5. Experiment 2: growth performance Table 6 shows feed intake, body weight gain and feed efficiency in chickens fed 0, 0.2, 0.5 and 1% dietary B. subtilis natto for 28 days. Growth performance tended to be higher in the 0.2 and 0.5% groups than the 0% group.
Table 5 Ammonia and glucose concentration in the blood (ns5), and amylase and lipase activity in the intestinal content (ns7) of chickens fed dietary dried B. subtilis natto at 0, 0.5, 1 and 3% levels for 3 and 28 days (mean"S.E.) Parameter
Level of B. subtilis natto 0%
0.5%
1%
3%
Fed for 3 days Ammonia (mgydl) Glucose (mgydl)
401.7"9.2a 213.2"5.2
345.1"5.4b 217.2"7.2
362.9"21.3ab 221.7"2.8
359.7"12.2ab 212.8"2.7
Fed for 28 days Ammonia (mgydl) Glucose (mgydl) Amylase (IU) Lipase (IU)
424.5"16a 218.9"3.6 353.6"17.6 99.1"21.8
368.1"3b 217.5"7.5 375.4"42 108.4"24.2
341.6"16.1b 218.5"5.6 363.9"29.9 99.1"19.2
347.7"7.3b 228.4"5.1 388.2"18.5 115.4"25.9
a b
Means within each grouping with different letter designations differ (P-0.05).
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Table 6 Feed intake, body weight gain and feed efficiency in chickens fed dietary dried B. subtilis natto at 0, 0.2, 0.5 and 1% levels for 28 days (mean"S.E.; ns13) Parameter
Feed intake (g) Body weight gain (g) Feed efficiency
Level of B. subtilis natto 0%
0.2%
0.5%
1%
2190"73.79 144.3"13.57 0.066"0.006
2208"44.97 164.7"12.94 0.075"0.006
2279"113.9 174.6"18.72 0.075"0.007
2279"113.9 148.3"13.08 0.067"0.005
3.7. Villus tip surface
4. Discussion
Fig. 2 illustrates the duodenal villus tip surface of chickens fed dietary B. subtilis natto at (a) 0, (b) 0.2, (c) 0.5 and (d) 1% levels. The flat cell outline in the 0% group (arrows in a) developed cell protuberances (arrows in b), and large protruded cell clusters (stars in b) were frequently observed in the 0.2% group. In the 0.5% group, cell clusters (stars in c) became smaller, but cell protuberances remained (arrows in c). However, in the 1% group, cell clusters disappeared, and only cell protuberances were found (arrows in d).
The improved growth performance of domestic fowl fed probiotics (Jiraphocakul et al., 1990; Santoso et al., 1995; Cavazzoni et al., 1998) is thought to be induced by the total effects of probiotic action, including the maintenance of normal intestinal microflora, increased digestive enzyme activity and decreased ammonia production (Jin et al., 1997, 2000). The histological changes in chicken intestines reported herein provide new information regarding the potential for using probiotics in chicken feed.
Fig. 1. Intestinal villus height, cell area and cell mitosis number in each intestinal segment of chickens fed dietary dried B. subtilis natto at 0, 0.2, 0.5, and 1% levels (mean"S.E.; ns4). All parameters tended to be activated in all dietary groups. abMeans within each grouping with different letter designations differ (P-0.05).
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Fig. 2. Duodenal villus tip surface of chickens fed dietary dried B. subtilis natto at (a) 0, (b) 0.2, (c) 0.5 and (d) 1%. Arrows indicate cell protuberances; stars indicate protruded cell clusters. It is evident that only flat cell outlines (arrows in a) are present in the 0% group, cell protuberances (arrows in b) and large, protruded cell clusters (stars in b) in the 0.2% group, cell protuberances (arrows in c) and small, protruded cell clusters (stars in c) in the 0.5% group, and only cell protuberances (arrows in d) in the 1% group. Scale bar (23 mm) is common to all pictures (300=).
In experiment 1, blood ammonia concentration was depressed in chickens fed 0.5% dietary B. subtilis natto for 3 days, and in all dietary groups for 28 days. It has been reported that uric acid produced in the chicken liver is partially excreted into the intestine and hydrolyzed into ammonia by microbial urease (Wrong, 1981; Karasawa et al., 1988). In addition, urease-producing bacteria are known to inhibit chicken growth (Lev et al., 1957) and the ammonia produced by these bacteria is toxic to chickens (Visek, 1978). Dietary probiotics have been shown to suppress the growth of urease-
producing bacteria (Yeo and Kim, 1997) and have subsequently been shown to reduce ammonia levels in chicken ceca (Endo and Nakano, 1999; Endo et al., 1999). The present results suggest that B. subtilis natto is also an effective microorganism for suppressing ammonia production. In addition, the results may mean that B. subtilis natto would be beneficial for improving chicken health. In experiment 1, we found depressed ammonia levels in the blood after feeding dietary B. subtilis natto. The effects of dietary B. subtilis natto on the histological stimulation of intestinal villi were
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observed in experiment 2. We think that the depressed blood ammonia concentration may significantly relate to a decrease in ammonia concentration in the intestine after feeding a probiotic. Reduced blood ammonia concentrations indicated increased fixation of ammonia in the intestinal lumen of pigs fed probiotics (Scheuermann, 1993). Yeo and Kim (1997) reported that ammonia produced in the intestine could enter the blood stream. Decreased ammonia concentration in small intestinal content was found in pigs fed Bacillus cereus (Kirchgessner et al., 1993) and in ceca of chicken fed probiotics (Endo and Nakano, 1999; Endo et al., 1999). Ammonia is known to be toxic to villus histology, and reduces gastric mucosal DNA synthesis (Warzecha et al., 2000) and cell proliferation (Rabkin et al., 1993). The activated cell mitosis observed here in chickens fed dietary B. subtilis natto might be induced by depression of ammonia production. In addition, ammonia was reported to cause histological damage, loss of mucus and DNA in the colon (Lin and Visek, 1991), histological damage in the gastric mucosa (Murakami et al., 1990) and acute gastric lesions (Warzecha et al., 2000). Such histological damage was not observed in the present B. subtilis natto-fed birds showing depressed levels of ammonia. As intestinal epithelial cells originating in the crypt migrate along the villus surface upward to the villus tip, and are extruded into the intestinal lumen within 48 to 96 h (Imondi and Bird, 1966; Potten, 1998), the longer villi are induced by activated cell mitosis, and no mucosal damage would result due to the depressed ammonia concentration. This corresponds with the long villi in the ileum of chicks and turkeys treated with Lactobacillus reuteri (Dunham et al., 1993), and probiotics have been shown to induce gut epithelial-cell proliferation in rats (Ichikawa et al., 1999). In addition, long villi were induced in the jejunum and ileum of turkeys fed dietary amylase (Ritz et al., 1995). Amylase is secreted by Bacillus subtilis (Hao et al., 1989; Haddaoui et al., 1999; Duran-Paramo et al., 2000). Amylase genes were also cloned in B. subtilis natto (Emori et al., 1990). It is not clear at present if the high villi observed here were induced by amylase, but it would appear to be related to amylase. Caspary (1992) has described that increased villus height suggests an increased surface area capable of greater absorption of available nutrients. It is understood that greater villus height and numerous cell mitoses in the intestine are
indicators that the function of the intestinal villi is activated (Langhout et al., 1999; Yasar and Forbes, 1999; Shamoto and Yamauchi, 2000). These facts suggest that the villus function might be activated after feeding dietary B. subtilis natto. Activated cell mitosis under the light microscope corresponds with large protruded cell clusters and cell protuberances on the villus tip surface under the depressed ammonia condition of chickens fed dietary B. subtilis natto. Cell clusters and protuberances appear to have occurred due to activation of the cell mitosis rate more so than the cell extrusion rate; this has resulted in an accumulation of cells on the villus tip. Furthermore, although ammonia was closely associated with mucosal histological damage, lesions and loss (Lin and Visek, 1991; Warzecha et al., 2000), cell damage was not found on the villus surface. This also suggests that cell accumulation on the villus tip showing no damage or loss was due to the depression of ammonia production. These cell protuberances have been morphologically demonstrated as displaying activated function in the epithelial cells (Shamoto and Yamauchi, 2000; Tarachai and Yamauchi, 2000; Samanya and Yamauchi, 2001), which suggests that the function of absorptive epithelial cells might be activated in the depressed ammonia condition of chickens fed dietary B. subtilis natto. After being fed dietary B. subtilis, an increase in body weight gain and improved feed efficiency have been reported in heavy meat-type poultry, such as turkeys (Jiraphocakul et al., 1990) and broiler chicks (Santoso et al., 1995, 2001). Yeo and Kim (1997) reported that daily body weight gain was increased only in the early life stage of broiler chicks, but was not different during the second 3-week period after feeding the dietary probiotics. In addition, in laying pullets (Nahashon et al., 1994) and young layer pullets (Nahashon et al., 1996) fed Lactobacillus, these diets improved body weight gain. In contrast, Goodling et al. (1987) described no increase in egg production or feed efficiency. In the layer cocks fed dietary B. subtilis natto in the present work, a significant improvement in growth performance could not be obtained, although feed efficiency tended to be slightly elevated in the 0.2 and 0.5% groups. The reason for this ineffectiveness of B. subtilis natto may be attributed to the fact that adult male layers were used, because these birds do not require as much metabolic energy. However, slightly
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improved feed efficiency in the 0.2 and 0.5% dietary groups suggests that B. subtilis natto may be a useful microorganism in adult layer cocks, and might induce a tendency towards improved growth performance in adult male layers. In conclusion, B. subtilis natto depresses ammonia concentration in blood, resulting in an activation of intestinal function. This suggests that B. subtilis natto has potential to be a useful microorganism in chickens. References Andremont, A., 2000. Consequences of antibiotic therapy to the intestinal ecosystem. Ann. Fr. Anesth. Reanim. 19, 395–402. Ashiuchi, M., Tani, K., Soda, K., Misono, H., 1998. Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate. J. Biochem. 123, 1156–1163. Burgat, V., 1991. Residues of drugs of veterinary use in food. Rev. Prat. 41, 985–990. Caspary, W.F., 1992. Physiology and pathophysiology of intestinal absorption. Am. J. Clin. Nutr. 55, 299S–308S. Cavazzoni, V., Adami, A., Castrovilli, C., 1998. Performance of broiler chickens supplemented with Bacillus coagulans as probiotic. Br. Poult. Sci. 39, 526–529. Dunham, H.J., Williams, C., Edens, F.W., Casas, I.A., Dobrogosz, W.J., 1993. Lactobacillus reuteri immunomodulation of stressor-associated diseases in newly hatched chickens and turkeys. Poult. Sci. 72, 103. Duran-Paramo, E., Garcia-Kirchner, O., Hervagault, J.F., Thomas, D., Barbotin, J.N., 2000. Alpha-amylase production by free and immobilized Bacillus subtilis. Appl. Biochem. Biotechnol. 84-86, 479–485. Emori, M., Takagi, M., Maruo, B., Yano, K., 1990. Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633. J. Bacteriol. 172, 4901–4908. Endo, T., Nakano, M., 1999. Influence of a probiotic on productivity, meat components, lipid metabolism, caecal flora and metabolites, and raising environment in broiler production. Anim. Sci. J. 70, 207–218. Endo, T., Nakano, M., Shimizu, S., Fukushima, M., Miyoshi, S., 1999. Effect of a probiotic on the lipid metabolism of cocks fed on a cholesterol-enriched diet. Biosci. Biotechnol. Biochem. 63, 1569–1575. Fuller, R., 1989. Probiotic in man and animal. J. Appl. Bacteriol. 66, 365–378. Goodling, A.C., Cerniglia, G.J., Hebert, J.A., 1987. Production performance of white leghorn layers fed Lactobacillus fermentation products. Poult. Sci. 66, 480–486. Haddaoui, E., Chambert, R., Petit-Glatron, M.F., Lindy, O., Sarvas, M., 1999. Bacillus subtilis alpha-amylase: the ratelimiting step of secretion is growth phase-independent. FEMS Microbiol. Lett. 173, 127–131.
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