Histological alterations of intestinal villi in chickens__ fed dried Bacillus subtilis var. natto

Comparative Biochemistry and Physiology Part A 133(2002)95–1041095-6433/02/$-see front matter ᮊ2002Elsevier Science Inc.All rights reserved.PII:S1095-6433Ž02.00121-6Histological alterations of intestinal villi in chickens fed driedBacillus subtilis var.nattoMongkol Samanya,Koh-en Yamauchi*Laboratory of Animal Science,Faculty of Agriculture,Kagawa University,Miki-cho,Kagawa-ken 761-0795,JapanReceived 5February 2002;received in revised form 10April 2002;accepted 15April 2002AbstractTwo experiments were conducted.In experiment 1,chickens were fed dried Bacillus subtilis var.natto for 3or 28days.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 28days.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.ᮊ2002Elsevier Science Inc.All rights reserved.Keywords:Layer chickens;Bacillus subtilis var.natto ;Probiotic;Ammonia;Villus histology;Light microscope;Electron microscope1.IntroductionIn the poultry industry,antibiotics are in wide-spread use to prevent poultry pathogens and dis-ease so as to improve meat and egg production.However,continued use of dietary antibiotics has resulted in common problems,such as the devel-opment of drug-resistant bacteria (Sorum and Sun-de,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 recom-mended as an effective alternative to antibiotics*Corresponding author.Tel.y fax:q 81-87-891-3053.E-mail address:yamauchi@ag.kagawa-u.ac.jp (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 is96M.Samanya,K.Yamauchi /Comparative Biochemistry and Physiology Part A 133(2002)95–104Table 1Composition of dried B.subtilis natto Nutrient Composition (%)Crude protein 52Crude fat 0.5Crude fiber 4.5Ash 6.5Water7Table 2Composition of the basal diet Ingredients and nutrients Composition (%)Ground corn q milo 64Soybean meal 19Fish meal 6Rice bran3Concentrate mixture a 8Crude protein 17Crude fat 3Crude fiber 6Ash 12.5Calcium 2.6Phosphorus 0.55ME (kcal y kg )2800Provided the following per kg of diet:vitamin A,8000IU;avitamin D ,1500IU;vitamin E,7.5mg;vitamin K ,1mg;33vitamin B ,1.05mg;vitamin B ,5mg;vitamin B ,3.75mg;126vitamin B ,0.0045mg;biotin,0.15mg;folic acid,0.375mg;12pantothenate,3mg;niacin,20mg;choline,3150mg;iodine,0.4mg;manganese,75mg;iron,180mg;and zinc,52.5mg.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.2.Materials and methods2.1.Experiment 1:animals,housing and experi-mental designAdult male white leghorn chickens (Gallus gal-lus domesticus )(Julia strain )were fed dried B.subtilis natto (Table 1;Bacillus subtilis Natto Powder-710,Kitamura Co Ltd,Aichi,Japan )at ᭨0,0.5,1and 3%levels to the basal mash diet (Table 2;Nippon Formula Feed Manufacturing Co Ltd,Kanagawa,Japan ).The B.subtilis natto cul-ture contained approximately 1=10–1=10810microorganisms y g.Birds were placed into individ-ual cages in a controlled environment with a 14-h light photoperiod (06:00–20:00h )at a mean environmental temperature of 218C.Birds were given ad libitum access to water and each diet for 3or 28days.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 experi-ment for the 3-day period,and weekly for the 28-day period.At the end of each experimental feeding period,blood was collected between 09:00and 11:00h 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 48C for 15min.Blood serum was then kept at y 208C until used.In the case of the 28-day feeding period,internal organs were col-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 y 208C 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 bloodVenous blood was used for ammonia analysis,and serum,separated from the blood by centrifu-gation,was used for glucose analysis.Ammonia and glucose were spectrophotometrically analyzed with commercial kits (Wako Pure Chemical Indus-tries,Tokyo,Japan ).2.4.Amylase and lipase in the intestinal contents The intestinal digesta samples were diluted 10-fold (w y v )in 2mM sodium phosphate buffer (pH 7.0)containing 6mM NaCl and homogenized for 1min,followed by sonication for 1min.The sample was then centrifuged at 19000=g for 20min at 48C.To prevent possible enzyme degra-97 M.Samanya,K.Yamauchi/Comparative Biochemistry and Physiology Part A133(2002)95–104dation,the supernatants from digesta were kept on ice throughout the preparation.Supernatants were stored at y208C 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 Maru-ko Seiyaku Co Ltd(Nagoya,Japan).When the amylase activity of samples was too high,2mM sodium phosphate buffer(pH7.0)containing6 mM sodium chloride was added to dilute the samples.2.5.Experiment2:animals,housing and experi-mental designAdult male white leghorn chickens were ran-domly divided into four groups of13birds each, as follows:dietary addition of dried B.subtilis natto to basal mash diet at was carried out at levels of0,0.2,0.5and1%.Birds were placed into individual cages in a controlled environment with a14-h light photoperiod(06:00–20:00h)at a mean temperature of158C.Birds were given ad libitum access to water and each diet for4weeks. 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 samplingAt the end of each experimental period,four birds from each group were randomly killed by decapitation under light anesthesia with diethyl ether.A mixture of3%glutaraldehyde and4% paraformaldehyde fixative solution in0.1M cacod-ylate buffer(pH7.4)was injected into the intes-tinal 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 diver-ticulum to the ileo-cecal-colonic junction.The tissue samples were taken from the middle part of each intestinal segment.A2-cm length of duode-num was excised for scanning electron microscopic observations and placed in the same fixative solu-tion described above.Another2-cm length of each intestinal segment was excised for light microscop-ic observations and kept in Bouin’s solution.Light microscopic samples were taken immediately prox-imal to the section collected for scanning electron microscopic.2.7.Light microscopyThe fixed samples were embedded in paraplast. Transverse sections were cut into5-m m samples, and every10th section was collected.After stain-ing with hematoxylin–eosin,the following values were measured using a Nikon Cosmozone1S image analyzer(Nikon).2.7.1.Measurement of villus heightFor villus height measurement,10villi 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 these10villi 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 areaFor measurement of a single cell area on a5-m m 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 different98M.Samanya,K.Yamauchi/Comparative Biochemistry and Physiology Part A133(2002)95–104Table3Feed intake,body weight gain and feed efficiency in chickens fed dietary dried B.subtilis natto at0,0.5,1and3%levels for3(n s 5)and28days(n s7)(mean"S.E.)Parameter Level of B.subtilis natto0%0.5%1%3%Fed for3daysFeed intake(g)177.5"15.41174.3"7.35187.3"13.25171.4"8.21 Body weight gain(g)23.0"4.6323.0"4.3523.8"3.1420.0"4.47 Feed efficiency0.12"0.0220.13"0.0220.13"0.0150.12"0.009 Fed for28daysFeed intake(g)1908.7"36.71944.1"47.61903.4"491929.4"45 Body weight gain(g)150.0"13.78163.3"18.6148.6"12.0164.3"12.5 Feed efficiency0.073"0.0070.083"0.0080.075"0.0050.085"0.006sections 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 microscopyThe samples were longitudinally slit at the non-mesenteric side along the entire length,and intes-tinal contents were washed away with0.01M phosphate-buffered saline,pH7.4.Tissue samples were pinned flat to prevent curling within the same fixative used above at room temperature for1h.A block was cut into a4=7-mm squares and2fixed for a further1h.The pieces were rinsed with10mM sodium cacodylate buffer(pH7.4) and post-fixed with1%osmium tetroxide in ice-cold buffer for2h.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 exam-ined with a scanning electron microscope(Hitachi S-800,Japan)at8kV.Morphological alterations of the villus tip surface were compared among groups.2.9.Statistical analysisThe average of all parameters for each bird from each treatment group was analyzed across all treatment groups by ANOV A analysis with a Dun-can’s multiple range test(S TAT V IEW program; Abacus Concepts Inc).Differences at P-0.05 were considered as significant.3.Results3.1.Experiment1:growth performanceTable3shows feed intake and body weight gain of chickens fed dietary B.subtilis natto at0,0.5, 1and3%levels for3or28days.Growth perform-ance was not different amongst the groups,except that feed efficiency in all chickens fed dietary B. subtilis natto for28days tended to be improved.3.2.Gross anatomical observations of internal organsWet weight of gizzard,pancreas,liver,heart, kidney,each intestinal part and ceca was not different among groups of chickens fed dietary B. subtilis natto at0,0.5,1and3%levels for28 days(Table4).3.3.Determination of ammonia and glucose in bloodTable5shows ammonia and glucose concentra-tions in blood of chickens fed dietary B.subtilis natto at0,0.5,1and3%levels for3or28days. In the case of the3-day dietary period,ammonia concentration in the blood tended to be depressed in all groups fed B.subtilis natto compared to concentrations found in the0%group;even the ammonia level in the0.5%B.subtilis natto-fed group was lower than the0%group(P-0.05). The concentration of glucose did not differ from one group to another.In the case of the28-day dietary period,the ammonia concentration in blood was depressed in all dietary B.subtilis natto groups compared to levels in the0%group(P-0.05).99M.Samanya,K.Yamauchi /Comparative Biochemistry and Physiology Part A 133(2002)95–104Table 4Wet weight of internal organs in chickens fed dietary dried B.subtilis natto at 0,0.5,1and 3%levels for 28days (mean "S.E.;n s 7)Internal organLevel of B.subtilis natto 0%0.5%1%3%Gizzard (g )24.19"1.0226.78"0.9828.60"1.6425.20"0.75Pancreas (g ) 2.54"0.17 2.67"0.06 2.84"0.22 2.51"0.12Liver (g )27.24"1.3026.73"0.9229.97"1.8227.51"0.65Heart (g )11.59"0.3711.30"0.4012.93"1.2310.90"0.22Kidney (g ) 3.16"0.36 3.37"0.26 3.82"0.56 3.48"0.17Duodenum (g )7.23"0.397.84"0.457.80"0.647.29"0.31Jejunum (g )12.20"0.7913.83"0.4214.69"1.0313.10"0.50Ileum (g )7.36"0.437.87"0.479.09"0.777.51"0.30Ceca (g )4.00"0.143.94"0.274.74"0.493.61"0.17Table 5Ammonia and glucose concentration in the blood (n s 5),and amylase and lipase activity in the intestinal content (n s 7)of chickens fed dietary dried B.subtilis natto at 0,0.5,1and 3%levels for 3and 28days (mean "S.E.)ParameterLevel of B.subtilis natto 0%0.5%1%3%Fed for 3daysAmmonia (m g y dl )401.7"9.2a 345.1"5.4b 362.9"21.3ab 359.7"12.2ab Glucose (mg y dl )213.2"5.2217.2"7.2221.7"2.8212.8"2.7Fed for 28daysAmmonia (m g y dl )424.5"16a 368.1"3b 341.6"16.1b 347.7"7.3b Glucose (mg y dl )218.9"3.6217.5"7.5218.5"5.6228.4"5.1Amylase (IU )353.6"17.6375.4"42363.9"29.9388.2"18.5Lipase (IU )99.1"21.8108.4"24.299.1"19.2115.4"25.9Means within each grouping with different letter designations differ (P -0.05).a bThe concentration of glucose in serum was not altered in any group.3.4.Determination of amylase and lipase in intes-tinal digestaAmylase and lipase activity in the intestinal digesta was not significantly different among groups fed dietary B.subtilis natto at 0,0.5,1and 3%levels for 28days (Table 5).3.5.Experiment 2:growth performanceTable 6shows feed intake,body weight gain and feed efficiency in chickens fed 0,0.2,0.5and 1%dietary B.subtilis natto for 28days.Growth performance tended to be higher in the 0.2and 0.5%groups than the 0%group.3.6.Intestinal villus height,cell area and cell mitosisFig.1shows intestinal villus height,cell area and cell mitosis in each intestinal segment in chickens fed 0,0.2,0.5and 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%pared 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 0.2and 0.5%groups,the jejunum in all groups,and the ileum in the 0.5and 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.100M.Samanya,K.Yamauchi /Comparative Biochemistry and Physiology Part A 133(2002)95–104Table6Feed intake,body weight gain and feed efficiency in chickens fed dietary dried B.subtilis natto at 0,0.2,0.5and 1%levels for 28days (mean "S.E.;n s 13)ParameterLevel of B.subtilis natto 0%0.2%0.5%1%Feed intake (g )2190"73.792208"44.972279"113.92279"113.9Body weight gain (g )144.3"13.57164.7"12.94174.6"18.72148.3"13.08Feed efficiency0.066"0.0060.075"0.0060.075"0.0070.067"0.005Fig.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.;n s 4).All parameters tended to be activated in all dietary groups.Means within ab each grouping with different letter designations differ (P -0.05).3.7.Villus tip surfaceFig.2illustrates the duodenal villus tip surface of chickens fed dietary B.subtilis natto at (a )0,(b )0.2,(c )0.5and (d )1%levels.The flat cell outline in the 0%group (arrows in a )developed cell protuberances (arrows in b ),and large pro-truded 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 ).4.DiscussionThe 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 produc-tion (Jin et al.,1997,2000).The histological changes in chicken intestines reported herein pro-vide new information regarding the potential for using probiotics in chicken feed.101M.Samanya,K.Yamauchi /Comparative Biochemistry and Physiology Part A 133(2002)95–104Fig.2.Duodenal villus tip surface of chickens fed dietary dried B.subtilis natto at (a )0,(b )0.2,(c )0.5and (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 (23m m )is common to all pictures (300=).In experiment 1,blood ammonia concentration was depressed in chickens fed 0.5%dietary B.subtilis natto for 3days,and in all dietary groups for 28days.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 lev-els 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 were102M.Samanya,K.Yamauchi/Comparative Biochemistry and Physiology Part A133(2002)95–104observed in experiment 2.We think that thedepressed blood ammonia concentration may sig-nificantly relate to a decrease in ammonia concen-tration in the intestine after feeding a probiotic.Reduced blood ammonia concentrations indicatedincreased fixation of ammonia in the intestinallumen of pigs fed probiotics(Scheuermann,1993).Yeo and Kim(1997)reported that ammonia pro-duced in the intestine could enter the blood stream.Decreased ammonia concentration in small intes-tinal content was found in pigs fed Bacillus cereus(Kirchgessner et al.,1993)and in ceca of chicken fed probiotics(Endo and Nakano,1999;Endo etal.,1999).Ammonia is known to be toxic to villushistology,and reduces gastric mucosal DNA syn-thesis(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 epithe-lial cells originating in the crypt migrate along the villus surface upward to the villus tip,and are extruded into the intestinal lumen within48to96 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 corre-sponds 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 sur-face 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 isactivated(Langhout et al.,1999;Yasar and Forbes,1999;Shamoto and Yamauchi,2000).These factssuggest that the villus function might be activatedafter feeding dietary B.subtilis natto.Activated cell mitosis under the light micro-scope corresponds with large protruded cell clus-ters and cell protuberances on the villus tip surfaceunder the depressed ammonia condition of chick-ens fed dietary B.subtilis natto.Cell clusters andprotuberances appear to have occurred due toactivation of the cell mitosis rate more so than thecell extrusion rate;this has resulted in an accu-mulation of cells on the villus tip.Furthermore,although ammonia was closely associated withmucosal histological damage,lesions and loss(Linand Visek,1991;Warzecha et al.,2000),celldamage was not found on the villus surface.Thisalso suggests that cell accumulation on the villustip showing no damage or loss was due to thedepression of ammonia production.These cellprotuberances have been morphologically demon-strated as displaying activated function in theepithelial cells(Shamoto and Yamauchi,2000;Tarachai and Yamauchi,2000;Samanya andYamauchi,2001),which suggests that the functionof absorptive epithelial cells might be activated inthe depressed ammonia condition of chickens feddietary B.subtilis natto.After being fed dietary B.subtilis,an increasein body weight gain and improved feed efficiencyhave been reported in heavy meat-type poultry,such as turkeys(Jiraphocakul et al.,1990)andbroiler chicks(Santoso et al.,1995,2001).Yeoand Kim(1997)reported that daily body weightgain was increased only in the early life stage ofbroiler chicks,but was not different during thesecond3-week period after feeding the dietaryprobiotics.In addition,in laying pullets(Nahashonet al.,1994)and young layer pullets(Nahashon etal.,1996)fed Lactobacillus,these diets improvedbody 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 beobtained,although feed efficiency tended to beslightly elevated in the0.2and0.5%groups.Thereason for this ineffectiveness of B.subtilis nattomay be attributed to the fact that adult male layerswere used,because these birds do not require asmuch metabolic energy.However,slightly103 M.Samanya,K.Yamauchi/Comparative Biochemistry and Physiology Part A133(2002)95–104improved feed efficiency in the0.2and0.5% 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 ammo-nia concentration in blood,resulting in an activa-tion of intestinal function.This suggests that B. subtilis natto has potential to be a useful micro-organism in chickens.ReferencesAndremont,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 IFO3336 producing poly-gamma-glutamate.J.Biochem.123, 1156–1163.Burgat,V.,1991.Residues of drugs of veterinary use in food. 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