Medycyna Wet. 2007, 63 (3) 290
Praca oryginalna Original paper
Fructooligosaccharides (FOS) reportedly can be substituted for subtherapeutic levels of antibiotics (banned in EU) to enhance the growth and production efficiency of poultry (2, 8). FOS are short-chain fruc-tan-type oligosaccharides with D-fructose attached by b(2®1) linkages that are attached to a D-glucosyl residue at the end of the chain (18). Carbohydrates, which are not digested in the small intestine, reach the large intestine where they can be partly or totally degraded by the microflora (3). These compounds usually have a high bulking effect, thus reducing a risk of constipation (6). Bulking index (an increase in faecal wet weight in gram per gram of added indi-gestible carbohydrate) for oligofructose and inulin is estimated at 1.1-1.2 (13). It has been reported that an elevated level of FOS supplementation (more than 10 g/kg) in diets for poultry could be associated with a risk of diarrhoea occurrence (17). In earlier study on rats that the occurrence of diarrhoea was found to significantly deteriorate the functioning of the caecal ecosystem after feeding with a lactulose-rich diet (9).
During a long-term experiment on turkeys was obser-ved that some birds (ca. one fifths of the group fed with a diet containing 1% of fructooligosaccharides) had symptoms of diarrhoea. Those slight symptoms, chiefly more watery faeces, were not of infectious ori-gin and should be connected with the intake of large amounts of fructooligosaccharides.
The purpose of this study was to estimate differen-ces in caeca development, metabolism and activity of microbial enzymes between turkeys fed FOS-diet with or without diarrhoea symptoms.
Material and methods
The main experiment with dietary fructooligosacchari-des was conducted on 3-day-old BUT-9 (Hatchery Grelavi Co., Kêtrzyn, Poland) male turkey poults, randomly assigned to dietary treatments, each of which had 3 pens of 20 birds per pen. The poults were raised in floor bedding pens and given 16 : 8 hours light : dark per day. The rations composition was changed every four weeks (tab. 1) according to the requirements of the birds (NRC, 1994).
Occurrence of mild-diarrhoea in turkeys fed
a FOS-rich diet and its impact on caecal parameters
JERZY JUKIEWICZ, ZENON ZDUÑCZYK, JAN JANKOWSKI*, MAREK JUKIEWICZ**
Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland *Department of Poultry Science, **Department of Diary Science and Quality Management, University of Warmia and Mazury,
Oczapowskiego 5, 10-718 Olsztyn, Poland
Jukiewicz J., Zduñczyk Z., Jankowski J., Jukiewicz M.
Occurrence of mild-diarrhoea in turkeys fed a FOS-rich diet and its impact on caecal parameters Summary
The aim of the study was to estimate the tendency and scope of changes in caecal development and meta-bolism when affected by a fructooligosaccharide-rich diet evoking diarrhoea. In the present study one fifth of a group of commercial male turkeys fed for 16 weeks with a diet containing 1% of fructooligosaccharides had mild symptoms of diarrhoea. Because the main activity of fructooligosaccharides is in the lower part of the gastrointestinal tract, attention was paid to the amount and composition (ammonia, dry matter, protein, short--chain fatty acids and pH value) of caecal digesta and the activity of microbial enzymes. The occurrence of mild diarrhoea, observed in the study, did not influence the productivity of the birds nor did it alter the relative weight of liver, pancreas, stomach, small intestine and colon. Diarrhoea significantly influenced the caecal metabolism of the birds by causing an enlargement of the caeca (digesta and wall), and the caecal digesta/ tissue index was almost twice as high in the case of these turkeys. Dry matter of the ileal and caecal digesta was significantly decreased in the group with diarrhoea. Diarrhoea lowered concentrations of Lowrys protein and short-chain fatty acids in the caeca, and the activity of bacterial b-glucuronidase, a- and b-galactosidase, a- and b-glucosidase in caecal digesta, compared to turkeys without diarrhoea. The ammonia production in the caeca was enhanced by diarrhoea symptoms. It seems that the 1% FOS included in the turkeys feed mixture poses a risk of diarrhoea occurring, which in turn might limit potential benefits of diet supplementa-tion with fructooligosaccharides.
Medycyna Wet. 2007, 63 (3) 291
During the experiment we observed that some turkeys fed 1% of fructooligosaccharides had more watery faeces than the rest of the group. At the age of 7 weeks, the birds were kept on the bedding in individual metabolic cages, and at that time the turkeys with diarrhoea symptoms were marked.
All procedures were approved by the University of Warmia and Mazury Institutional Animal Care and Use Committee. After 16 weeks of feeding, the birds were weighed, and then those with diarrhoea symptoms (10 toms) and 10 toms with body weights close to the group average were taken from the group and killed according to the re-commendations for euthanasia of experimental animals (4). The main parts of their digestive tract (stomach, small inte-stine, caeca and colon), liver and pancreas were removed and weighed. Caecal (ileal) pH was measured using a mi-croelectrode and a pH/ION meter (model 301, Hanna In-struments). Samples of ileal (from the middle part of ileum) and caecal contents were transferred to microfuge tubes which were immediately stored at 70°C. The caecum wall was flushed clean with ice-cold saline, blotted on filter paper and weighed (caecal wall weight). The same was done with the stomach, small intestine and colon tissues. Calcu-lated caecal digesta/tissue index means how much grams of digesta coincided with 1 g of tissue. Dry matter of the caecal and ileal digesta was determined at 105°C. Ammo-nia extracted and trapped in a solution of boric acid was determined by direct titration with sulfuric acid (according to the standard Conways method). Protein content in the caecal digesta was determined by a colorimetric method using the Folin-Ciocalteu phenol reagent and BSA as the standard (11). Caecal content was analysed for SCFA con-centration with gas chromatography (Shimadzu GC-14A with a glass column 2.5 m × 2.6 mm, containing 10% SP-1200/1% H3PO4 on 80/100 Chromosorb W AW, column temperature 110°C, detector FID temperature 180°C, injector temperature 195°C). The caecal digesta were weighed (a sample of about 0.2 g), mixed with 0.2 mL of formic acid, diluted with deionised water and centrifuged at 10 000 × g for 5 min. Supernatant was decanted for injection into the gas chromatograph.
Glycolytic activity in the caecal (ileal) digesta was measured by the rate of release of p- or o- nitrophenol from their nitrophenylglucosides according to the modified method of Djouzi and Andrieux described by Jukiewicz et al. (10). The following substrates were used: for b-glu-curonidase: p-nitrophenyl-b-D-glucuronide, for a-galacto-sidase: p-nitrophenyl-a-D-galactopyranoside, for b-galac-tosidase: o-nitrophenyl-b-D-galactopyranoside, for a-glu-cosidase: p-nitrophenyl-a-D-glucopyranoside, and for b-glucosidase: p-nitrophenyl-b-D-glucopyranoside. The reaction mixture contained 0.3 mL of substrate solution (5 mM) and 0.2 mL of a 1 : 10 (v/v) dilution of the caecal sample in 100 mM phosphate buffer (pH 7.0) after centri-fugation at 10 000 × g for 15 minutes. Incubation was carried out at 37°C and p-nitrophenol was quantified at 400 nm and at 420 nm (o-nitrophenol concentration) after the addition of 2.5 mL of 0.25 M cold sodium carbonate. Enzymatic activity (a- and b-glucosidase, a- and b-galac-tosidase, and b-glucuronidase) was expressed as µmol
pro-duct formed per min (IU) per g digesta) in the caecal (ileal) sample.
The results of the experiment were analysed using the one-way ANOVA test, and significant differences between groups were determined by the Duncans multiple range test. Differences were considered significant at p £ 0.05.
Results and discussion
One fifths of the turkeys fed dietary 1% fructooligo-saccharides had slight indications of diarrhoea. The occurrence of diarrhoea might be connected with a high amount of dietary FOS used in that study as well as ontogenetic predispositions of some birds. The appearance of that ailment had no negative effect on
s t n e n o p m o C Feedingpeirod,weeks 4 -1 5-8 9-12 13-16 e t a rt n e c n o c l a s a B t a e h W 24.56 28.13 21.00 40.54 n r o C 20.00 20.00 31.78 25.00 l a e m n a e b y o S 42.00 41.00 34.00 26.00 l a e m h s i F 33.00 33.00 33.00 l a e m t a e M 35.00 li o n a e b y o S 31.90 3.50 5.40 4.50 * x i m l a r e n i m d n a n i m a ti V 31.00 1.00 1.00 1.00 l C a N 30.13 0.25 0.28 0.25 e n o t s e m i L 30.62 0.80 0.95 1.10 e t a h p s o h p m u i c l a c o n o M 32.03 2.60 2.70 1.90 ) % 9 9 ( e n i n o i h t e m -L D 30.30 0.27 0.27 0.12 ) % 9 9 ( l C H e n i s y l-L 30.37 0.40 0.31 0.39 e n i n o e r h t-L 30.09 0.05 0.11 0.02 : n o it i s o p m o c l a c i m e h c d e t a l u c l a C g k /l a c k , E M 2725 2797 2981 3004 % , n i e t o r p e d u r C 28.771 25.931 22.921 18.781 % , e r b if e d u r C 3.40 3.67 3.08 3.00 % ,t a f e d u r C 4.40 5.51 7.64 6.56 % , s y L 1.80 1.65 1.40 1.20 % , s y C + t e M 1.16 1.06 10.978 0.73 % , a C 1.30 1.14 1.19 1.01 % , e l b a li a v a P 0.72 0.69 0.67 0.50 % , a N 0.15 0.15 0.16 0.14 % ,l C 0.21 0.27 0.28 0.23
Tab. 1. Composition (%) and nutritive value of basal diet
Explanation: for the 1-4, 5-8 and 9-16 weeks of feeding the vita-min and vita-mineral premix supplied per kg of diet: vitavita-min A, 15000, 13000 and 12000 IU; vitamin E, 40, 35 and 30 mg, respectively. For the 1-16 weeks of feeding the vitamin and mineral premix supplied per kg of diet: Se, 0.3 mg; Mn, 150 mg; Zn, 90 mg; Fe, 60 mg; Cu, 15 mg; I, 1 mg; Diclazuril, 1 mg; vitamin D3, 4500 IU;
vitamin K3, 2.5 mg; vitamin B1, 3.5 mg; vitamin B2, 10 mg;
vita-min B6, 6 mg; vitamin B12, 0.03 mg; folic acid, 2 mg; biotin,
Medycyna Wet. 2007, 63 (3) 292
bird growth nor the relative weight of the liver, pancreas, small intestine or colonic tissue (tab. 2). The pH value of ileal digesta was unaffected while dry matter concentration was significantly decreased by the occurrence of diarrhoea. The SCFA were not iden-tified in the ileal digesta and the activity of bacterial enzymes in the ileum was rather low and not influen-ced by diarrhoea.
In the FOS-fed turkeys with diarrhoea manifesta-tion, the weight of caecal tissue and digesta was sig-nificantly higher compared to the birds without diar-rhoea (tab. 2). The caecal digesta/tissue index was two times higher in the group with diarrhoea and it sugge-sted that the caecal wall got thinner and weaker in that case. When a higher amount of fermentable oligosac-charides was used in animal feeding, the increased caecal weights (full and empty) were considered a phy-siological response of no toxicological significance (1) and often associated with some beneficial changes in the blind gut. In the case of a diarrhoea group, caecal wall enlargement may be a simple adaptive response to an increased bulk of contents. The excessive enlar-gement of caecal digesta observed in that group may cause hurtful and uncomfortable feelings to the host.
It is well known that the intake of a large amount of non-digestible oligosaccharides causes diarrhoea (9, 14). On the other hand some oligosaccharides are used as prebiotics and have been examined in animal and human trials for their effectiveness in the prevention and treatment of several types of diarrhoea, including antibiotic-associated diarrhoea, bacterial and viral diar-rhoea, as well as that caused by nutrition intolerance (5, 7). The final effect of oligosaccharides consump-tion strongly depends on their dose in the diet.
The caecal pH value was considerably increased in the turkeys with diarrhoea. Dry matter of caecal dige-sta was significantly decreased in the birds with diar-rhoea and consequently the stool consistency showed visible differences among the groups. The caecal pro-tein concentration was greater in turkeys without diar-rhoea. It suggested the lowering of caecal microflora population in birds with diarrhoea ailment compared with the group without diarrhoea. In the case of diar-rhoea turkeys produced more ammonia per caeca. The production of ammonia is closely related to bacterial breakdown of urea and undigested protein as well as to certain toxic events in the gastrointestinal tract, hence ammonia is considered to be a potential cancer pro-moter in the hindgut (5). In the experiment conducted by Jukiewicz and Zduñczyk (9) the rats with lactulo-se-induced diarrhoea had a significantly higher caecal ammonia and lower Lowrys protein concentration in the caecum compared to the rats without diarrhoea occurrence. Not only the lower concentration of Lowrys protein but also significantly decreased activity of bac-terial enzymes reflected the detrimental effect of diar-rhoea on microflora population in the caeca (tab. 3).
In the turkeys with diarrhoea, the concentration of total and individual SCFA in the caeca was signifi-Tab. 2. Body weight and intestinal tract parameters of
turkeys fed diet containing 1.0% fructooligosaccharides with or without diarrhoea (n = 10; x ± SEM)
Explanation: values with different superscript letters differ signi-ficantly at p £ 0.05 r e t e m a r a P dWiatirrhhooueta diaWrrhtihoea SEM g k ,t h g i e w y d o B 13.81a 13.83a 0.137 W B g k / g ,t h g i e w r e v i L 12.21a 12.77a 0.296 W B g k / g ,t h g i e w s a e r c n a P 1.20a 1.14a 0.026 W B g k / g , e u s s it h c a m o t S 9.11a 8.94a 0.266 m c , h t g n e l e n it s e t n i ll a m S 303a 311a 2.860 W B g k / g , e u s s it e n it s e t n i ll a m S 12.92a 12.82a 0.244 H p 6.34a 6.35a 0.046 % ,r e tt a m y r d 17.86a 14.83b 0.723 s A F C S nodetected m c , h t g n e l a c e a C 88a 91a 1.034 W B g k / g , e u s s it a c e a C 2.97a 3.42b 0.077 W B g k / g , a t s e g i d l a c e a C 2.31a 4.63b 0.454 g / g , x e d n i e u s s it / a t s e g i d l a c e a C 0.777a 1.354b 0.035 H p 16.87a 17.00b 0.038 % ,r e tt a m y r d 17.99a 10.80b 1.047 g 0 0 1 / g m , a i n o m m a 110.6a 107.8a 2.614 g / g m , n i e t o r p 0.576a 0.326b 0.030 W B g k / g m , a i n o m m a 2.550a 4.990b 0.310 W B g k / g m , n i e t o r p 1.331a 1.509a 0.080 W B g k / g , e u s s it n o l o C 1.060a 1.090a 0.044
Tab. 3. Bacterial enzymes activity (U/g wet digesta) in ileal and caecal digesta of turkeys fed diet containing 1.0% fructo-oligosaccharides with or without diarrhoea (n = 10; x ± SEM)
Explanation: as in tab. 1. e m y z n E dWiatirrhhooueta diaWrrhtihoea SEM : a t s e g i d l a e lI a-glucosidase 1.09a 0.94a 0.04 b-glucosidase 0.07a 0.08a 10.008 a-galactosidase 0.49a 0.44a 0.07 b-galactosidase 0.35a 0.37a 0.03 b-glucuronidase 0.48a 0.51a 0.03 a t s e g i d l a c e a C a-glucosidase 8.33a 3.72b 0.49 b-glucosidase 2.61a 1.20b 0.19 a-galactosidase 35.1a 15.1b 2.61 b-galactosidase 48.7a 15.0b 4.35 b-glucuronidase 27.3a 10.4b 2.23
Medycyna Wet. 2007, 63 (3) 293 Explanation: as in tab. 1. r e t e m a r a P dWiatirrhhooueta diaWrrhtihoea SEM A F C S l a t o T 35.06a1 20.49b1 2.73 e t a t e c A 23.34a1 12.83b1 2.38 e t a n o i p o r P 3.47a 2.03b 0.21 e t a r y t u b o s I 0.64a 0.50b 0.03 e t a r y t u B 5.13a 3.32b 0.31 e t a r e l a v o s I 1.01a 0.70b 0.06 e t a r e l a V 1.47a 1.11b 0.08 e li f o r p 4 C : 3 C : 2 C ) A F C S l a t o t l o m µ 0 0 1 /l o m µ ( 67:10:15 63:10:16
Tab. 4. SCFAs concentration (µmol/g) in caecal digesta of turkeys fed diet containing 1.0% fructooligosaccharides with or without diarrhoea (n = 10; x ± SEM)
cantly lower than in the group without diarrhoea (tab. 4). Briefly, the end products of fermentation of carbo-hydrates by caecal and colonic bacteria are short--chain fatty acids, and they are thought to be essential in the maintenance of a healthy caeca and colon (16), hence the lower concentration of SCFA in turkeys with diarrhoea should also be considered as a detrimental effect on the caecal metabolism. In the present study, the composition of the major short-chain fatty acids, despite the differences in experimental groups with-out and with diarrhoea, was in agreement with the results of other authors (15) indicating that at all ages of birds acetic acid is predominating followed by butyric and propionic acid.
Conclusions
In this study, an individual sensibility of turkeys to feeding with 1% FOS-diet was observed. One fifths of birds fed fructooligosaccharides showed diarrhoea symptoms, however the intensity of that ailment was rather slight. The diarrhoea occurrence did not have any influence on the productivity of turkeys, but had a strong negative effect on the caecal metabolism. An increased pH range and a decrease in the value of the caecal digesta/tissue index, dry matter, protein and SCFA concentrations in the caecal digesta, as well as significant suppression in the activity of bacterial enzymes were found in turkeys with diarrhoea mani-festation.
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Authors address: Dr hab. Jerzy Jukiewicz, Institute of Animal Repro-duction and Food Research of Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland; e-mail: glebczo@pan.olsztyn.pl
