Abstract
Non-starch-polysaccharide(NSP)-hydrolyzing enzymes are commonly used in broiler fattening. The
possible mode of action of soluble non-starch-polysaccharides on growth depressing effects and the
effect of NSP-hydrolyzing enzymes has been investigated for a long time. However, there is still a
lack of knowledge concerning the role of intestinal microbial communities, which is often pointed
out even though its role is not known in detail yet.
The aim of this thesis was to contribute to a better knowledge and understanding of microbial com-munities
in the gastrointestinal tract of broiler chickens and their changes due to different diets.
A feeding trial was carried out to provoke changes in intestinal microbial communities by modifica-tion
of the NSP content in different diets and the use of a xylanase preparation. Birds were fed ei-ther
a maize based diet with low content of soluble NSP or diets based on wheat and rye with high
contents of soluble NSP. One of the wheat and rye based diets was supplemented with a xylanase.
Tallow was used as fat source in all diets.
Performance of birds was recorded and animals were killed on day 7, 14, 21, and 28. Digesta were
sampled from the duodenum, jejunum, upper and lower part of the ileum and caeca. Samples from
the duodenum, jejunum, upper and lower ileum from day 14 to day 28 were used for further exami-nations.
Supernatants of digesta samples were collected by centrifugation and used for agar diffu-sion
assays to examine selected enzyme activities. For molecular studies digoxygenin labeled oli-gonucleotide
probes were tested for specific and sensitive results in hybridization experiments and
relations between optical density and amount of used RNA were examined. Only oligonucleotide
probes with reproducible specific results were used for further examinations. Oligonucleotide
probes were taken from literature references or developed at the Institute of Animal Nutrition, Free
University Berlin. Nucleid acids were extracted from digesta samples using a modified phe-nol:
chloroform protocol and raw extractions were divided in RNA and DNA and purified using
silica gel columns. The amount of ribosomal RNA of selected bacterial groups and species was de-termined
by hybridization of samples with specific 16S/23S oligonucleotide probes and the
achieved optical density was converted in specific RNA in ng per ýg total RNA. For statistical in-terpretation,
mean values and standard deviations of triplicate samples were calculated. Statistical
analysis was performed by using a data pool based on the total trial period.
The performance of the birds showed antinutritive effects of soluble NSP in broiler diets, which
have been well documented in literature.
Xylanase and β -glucanase activities in digesta supernatants were determined by agar diffusion as-says.
The assays showed that an increasing content of NSP in broiler diets promotes the activity of
those bacteria which are able to hydrolyze β -glucans. Partial hydrolysis of NSP by xylanase sup-plementation
led to a further increase. An influence of soluble NSP and/or xylanase supplementa-Non-starch-polysaccharide(NSP)-hydrolyzing enzymes are commonly used in broiler fattening. The
possible mode of action of soluble non-starch-polysaccharides on growth depressing effects and the
effect of NSP-hydrolyzing enzymes has been investigated for a long time. However, there is still a
lack of knowledge concerning the role of intestinal microbial communities, which is often pointed
out even though its role is not known in detail yet.
The aim of this thesis was to contribute to a better knowledge and understanding of microbial com-munities
in the gastrointestinal tract of broiler chickens and their changes due to different diets.
A feeding trial was carried out to provoke changes in intestinal microbial communities by modifica-tion
of the NSP content in different diets and the use of a xylanase preparation. Birds were fed ei-ther
a maize based diet with low content of soluble NSP or diets based on wheat and rye with high
contents of soluble NSP. One of the wheat and rye based diets was supplemented with a xylanase.
Tallow was used as fat source in all diets.
Performance of birds was recorded and animals were killed on day 7, 14, 21, and 28. Digesta were
sampled from the duodenum, jejunum, upper and lower part of the ileum and caeca. Samples from
the duodenum, jejunum, upper and lower ileum from day 14 to day 28 were used for further exami-nations.
Supernatants of digesta samples were collected by centrifugation and used for agar diffu-sion
assays to examine selected enzyme activities. For molecular studies digoxygenin labeled oli-gonucleotide
probes were tested for specific and sensitive results in hybridization experiments and
relations between optical density and amount of used RNA were examined. Only oligonucleotide
probes with reproducible specific results were used for further examinations. Oligonucleotide
probes were taken from literature references or developed at the Institute of Animal Nutrition, Free
University Berlin. Nucleid acids were extracted from digesta samples using a modified phe-nol:
chloroform protocol and raw extractions were divided in RNA and DNA and purified using
silica gel columns. The amount of ribosomal RNA of selected bacterial groups and species was de-termined
by hybridization of samples with specific 16S/23S oligonucleotide probes and the
achieved optical density was converted in specific RNA in ng per ýg total RNA. For statistical in-terpretation,
mean values and standard deviations of triplicate samples were calculated. Statistical
analysis was performed by using a data pool based on the total trial period.
The performance of the birds showed antinutritive effects of soluble NSP in broiler diets, which
have been well documented in literature.
Xylanase and β -glucanase activities in digesta supernatants were determined by agar diffusion as-says.
The assays showed that an increasing content of NSP in broiler diets promotes the activity of
those bacteria which are able to hydrolyze β -glucans. Partial hydrolysis of NSP by xylanase sup-plementation
led to a further increase. An influence of soluble NSP and/or xylanase supplementa-
tion on xylan degrading microorganisms could not be shown. Suggestions to the negative effect of
NSP on digestibility of fat were also elaborated by agar diffusion assays. However, the obtained
results of lipase and bile salt hydrolase activity gave no evidence that a relation exists between the
activity of these enzymes and the well documented depression in fat digestibility when NSP are
added to broiler diets. Higher activities of lipases were expected in samples from birds fed the
maize based diet. However, higher activities were observed in animals fed the wheat and rye based
diets. This could indicate regulatory processes to compensate inferior fat digestion. Higher activities
of lipases in the duodenum of the enzyme supplemented group in comparison to the group with
wheat and rye diet indicate a better antiperistaltic movement of digesta. Higher activities of bacte-rial
bile salt hydrolases due to high intestinal viscosity as described in literature could not be ob-served.
Microbial communities of the digestive tract of broilers were characterized by hybridization of
RNA from digesta samples with specific oligonucleotide probes on the level of bacterial groups and
species.
Hybridization results of oligonucleotide probes have shown that specific hybridization parameters
could be achieved for all of the group specific oligonucleotide probes. According to these results the
oligonucleotide probes S-D-Bact-0338-a-A-18 (total bacterial 16S rRNA), S-G-Bact-0303-a-A-17
(Bacteroides-Pretovella-cluster), S-*-Chis-0150-a-A-23 (Clostridium histolyticum-group), 16E1
(E.coli and Shigella spp.), S-G-Enc-038-a-A-18 (Enterococcus spp.), S-F-Lact-0770-a-A-24 (Lac-tobacillus
spp.) and S-G-Bif-1432-a-A-21 (Bifidobacterium spp.) were used to screen RNA extracts
of digesta samples. However, only S-S-Ecaec-0181-a-A-24 (E.caecorum), S-S-Efaes-1237-b-A-17,
S-S-Efeas-203-a-A-20 (E.faecalis and E.faecium), and S-S-casflaga-0185-a-A-21 (comprising
E.casseliflavus, E.flavescens, E.gallinarum, but specific only for E.gallinarum) that were developed
at the Institute of Animal Nutrition, Free University Berlin, have shown specific results on species
level. Sequence candidates tested for their applicability as oligonucleotide probes to detect E.asini,
E.avium/E.raffinosus, E.columbae, E.faecium and E.hirae could not be used to screen RNA extracts
due to their lack of specificity. 23S rRNA oligonucleotide probes taken from literature references to
detect E.faecium (DB6), E.avium, E.malodoratus, E.pseudoavium and E.raffinosus (DB9), E.durans
and E.hirae (Eduhi9b), and E.gallinarum (Ega9b) have shown no signals with the tested conditions.
S-S-Lacet-0061-a-A-25 (L.acetolerans), S-S-Lacid-2519-a-A-20 (L.acidophilus), S-S-Lamy-0499-
a-A-24 (L.amylophilus), S-S-Lfer-061-a-A-26 (L.fermentum), S-S-Lgas-0054-a-A-24 (L.gasseri)
and S-S-Lreu-0485-a-A-23 (L.reuteri) had been used successfully in earlier experiments at the Insti-tute
of Animal Nutrition, Free University Berlin.
Most of the probes presented a linear relationship between chemiluminescence and amount of RNA.
This could be used to calculate the amount of specific RNA in the samples per ýg total RNA.
In hybridization experiments using RNA extracts from digesta samples S-S-Lfer-061-a-A-26
showed an exponential relationship between amount of RNA and intensity of signals. No relation-ship
was observed for the enterobacterial probe 16E1.
The lack of information about the content of ribosomal RNA of single bacterial cells made it diffi-cult
to compare results of hybridization experiments with data from cultivation methods published
in literature.
Diets with different contents of soluble NSP and supplementation of a wheat and rye based diet led
to different amounts of ribosomal RNA of selected bacterial groups and species. In comparison to
the maize fed broilers total bacterial metabolic activities and metabolic activities of enterobacteria
and enterococci were shifted distal parts of small intestine in birds fed the wheat and rye based diet.
Furthermore, metabolic activities of lactobacilli decreased. Xylanase supplementation led to lower
metabolic activities of enterobacteria and higher activities of lactobacilli than in the unsupple-mented
group with wheat and rye based diet. The shift of total bacterial activities to the lower parts
of the intestine was less pronounced. Hybridization of selected species of enterococci and lactoba-cilli
showed qualitative modifications within bacterial groups according to different diets.
Further examinations are necessary to evaluate and enumerate the microbial diversity in the intes-tine of broilers. |
