Impact of prebiotic substances on gut health of livestock animals [Elektronische Ressource] : inulin, lactulose and Pinus massoniana pollen / Sabine Masanetz

S.Masanetz

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 8 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Physiologie


Impact of prebiotic substances on gut health of livestock animals
Inulin, lactulose and Pinus massoniana pollen



Sabine Masanetz



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. Dr. H.-R. Fries
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Dr. H. H.D. Meyer
2. Univ.-Prof. Dr. D. R. Treutter
3. Priv.-Doz. Dr. M. W. Pfaffl

Die Dissertation wurde am 16.06.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 09.01.2011 angenommen.
CONTENT
CONTENT

ABBREVIATIONS IV
Names and abbreviations of genes investigated in the present study V
TABLES AND FIGURES VII
ABSTRACT VIII
ZUSAMMENFASSUNG X
INTRODUCTION 1
Calf rearing and feed additives 1
The intestinal microbiota in calves 2
Health promoting effects of prebiotic substances 5
AIMS OF THE STUDY 9
MATERIAL AND METHODS 11
in vitro investigation: effects of pine pollen extracts on porcine ileal cell culture 11
Pollen compound extraction 11
Determination of cell proliferation 11
Total RNA extraction and expression analysis 12
Pollen compound analysis 13
Statistical analysis 15
in vivo investigation: effects of inulin and lactulose on preruminant calves 16
Animals, husbandry, feeding and experimental procedures 16
Tissue sampling 16
Blood sampling 17
Histological analyses 18
Total RNA extraction and expression analysis 20
Statistical analysis 21
RESULTS 24
in vitro investigation: effects of pine pollen extracts on porcine ileal cell culture 24
Extraction yields 24
Cell proliferation 24
mRNA expression 28
Pollen compounds 28
in vivo investigation: effects of inulin and lactulose on preruminant calves 29
General animal performance 29
II CONTENT
Haematology 29
Histological analyses 31
RNA quality 33
Choice of reference gene pairs 33
mRNA expression changes of inflammation and immune modulating factors 33
mRNA expression changes of proliferation and apoptosis related genes 34
DISCUSSION 35
in vitro investigation: effects of pine pollen extracts on porcine ileal cell culture 35
Extraction yields 35
Cell proliferation 35
mRNA expression 36
Pollen compounds 37
in vivo investigation: effects of inulin and lactulose on preruminant calves 38
General animal performance 39
Haematology 39
Histological analyses 41
mRNA expression changes of inflammation and immune modulating factors 43
mRNA expression changes of proliferation and apoptosis related genes 45
CONCLUSION 47
REFERENCES 50
ACKNOWLEDGEMENTS 62
SCIENTIFIC COMMUNICATIONS 63
Publications 63
Presentations 63
Posters 64
CURRICULUM VITAE 65
APPENDIX 66

III ABBREVIATIONS
ABBREVIATIONS

AB-PAS Alcian blue-periodic acid staining
ANOVA analysis of variance
B. fragilis Bacteroides fragilis
B. thetaiotaomicron Bacteroides thetaiotaomicron
C. perfringens Clostridium perfringens
C cycle threshold T
DNA deoxyribonucleic acid
ECIS electronic cell impedance sensing
E. coli Escherichia coli
EDTA ethylendiamintetraacetic acid
E. faecium Enterococcus faecium
ESI electro spray ionization
FOS fructo-oligosaccharides
LC liquid chromatography
mRNA messenger RNA
MS mass spectrometry
m/z mass to charge ratio
n number
PBS phosphate buffered saline
PCR polymerase chain reaction
P. freudenreichii Propionibacterium freudenreichii
P. densiflora Pinus densiflora
P. massoniana Pinus massoniana
qRT-PCR quantitative RT-PCR
RIN RNA integrity number
RNA ribonucleic acid
RT reverse transcription
S. aureus Staphylococcus aureus
s.e.m. standard error of means
ToF time of flight


IV ABBREVIATIONS
Names and abbreviations of genes investigated in the present study
In the following all genes that were investigated in the present study are listed with
their full name, the abbreviation used in the text and tables. Additionally the
accession number of the sequence that was used for primer design is given.

Cell culture experiment (sus scrofa):
Caspase 3 (Casp3) NM_214131
Cyclin A2 (CCNA2)* Multi*
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) AF017079
Histone H3.3A (H3F3A) NM_213930
Interleukin 6 (IL6) NM_214399
Interleukin 8 (IL8) NM_213867
Transforming growth factor beta 1 (TGFB1) NM_214015
Ubiquitin B (UBB) NM_001105309

Feeding experiment (bos taurus):
antigen identified by monoclonal antibody Ki-67 (MKI67) XM_590872
actin beta (ACTB) NM_173979
BCL2-like 1 (BCL2L1) NM_001077486
BCL2-associated X protein (BAX) NM_173894
caspase 3 (CASP3) NM_001077840
CD4 molecule (CD4)* Multi*
CD8b molecule (CD8B) NM_001105344
CD69 molecule (CD69) NM_174014
epidermal growth factor receptor (EGFR) XM_592211
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) NM_001034034
G protein associated kinase (GAK) NM_001046084
interleukin 1 beta (IL1B) NM_174093
interleukin 2 receptor alpha (IL2RA) NM_174358
interleukin 8 (IL8) NM_173925
interleukin 10 (IL10) NM_174088.1
interferon gamma (IFNG) NM_174086
platelet/endothelial cell adhesion molecule (PECAM1) NM_174571
peptidase (mitochondrial processing) alpha (PMPCA) NM_001076964
V ABBREVIATIONS
ras-related protein Rab21 (RAB21) XM_001249323
receptor for Fc fragment of IgA (FCAR) NM_001012685
transforming growth factor beta 1 (TGFB1) NM_001166068
tumor necrosis factor (TNF superfamily, member 2) (TNF) NM_173966
ubiquitin (UBIQ) Z18245
vacuolar protein sorting 4 homolog A (VPS4A) NM_001046615

* In some cases mRNA sequences of the gene of interest were not available for the species used in
the study (sus scrofa or bos taurus). There primer design was performed on a consensus sequence
from different species.

VI TABLES AND FIGURES
TABLES AND FIGURES

Tables:
Table 1: Primer pairs and PCR product sizes for qRT-PCR in the cell culture
experiment
Table 2: Ingredients and analysis of nutrient and energy content of the diets
Table 3: Primer pairs, PCR product sizes and appropriate profiles for qRT-PCR
in the feeding experiment
Table 4: Numbers of LC-ESI-ToF-MS signals specific for pine pollen extracts
Table 5: Results of haematological examination of blood samples
Table 6: Results of histological analyses
Table 7: Changes in relative mRNA expression levels found in the in vivo study

Figures:
Figure 1: Chemical structure of inulin
Figure 2: Chemical structure of lactulose
Figure 3: Known effects of inulin on calf health and performance
Figure 4: Known effects of lactulose on calf health and performance
Figure 5: Total ion chromatograms of a Masson pine pollen and a control extract
Figure 6: m/z spectra of a Masson pine pollen and a control extract
Figure 7: Measurements on gut mucosal morphology in the intestine
Figure 8: Quantitative analysis of goblet cells
Figure 9: Masson pine pollen before and after breaking with a ball mill
Figure 10: Effects of water extract of pine pollen on cell proliferation
Figure 11: Effects of 50% ethanol extract of pine pollen on cell proliferation
Figure 12: Effects of 100% ethanol extract of pine pollen on cell proliferation
Figure 13: Effects of 80% methanol extract of pine pollen on cell proliferation
Figure 14: Effects of hexane extract of pine pollen on cell proliferation
Figure 15: Effects of 50% ethanol extract of pine pollen on mRNA expression
Figure 16: Current knowledge of inulin effects on calf health and performance
Figure 17: Current knowledge of lactulose effects on calf health and performance

VII ABSTRACT
ABSTRACT

Prebiotic substances such as inulin or lactulose have been propagated to improve
health of man and animal by stimulation of beneficial bacteria and their fermentation
products in the intestine. Consequently prebiotics have been suggested as
alternatives to antibiotics after the ban of antimicrobial growth promoters in food
producing animals.
So the current work was performed to investigate effects of feeding 2% of either
inulin or lactulose in milk replacer to male Holstein-Friesian calves. Therefore animal
performance and haematologic traits were investigated. Additionally intestinal
mucosa architecture and gut associated lymphoid tissue morphology were screened
by means of histological analyses. Furthermore the mRNA expression patterns of
selected genes involved in cell pr