Protocol In ovo work Task3.3( Investigation of timing and - ECO-FCE

Report
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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WP3 ECO-FCE PROTOCOL –
IN OVO WORK TASK 3.3
Partner: The University of Technology and Life
Sciences, Bydgoszcz
Project Manager: Prof. Marek Bednarczyk
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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UTP Team
• Team Leader: Prof. Marek Bednarczyk
• Manager Assistant: K.Stadnicka
• Leaders of Working Groups:
Prof. Gabriela Elminowska-Wenda (histology)
Prof. Maria Siwek (genetics)
Prof. Pawel Maćkowiak (Physiology, UP Poznań)
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
3
Tasks & Objectives
Presentation
scheme
Work Schedule
Main methodological aspects:
a. in ovo delivery of bioactives
b. Dosage optimisation Experiment
c. Main Experiment
d. Performance traits (UTP)
e. Bacteriology Panel (IBB)
f. Histology Panel (UTP)
g. Genetic Panel (UTP)
h. Physiology Panel (UP POZNAŃ)
Additional Qs
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
4
Tasks & Objectives
• ECO-FCE WP3-chickens
• Gut Structure, function,
microbiota and metagenomics
Task 3.3.2 Optimisation of synbiotic
doses for chicken in ovo trial
• PROTOCOL- IN OVO WORK
• Task 3.3: Investigation of timing
and delivery mechanism to
optimize gut microbial profile
and ensure persistence
• Partners involved:
• UTP, IBB, Poznan, Cobb
Task 3.3.3.Investigating the influence
of in ovo administered synbiotics on
chicken
performance
regarding
physiological
parameters,
meat
quality, host-microbiome interactions
and FCE.
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
5
Work schedule- estimated
IN OVO DOSAGE
OPTIMISATION
T.3.3.2
IN OVO + ANIMAL MODEL MAIN EXPERIMENT
T.3.3.3
WP3
10.2014- 02.2015
In ovoincubation,
hatch analysis,
bacteriology
03.2015- 03.2015
In ovo
&
growth
histology
bacteriology
physiology
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Main methodological aspects
dosage optimisation-3.3.2.
SYNBIOTICS DOSAGE OPTIMISATION
SCHEDULE
If possible: 13 EXPERIMENTAL TREATMENTS (2 synbiotics x
Two synbiotics:
1) SYN 1 (Lb. plantarum IBB3036
+ lupin RFOs)
2) SYN 2 (Lb. salivarius IBB3154 +
Bi2tos, Clasado Ltd.)
Chickens:
Chicken genetics: Cobb500 FF
3 000 fertilised eggs
n = 100 per repetition, 3 repetitions per
treatment
5 doses
6 doses +control)
Single Treatment= 100 embryos x 3 repetitions
SYN1
SYN2
•103 - 104 -105 bacteria cfu/egg * 2-5 mg prebiotic
•control: Ringer’s solution, 200 µl/embryo
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
7
Main Experiment- T3.3.3.
4 050 fertilised eggs
n = 75 per repetition,
8 additional repetitions, with n=10
chickens/pen will be subjected for
sampling as zootechnical batches,
 8 major repetitions will be assigned
to the performance experiment
(calculating FCR, total BW, total FI etc.)
The chickens will be group-housed
No. of
No. of
chickens
repetitions
(males) per
Group
pen
SYN1
8
75
SYN2
8
75
CONTROL
8
75
Controlled environmental housing conditions
Group housing in cages according to standards Experimental Farm
of Warmia and Mazury University in Olsztyn
Technology for automatic in ovo pre/synbiotic administration
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
8
Performance traits (UTP)
•Day-old chickens will be individually weighed and placed in groups of 75 chickens.
•Body weight of chickens will be recorded weekly (day 2 (arrival), 7, 14, 21, 28, 35, 42
of life) and on the respective slaughtering days.
•Feed amount provided and feed residuals will be recorded daily.
•FCE will be determined using residual feed intake (RFI) and residual feed intake and
residual growth (RIG) using SAS (version 9.3; SAS Inc., Cary, NC, USA). Data included in
regression analysis will be chicken weight and feed intake.
•Diet: recommended for Cobb500
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Sampling schedule
Feacal
samples
In ovo Hatch
-12
Feacal samples
BW
0
1 4
BW
7
BW
Feacal samples
BW
14
21
BW
28
42
blood blood blood
blood
blood
blood
pancr pancr pancr
pancr
pancr
transcript.
pancr
transcript.
LEGEND:
performance
bacteriology
physiology
histology
genetics
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Bacteriology Panel
Feacal sampling
• Fresh fecal samples in 1-3-6 week of growth will be obtained
randomly from each pen and processed within 1 hour after
defecation.
• One faecal sample per pen is a representative (group-housing)
Detailed protocols: IBB Partner
[Task 3.3.3] Bacteriological analysis
1. Quantitative assessment of the main groups of bacteria Plate-test
present in the gastrointestinal tract of chicken (e.g.
Enterobacteriaceae, Enterococcus, Lactobacillus and the
2 probiotic bacteria
2. Qualitative monitoring of probiotic bacteria and
PCR
endogenous microbiota (identification of main groups of
technique
bacteria).
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Histology Panel
Gut segments will be separated and measured (length, weight) individually
1.Duodenum: from pylorus to end of pancreatic loop
2.Jejunum: part between duodenum and ileum
a.Meckel’s diverticulum indicates the proximal (end) section of jejunum
[Smirnov et al., 2006; Uni et al., 2003].
3.Ileum: end of mesentery arteries to ileo-caecal junction
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
12
Genetic Panel
Adressed Q: Do SYN1 or SYN2 change the gene expression profile in organs involved in
immune response?
Gene expression
(Spleen, jejunum, caecal tonsils.)
RNA isolation & quality check
GeneMATRIX Universal RNA Purification Kit
Experion RNA StdSens Analysis Kit with
Experion Automated Electrophoresis System
Selection of the most informative time point
21st, 35th day, by RT-qPCR,
using:
Microarray
Gene 1.1 ST Array (Affymetrix)
panel of eight target genes, that indicate
the level of gene regulation in the tissues
a panel of differentially expressed genes in chickens
treated in ovo with synbiotics will be determined +
validation study
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
13
Physiology Panel
Adressed Q: Do SYN1 or SYN2 have beneficial effect on the level of metabolic parameters?
BW
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Physiology Panels (1-3)
Pancreatic enzymes activity
Frozen samples; homogenised
after thawing
• - Trypsin (protein)
• - Amylase (starch)
• - Lipase (dietary lipids)
• Colorimetric assays
Concentration of hormones
regulating the metabolism
and feed consumption
• Insulin (anabolism)
• thyroid hormones (T4; T3;
fT4; fT3) – metabolic
turnover/ catabolism
• Corticosteroneglukoneogenesis/stress/feed
conversion
• Leptin – feed intake/ fatness
• RIA i EIA tests on serum, cell
lysate and whole blood
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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Physiology Panel
Blood biochemical indices
static and enzymes
- Albumins – osmotic rate
• - Total protein– nutrition
•
•
•
accuracy/osmotic rate
- Glucose (carbohydrate
regulation accuracy)
Triglicerides (fatness measure,
health status)
- Free fatty acids (lipolysis
measure)
- Total/free cholesterol–
health status, meat quality
-Aminotransferases activity
(Aspat; AlAT) – general
health status measure (liver,
heart muscle), nitrogen
turnover, gluconeogenesis
Colorimetric assays + UV
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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- apetite
leptin
- Feed Convertion
corticosterone
insulin
leptin
BW
T3, T4, fT3,fT4
insulin
Hormones regulating FI and metabolism- to be tested in ECO FCE
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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www.meatingplace.com
THANK YOU
This project has received funding from the European Union‘s Seventh Framework Programme for
research, technological development and demonstration under grant agreement No. 311794.
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