1. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 1

2. WP3ECO-FCE PROTOCOL – IN OVO WORK TASK 3.3 Partner: The University of Technology and Life Sciences, Bydgoszcz Project Manager: Prof. Marek Bednarczyk 2

3. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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) 3

4. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Presentation scheme  Tasks & Objectives  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 4

5. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Tasks & Objectives ECO-FCE WP3-chickens Gut Structure, function, microbiota and metagenomics 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 3.3.2 Optimisation of synbiotic doses for chicken in ovo trial 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. 5

6. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Work schedule- estimated WP3 IN OVO DOSAGE OPTIMISATION T.3.3.2 IN OVO + ANIMAL MODEL MAIN EXPERIMENT T.3.3.3 In ovo- incubation, hatch analysis, bacteriology 10.2014- 02.201503.2015- 03.2015 In ovo & growth physiology bacteriology histology 6

7. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Main methodological aspects dosage optimisation-3.3.2. SYNBIOTICS DOSAGE OPTIMISATION Two synbiotics: 1) SYN 1 (Lb. plantarum IBB3036 + lupin RFOs) 2) SYN 2 (Lb. salivarius IBB3154 + Bi 2 tos, Clasado Ltd.) Chickens: Chicken genetics: Cobb500 FF 3 000 fertilised eggs n = 100 per repetition, 3 repetitions per treatment 5 doses SCHEDULE If possible: 13 EXPERIMENTAL TREATMENTS (2 synbiotics x 6 doses +control) Single Treatment= 100 embryos x 3 repetitions SYN1SYN2 10 3 - 10 4 - 10 5 bacteria cfu/egg * 2-5 mg prebiotic control: Ringer’s solution, 200 µl/embryo 7

8. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Main Experiment- T3.3.3. Group No. of repetitions No. of chickens (males) per pen SYN1875 SYN2875 CONTROL875 Technology for automatic in ovo pre/synbiotic administration Controlled environmental housing conditions Group housing in cages according to standards Experimental Farm of Warmia and Mazury University in Olsztyn  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 8

9. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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 9

10. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Sampling schedule Feacal samples In ovoHatchBW -1201 47 14 21 28 42 blood pancr transcript. LEGEND: performance bacteriology physiology histology genetics 10

11. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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) [Task 3.3.3] Bacteriological analysis 1. Quantitative assessment of the main groups of bacteria present in the gastrointestinal tract of chicken (e.g. Enterobacteriaceae, Enterococcus, Lactobacillus and the 2 probiotic bacteria Plate-test 2. Qualitative monitoring of probiotic bacteria and endogenous microbiota (identification of main groups of bacteria). PCR technique Detailed protocols: IBB Partner 11

12. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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 12

13. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Genetic Panel 13 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 21 st, 35 th day, by RT-qPCR, using: panel of eight target genes, that indicate the level of gene regulation in the tissues Microarray Gene 1.1 ST Array (Affymetrix) a panel of differentially expressed genes in chickens treated in ovo with synbiotics will be determined + validation study Adressed Q: Do SYN1 or SYN2 change the gene expression profile in organs involved in immune response?

14. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. Adressed Q: Do SYN1 or SYN2 have beneficial effect on the level of metabolic parameters? BW 14 CONSUMING ENERGY COLLECTING ENERGY Physiology Panel

15. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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 Corticosterone- glukoneogenesis/stress/feed conversion Leptin – feed intake/ fatness RIA i EIA tests on serum, cell lysate and whole blood 15

16. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 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 16 -Aminotransferases activity (Aspat; AlAT) – general health status measure (liver, heart muscle), nitrogen turnover, gluconeogenesis Colorimetric assays + UV

17. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. COLLECTING ENERGY CONSUMING ENERGY BW leptin insulin T3, T4, fT3,fT4 leptincorticosterone - apetite- Feed Convertion insulin 17 Hormones regulating FI and metabolism- to be tested in ECO FCE

18. This project has received funding from the European Union‘s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794. 18 www.meatingplace.com THANK YOU