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WP3 ECO-FCE Protocol – IN OVO WORK Task 3.3

WP3 ECO-FCE Protocol – IN OVO WORK Task 3.3. Partner: The University of Technology and Life Sciences, Bydgoszcz Project Manager: Prof. Marek Bednarczyk. UTP Team. Team Leader: Prof . Marek Bednarczyk Manager Assistant : K.Stadnicka Leaders of Working Groups :

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WP3 ECO-FCE Protocol – IN OVO WORK Task 3.3

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  1. WP3 ECO-FCE Protocol – IN OVO WORKTask 3.3 Partner: TheUniversity of Technology and Life Sciences, Bydgoszcz Project Manager: Prof. Marek Bednarczyk

  2. UTP Team • Team Leader: Prof. Marek Bednarczyk • Manager Assistant: K.Stadnicka • Leaders of WorkingGroups: Prof. Gabriela Elminowska-Wenda (histology) Prof. Maria Siwek (genetics) Prof. Pawel Maćkowiak (Physiology, UP Poznań)

  3. Presentationscheme • Tasks & Objectives • Work Schedule • Mainmethodologicalaspects: a. in ovo delivery of bioactives b. DosageoptimisationExperiment c. MainExperiment d. Performance traits (UTP) e. Bacteriology Panel (IBB) f. Histology Panel (UTP) g. Genetic Panel (UTP) h. Physiology Panel (UP POZNAŃ) • AdditionalQs

  4. Tasks & Objectives Task 3.3.2 Optimisation of synbiotic doses for chicken in ovo trial • 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 3.3.3.Investigating the influence of in ovoadministered synbiotics on chickenperformance regarding physiological parameters, meat quality, host-microbiome interactions and FCE.

  5. Workschedule- estimated IN OVO DOSAGE OPTIMISATION T.3.3.2 IN OVO + ANIMAL MODEL MAIN EXPERIMENT T.3.3.3 WP3 10.2014- 02.2015 03.2015- 03.2015 In ovo-incubation, hatchanalysis, bacteriology In ovo & growth histology bacteriology physiology

  6. Mainmethodologicalaspectsdosage optimisation-3.3.2. SYNBIOTICS DOSAGE OPTIMISATION SCHEDULE Two synbiotics: • SYN 1 (Lb. plantarum IBB3036 + lupin RFOs) • 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 • 103 -104-105 bacteriacfu/egg * 2-5 mg prebiotic • control: Ringer’ssolution, 200 µl/embryo

  7. MainExperiment- T3.3.3. • 4 050 fertilised eggs • n = 75 per repetition, • 8 additional repetitions, with n=10chickens/pen will be subjected for sampling as zootechnical batches, • 8majorrepetitions will be assigned to the performance experiment (calculating FCR, total BW, total FI etc.) • The chickens will be group-housed 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/synbioticadministration

  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

  9. Samplingschedule

  10. Bacteriology Panel Feacalsampling • Fresh fecal samples in 1-3-6week of growthwill be obtained randomlyfrom each pen and processed within 1 hour after defecation. • One faecal sample per pen is a representative (group-housing) Detailedprotocols: IBB Partner

  11. Histology Panel Gut segments will be separated and measured (length, weight) individually • Duodenum: from pylorus to end of pancreatic loop • Jejunum: part between duodenum and ileum • Meckel’sdiverticulum indicates the proximal (end) section of jejunum [Smirnov et al., 2006; Uni et al., 2003]. • Ileum: end of mesentery arteries to ileo-caecal junction

  12. Genetic Panel Adressed Q: Do SYN1 or SYN2 change the gene expression profile in organs involved in immune response? Gene expression (Spleen, jejunum, caecaltonsils.) • RNA isolation & quality check Experion RNA StdSens Analysis Kit with Experion Automated Electrophoresis System GeneMATRIX Universal RNA Purification Kit • Selection of the most informative time point 21st, 35th day, by RT-qPCR, using: panel of eight target genes, that indicate the level of gene regulation in the tissues • Microarray a panel of differentially expressed genes in chickens treated in ovo with synbiotics will be determined + validationstudy Gene 1.1 ST Array (Affymetrix)

  13. Physiology Panel Adressed Q: Do SYN1 or SYN2 have beneficial effect on the level of metabolic parameters? Pre- synbiotics?? BW CONSUMING ENERGY COLLECTING ENERGY

  14. PhysiologyPanels (1-3) Pancreaticenzymesactivity Concentration of hormonesregulatingthemetabolism and feedconsumption Insulin (anabolism) thyroidhormones (T4; T3; fT4; fT3) – metabolicturnover/ catabolism Corticosterone- glukoneogenesis/stress/feedconversion Leptin – feedintake/ fatness RIA i EIA tests on serum, celllysate and wholeblood Frozensamples; homogenisedafterthawing • - Trypsin(protein) • - Amylase(starch) • - Lipase(dietarylipids) • Colorimetricassays

  15. Physiology Panel Bloodbiochemicalindices static and enzymes • Albumins – osmoticrate • - Total protein– nutritionaccuracy/osmoticrate • - Glucose(carbohydrateregulationaccuracy) • Triglicerides(fatnessmeasure, health status) • - Freefattyacids(lipolysismeasure) • - Total/free cholesterol– health status, meatquality • Aminotransferasesactivity (Aspat; AlAT) – general health status measure (liver, heartmuscle), nitrogenturnover, gluconeogenesis • Colorimetricassays + UV

  16. - apetite - FeedConvertion leptin corticosterone insulin leptin BW CONSUMING ENERGY T3, T4, fT3,fT4 insulin COLLECTING ENERGY Hormonesregulating FI and metabolism- to be testedinECO FCE

  17. www.meatingplace.com THANK YOU

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