This project has received funding from the European Union s Seventh Framework Programme for research, technological development and demonstra on

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2 Improving feed efficiency by understanding the intes nal bacterial network in pigs and poultry Dr. Barbara Metzler- Zebeli Mr. Andor Molnar Ms. Janine Scholz University of Veterinary Medicine Vienna Dr. Stefan G. Buzoianu Dr. Peadar G. Lawlor Ms. Ursula McCormack Moorepark Research Centre, Teagasc, Ireland

3 Introduc on

4 Feb 2013 Feb partners 7 WPs Overall objec ves ECO- FCE overview improve food security by op mising the feed efficiency in pigs and broilers without nega vely affec ng animal welfare and meat quality reduce the ecological footprint of the pig and broiler produc on systems WP 3 objec ve to examine the gut structure, func on, microbiota and metagenomics in animals divergent for feed efficiency

5 Work Package 3 Intes nal structure & func on Genomics Gene cs Low RFI Intes nal health High Medium Health & welfare Meat quality Animal performance Intes nal microbiota

6 Determina on of feed efficiency Selec on of high and low feed efficient animals - based on Residual Feed Intake Residual Feed Intake (RFI) = difference between observed and predicted feed intake, with lower RFI values indica ng greater energy efficiency RFI = FI [a + b 1 * BW b 2 * BWG] Where a is the intercept and b 1 and b 2 are par al regression coefficients of feed intake (FI) on BW 0.75 and body weight gain (BWG), respec vely. Other measures of feed efficiency Feed efficiency = gain (g) / feed intake (g) Feed conversion ra o (FCR) = feed intake (g) / gain (g) RG = BWG [a + b 1 * BW b 2 * FI] RIG = (RG/SD RG) - (RFI/SD RFI)

7 Feed efficiency in monogastric livestock species Feed efficiency Gene cs Diet Rearing environment Age Gut commensal microbiota Substan al varia on in feed efficiency between individual animals. Great varia on in gut commensal microbiota between individuals.

8 Role of the intes nal microbiota Benefits to the host intes nal matura on inhibi on of pathogen growth nutrient salvaging detoxifica on produc on of vitamins Costs to the host compe on for nutrients immune ac va on produc on of toxins opportunis c toxin reabsorp on mucoly c ac vity

9 Effect of host microbiota on host metabolism and hormone secre on Intes nal microbiota can redirect energy par oning to adipose ssue and reduce fa y acid oxida on. Bäckhed (2011) Ann Nutr Metab 58(suppl 2):44 Implica ons for feed use efficiency and carcass composi on in livestock animals?

10 Effect of gut microbiota composi on on body weight Obese humans & mice: Firmicutes á Bacteroidetes â Low- calorie diet Firmicutes â Bacteroidetes á wikipedia.org Ac nobacteria á Bacteroidetes â no difference in Firmicutes Changes in Lactobacillus and Bifidobacterium species Methanogenic archaea á Requena et al. (2013) Trends Food Sci Tech 34:44 Meat- producing monogastric livestock species are young, fast growing and lean animals Are the key players the same as in human obesity models?

11 Chickens

12 Diet- related cecal microbiota and performance in male chickens Caecal microbial communi es by diet Caecal microbial communi es iden fied as being from birds with improved performance or poorer performance Diet is the most influencing factor affec ng feed efficiency. Torok et al. (2011) AEM 77: 5868

13 Batch to batch varia on in caecal microbiota of chickens 3 different batches of chickens Very different microbiota profiles across chicken batches PCA plot of caecal microbiota. The plot is based on between groups (trials) analysis. Very different feed use efficiencies across chicken batches Stanley et al. (2013) PloS ONE 8(12): e84290 High varia on in caecal microbiota partly due to lack of colonisa on of the chickens by maternally derived bacteria High hygiene levels in modern commercial hatcheries remove natural bacteria Environmental microbiota from transport boxes, first feed and staff people

14 Fecal community of high and low feed efficient broiler chickens Low efficient chickens High efficient chickens 12 7 Proteobacteria (%) Firmicutes (%) Bacteroidetes (%) Singh et al. (2014) J Appl Genet 55: 145

15 Characterisa on of differences in gut microbiota and gut func on of chickens with good and poor feed efficiency Experimental design: 2 partner ins tu ons (AFBI & Vetmeduni) performed iden cal chicken experiments with 3 batches of 50/64 chicks Similar chicken gene c: Cobb 500FF Similar maize- soybean meal diets (starter, grower, and finisher diets) No in- feed an bio cs and any other gut health- related addi ves Chickens were individually housed Best and worst feed efficient chickens were iden fied using Residual Feed Intake Ammonia Metagenomics Metagenomics Metagenomics emission (faeces) (faeces) (faeces) (faeces) Weighing Weighing Weighing Weighing Weighing Weighing d1 d7 d14 d21 d28 d35 d42 Daily feed intake On day 42, samples were collected for: Ileal and caecal digesta for metagenomics and microbial metabolites Tissue of duodenum, jejunum, ileum, caeca for gut func on and structure

16 Residual feed intake of good and poor feed efficient broiler chickens Residual feed intake Male Female (g) Great varia on in residual feed intake and thus in feed use efficiency Feed efficiency, P< Batch, P>0.1 Good Average Poor

17 Microbial metagenome of good and poor feed efficient chickens Shotgun sequencing using MiSeq Technology (Illumina) Under construc on

18 Jejunal electrophysiological characteris cs of good and poor feed efficient broiler chickens Gut electrophysiology was performed using Ussing chamber technique. Tissue originated from the distal jejunum Tissue resistance (Ω/cm 2 ) Females Feed efficiency, P=0.020 b a Good Average Poor a Tissue conductance (ms x cm 2 ) a b Females Feed efficiency, P=0.002 b Good Average Poor Short- circuit current (I sc ; µeq/cm 2 x hour) a ab Females Feed efficiency, P=0.076 Good Average Poor b Good feed efficient females showed lower ssue resistance, higher conductance and short- circuit current indica ng a higher ion flux and permeability of the jejunal mucosa Influencing factors: Host genome or gut microbiota?

19 Pigs

20 Literature Li le data available in pigs Bacteroidetes & Firmicutes in obese pigs (Pedersen et al., 2013) Firmicutes & β- Proteobacteria in ERS- fed pigs (Haenen et al., 2013) Protein, CHO and lipid metabolic pathways affected by intes nal microbial profile mice (Antunes et al., 2011) pigs (Mulder et al., 2009)

21 Screening on feed efficiency in pigs 46 li ers Pigs divergent for RFI Common protocols Teagasc 3 AFBI Vetmeduni Common diets Common gene cs Common & site- specific boars weaning d 42 d 84 P F F P d 112 F I C F faecal I ileal digesta C caecal digesta P performance

22 Microbiota profiling d 0 (weaning) d 42 d 84 d 126 d 139 P P P P F F F F F I C Composi onal analysis 16S rrna gene sequencing Illumina Func onality Shotgun metagenomics F faecal; I ileal digesta; C caecal digesta; P performance

23 Progress on microbiota profiling Samples collected DNA extracted 16S rrna gene sequencing results being analysed Shotgun metagenomics samples being prepared results ~ Oct 2014

24 Manipula on of GIT microbial profile Addi ves Microbial inocula on Low RFI Management Nutri on

25 Inocula on with faecal inoculum from good feed converters Anaerobically processed diluted 1:6 strained centrifuged (6000 G for 15 minutes) frozen at - 80 C in 10% glycerol No inoculum Sows Inoculum No inoculum Single inocula on Mul ple inocula on Offspring No inoculum Single inocula on Mul ple inocula on

26 Nutri onal interven on Op mum strategy inoculum Prebio cs alone or in combina on Monitoring and sampling of offspring through their life me performance health intes nal microbiota

27 Acknowledgements ECO- FCE has received funding from the European Union s Seventh Framework Programme for research, technological development and demonstra on (FP7 2007/2013) under grant agreement No Teagasc Walsh Fellowship Programme

28 Thank you

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30 ECO- FCE Gut structure, func on, microbiota and metagenomics Hypothesis: It is assumed that the gut microbiome of pigs and broiler chickens with good and poor feed use efficiency differs in key members, thereby influencing the intes nal and metabolic host response, produc on efficiency and host health. Objec ves: 1. To enhance our understanding of the interac ons between gut microbiome and host genome in pigs and chickens. This task will be achieved by employing cu ng- edge 16S rrna- specific and shotgun metagenomics. 2. Using this improved understanding, strategies to improve feed conversion efficiency through gut microbiome manipula on in embryonic and subsequent developmental stages will be developed.

31 Interac ons between gut microbiome and host physiology and health NF- κb Bäckhed (2011) Ann Nutr Metab 58(suppl 2): 44; Twarziok et al. (2014) Mol Inf 33: 171