Sponsor Day on animal feeding: Studies of feed additives in experimental conditions
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This document summarizes research on studying feed additives in experimental conditions. It describes various experimental infection models used to study Salmonella, E. coli, and Clostridium perfringens. It also discusses analyzing the gut microbiota using cloning, sequencing, and ion torrent analysis. Key findings include that the gut microbiota plays an essential role in digestive physiology and animal health, and can be modified by feed composition and additives, which can help reduce variance in productive parameters and improve farm economics.
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Sponsor Day on animal feeding: Studies of feed additives in experimental conditions
- 1. Studies of feed additives in experimental conditions Dr. Ignacio Badiola Head of the BACPAR SubProgram, IRTA-CReSA SPONSOR DAY SEMINAR ON ANIMAL FEEDING
- 2. Introduction Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Maisons-Alfort Departamento de Ciencia Animal, ETSI Agrónomos, UPV Departamento de Producción Animal, ETSI Agrónomos, UPM Department of Agronomy, Food, Natural Resources, Animal and Environment, Università de Padova Department of Comparative Biomedicine and Food Science, Università de Padova Department of Genetics and Microbiology, UAB Faculty of Animal Sciences of the Agricultural University of Athens Gut and Immunology Group, Rowett Institute of Nutrition and Health, University of Aberdeen Institut National de la Recherche Agronomique, Toulouse Institut National de la Recherche Agronomique, Tours Instituto de Estudios Biofuncionales. Facultad de Farmacia. UCM Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC-UCLM-JCCM) IRTA, Centre de Sant Carles de la Ràpita, Unitat de Cultius Aqüícoles IRTA, Centre Mas de Bover, Nutrició de Monogàstrics National Veterinary School, Toulouse Research Institute for Animal Breeding and Nutrition, Godollo University Royal Veterinary and Agricultural University (KVL), Frederiksberg Statens Veterinärmedicinska Anstalt, Uppsala University ADISSEO France S.A.S. DSM Nutritional Products Elanco Animal Science Research Indukern, S.A. Laboratorios Andrés Pintaluba, S.A. Laboratorios Calier, S.A. Laboratorios CENAVISA Laboratorios Esteve Veterinaria, S.A. Laboratorios Intervet, S.A. Laboratorios Lamons, S.A. Laboratorios Maymo S.A. Laboratorios S.P. Veterinaria, S.A. NANTA, S.A. Novartis Animal Health Novozymes Biologicals FR S.A. ONDAX Scientific Rubinum Animal Health • Public projects with in vivo feed additive trials (9) EU: 5 Nationals: 4 • Private company contracts with in vivo feed additive trials (26) International Antimicrobials: 3 Prebiotics and probiotics: 10 Nationals Antimicrobials: 12 Prebiotics and probiotics: 1
- 3. Introduction • Feed additives are used to supplement essential components in the diet of farm animals (essential aminoacids, vitamins). • Another important role of feed additives is to increase the digestibility of raw materials (enzymes). • After the ban of antimicrobials growth promoters, the interest for feed additives with antimicrobial activity has been increased (plant extracts, organic acids...) • To reduce pathogenic and to increase saprophytic microorganisms are fundamental aims for feed additives (probiotics, prebiotics). • To stimulate the immunologic system is another point of interest for feed additives. • To improve the knowledge of intestinal physiology, and specially changes in the intestinal microbiota (components and metabolism), is essential for the development of new feed additives.
- 4. Experimental infection models Salmonella Enteritidis in chickens. Salmonella Enteritidis in hens. Salmonella Typhimurium in pigs. Escherichia coli in pigs. Escherichia coli in chickens. Clostridium perfringens in chickens.
- 6. The gut microbiota contributions to host physiology Sekirov et al., Physiol. Rev. 2010;90:859-904 DC tolerization Cellular immunity Lymphoid organogenesis Mucosal Immunity ↑Bacteroidetes B. fragilis (PSA) SCFAs AMPs Gm-PG GM-LPS ↑IgA ↑Iap activation NFKB inactivation Lactobacillus spp. E.coli Barrier maintenance B. thetaiotaomicron Bifidobacterium spp. Clostridium spp. SCFA metabolism Conjugation of linoleic acid Nutrition Xenobiotics metabolism Drug disposition ↓Oxalate excretion Lipid metabolism Behavior O.formigenes Normalization of HPA stress response B.infantis Angiogenesis Presistalsis Glycosylation GIT surface maturation GIT functional maturation Immunocompetence Tolerance
- 7. The intestinal microbiota of productive animals is composed by: 16-23% cultured bacteria. 77-84% uncultured bacteria. To analyse the cultured and the uncultured intestinal bacteria we initially used the Restriction Fragment Length Polymorphism (RFLP) method
- 9. 0 500 1000 1500 2000 2500 0 100 200 300 400 500 600 700 800 T1-D27-A1403-Ce T3-D27-A1401-CeLactobacillus helveticus Uncultured alpha proteobacterium Uncultured gamma proteobacterium Parabacteroides sp. Prevotella nigrescens No sequence homology No sequence homology Uncultured bacterium Uncultured Desulfovibrio Uncultured Succinivibrio Uncultured Veillonella No sequence homology Lactobacillus spp. Rodobacter spp. Uncultured Desulfovibrio No sequence homology Lactobacillus salivarius No sequence homologyParabacteroides sp. No sequence homology Identification of Bacteria Species by Cloning and Sequencing
- 10. Effect of strains L-92 and L-94 (Bacteroides spp.) on mortality rate by ERE 0 10 20 30 40 50 L-94 Control - Trial 1 23.33 46.66 Mortality by ERE At weaning At the beginning of ERE 0 10 20 30 40 50 60 L-92 L-94 Control - Trial 2 29.17 31.25 58.33 Mortality by ERE OT00-040-C2-2
- 11. Effect of fiber source and enzyme supplementation on digesta’s and mucosa’s biodiversity of ileum and caecum C = Corn WBR = Wheat+Barley+Rye - = Without enzyme + = With enzyme
- 12. New molecular biology methods RNA-Later Etanol Etanol 0 2 4 6 0 5 10 15 20 C-C-C/L C-C-L/L L-C-C/L L-C-L/L C-C-C/L C-C-L/L L-C-C/L L-C-L/L 1st period 2nd period Q-PCR and 2-ΔΔCт 22d 30d RNA-Later Ion Torrent 316™ Chip Kit
- 13. Summary of Ion-Torrent analysis of Ileal Microbiota First Period Second Period C-C/GFC C-C/BFC L-C/GFC L-C/BFC C-C-C/GFC C-C-C/BFC C-C-L/GFC C-C-L/BFC L-C-C/GFC L-C-C/BFC L-C-L/GFC L-C-L/BFC Number of sequences 345221 353196 382219 311926 125949 181309 173262 201476 197613 200657 166206 155863 % Lactobacillus 91.74 86.38 83.36 93.99 91.19 83.62 91.07 90.61 86.99 84.98 90.82 93.75 % Other bacteria 8.26 13.62 16.64 6.01 8.81 16.38 8.93 9.39 13.01 15.02 9.18 6.25 Biodiversity 78 60 89 57 84 93 71 50 86 135 101 68
- 14. Kruskal-Wallis rank sum test & Period P-value Kruskal-Wallis rank sum test & FCR P-value Kruskal-Wallis rank sum test & SD P-value Kruskal-Wallis rank sum test & FD P-value Bacillus 0.001 Anaerostipes 0.027 Jeotgalicoccus 0.027 Butyricicoccus 0.006 unclas_Bacillales 0.001 Aerococcus 0.035 unclas_Bacteroidetes 0.044 unclas_Betaproteobacteria 0.011 unclas_Porphyromonadaceae 0.003 unclas_Burkholderiales 0.044 Catenibacterium 0.064 unclas_Clostridiales 0.016 Prevotella 0.003 unclas_Lactobacillaceae 0.059 Comamonas 0.064 unclas_Clostridia 0.019 unclas_Prevotellaceae 0.003 Brachybacterium 0.064 Deinococcus 0.064 Bacteroides 0.020 unclas_Bacillaceae.1 0.004 Curvibacter 0.064 Finegoldia 0.064 Clostridium.XI 0.021 unclas_Chitinophagaceae 0.004 Delftia 0.064 Peptoniphilus 0.064 unclas_Bacteria 0.021 Paracoccus 0.006 Parabacteroides 0.064 unclas_Bradyrhizobiaceae 0.064 unclas_Lachnospiraceae 0.021 Enterococcus 0.006 Erysipelotrichaceae_incerta_sedis 0.074 Methylobacterium 0.025 Dorea 0.008 Neisseria 0.097 Streptophyta 0.027 Lactococcus 0.011 unclas_Rhodobacteraceae 0.097 Cloacibacterium 0.027 Roseburia 0.011 Fusobacterium 0.027 Sarcina 0.011 Ruminococcus 0.027 unclas_Acidaminococcaceae 0.011 unclas_Peptostreptococcaceae 0.027 Selenomonas 0.011 Clostridium.IV 0.037 unclas_Bacilli 0.012 Sphingomonas 0.043 unclas_Erysipelotrichaceae 0.020 unclas_Firmicutes 0.046 unclas_Alphaproteobacteria 0.025 unclas_Ruminococcaceae 0.046 Aquabacterium 0.027 Barnesiella 0.046 Parasutterella 0.027 Alistipes 0.061 unclas_Planococcaceae 0.027 Brachybacterium 0.064 unclas_Clostridiaceae.1 0.033 Coprobacillus 0.064 Chryseobacterium 0.033 Coprococcus 0.064 Barnesiella 0.035 Gemella 0.064 unclas_Lactobacillales 0.036 Granulicatella 0.064 Flavonifractor 0.037 Megasphaera 0.064 Phenylobacterium 0.037 Peptoniphilus 0.064 unclas_Enterococcaceae 0.045 Clostridium.XlVb 0.073 Clostridium.XVIII 0.061 unclas_Bacteroidales 0.073 Comamonas 0.065 Sarcina 0.074 Coprobacillus 0.065 Pelomonas 0.074 Deinococcus 0.065 unclas_Comamonadaceae 0.093 Granulicatella 0.065 Actinomyces 0.097 Lysinibacillus 0.065 Megasphaera 0.065 Sphingobacterium 0.065 unclas_Cytophagaceae 0.065 unclas_Leptotrichiaceae 0.065 unclas_Veillonellaceae 0.065 Clostridium.XlVb 0.073 Sphingomonas 0.073 unclas_Ruminococcaceae 0.074 unclas_Bacteroidetes 0.081 Anaerococcus 0.083 Veillonella 0.083 Clostridium.sensu.stricto 0.091 Oscillibacter 0.092 unclas_Neisseriaceae 0.093 Clostridium.XI 0.093 Staphylococcus 0.093 Neisseria 0.097 Significant differences for Ileal Microbiota vs Trial Factors
- 15. Effect of feed supplemented with marine algae extract on some immunological parameters of intestinal mucosa by RT-PCR CENIT VIDA (CEN-20101026)
- 16. Importance of variance homogeneity as productive parameter Clinical efficacy evaluation of the use of tylosin for the treatment and control of porcine proliferative enteropathy (ileitis) associated with Lawsonia intracellularis Mean Variance Control Tylosin P-value1 Control Tylosin P-value2 Animal weight (kg/pig) Day 0 28.42 29.20 0.77 25.40 15.10 0.16 Day 21 42.72 45.66 0.36 51.80 21.70 0.02 Day 31 50.90 53.98 0.35 76.10 23.00 0.002 Daily growth rate (kg/pig/day) Day 0 to 21 0.681 0.773 0.036 0.022 0.009 0.02 Day 21 to 31 0.818 0.833 0.76 0.064 0.018 0.001 Daily feed consumption (kg/pig/day) Days 0 to 21 1.471 1.569 0.21 0.005 0.008 0.65 Days 21 to 31 1.862 1.922 0.5 0.006 0.014 0.53 Feed conversion ratio Days 0 to 21 2.160 2.030 0.050 0.535 0.073 <0.001 Days 21 to 31 2.280 2.300 0.770 0.550 0.137 <0.001 1 Anova test 2 Bartlett test
- 17. Importance of variance homogeneity as productive parameter This batch will be punished if all animals are sent together to slaughterhouse Troubles for the correct cleaning and disinfection of farm facilities. Increasing the risk of maintenance of infectious agents. Higher prevalence of respiratory and digestive disorders in the next batch.
- 18. Conclusions • It is important to normalise experimental infection models. • The intestinal microbiota is essential for animal health and welfare. • The gut microbiota plays an essential role on digestive physiology. • Some microbial components have direct effect on intestinal mucosa (barrier effect and gut associated lymphoid tissue). • The intestinal microbiome can be modified by feed composition (raw materials and feed additives). • Feed additives can reduce the variance of productive parameters, representing a significant improvement on the economical balance of farm.
- 19. Edifici CReSA. Campus UAB. 08193 Bellaterra (Barcelona) Spain. Tel. (+34) 93 581 32 84 Fax. (+34) 93 581 44 90 e-mail: cresa@uab.cat - www.cresa.cat THANKS FOR YOUR INTEREST SPONSOR DAY SEMINAR ON ANIMAL FEEDING