UNIVERSITY OF IBADAN RESEARCH FOUNDATION PROPOSAL FOR COLLABORATIVE RESEARCH PROJECT

1. UNIVERSITY OF IBADAN RESEARCH FOUNDATIONPROPOSAL FOR COLLABORATIVE RESEARCH PROJECT INFLUENCE OF GARLIC (Allium sativum) FEED INCLUSION ON PRODUCTION PERFORMANCE AND HEALTH OF COMMERCIAL CHICKENS 19 November, 2013

2. Research Team • Dr. Omolade A. Oladele (Principal investigator) Avian Diseases Unit, Department of Veterinary Medicine, University of Ibadan. 2. Dr. O. A. Abu (Co-Principal Investigator) Department of Animal Science, University of Ibadan. • Dr. Olufunke O. Ezekiel (Co-Principal Investigator) Department of Food Technology, University of Ibadan. UIR-F Oladele, O.A.

3. Dr. O.O. Esan Department of Veterinary Medicine University of Ibadan. • Dr. OlutolaOyedele Agricultural Extensionist, National Horticultural Research Institute (NIHORT) Idi-Ishin, Ibadan. 6. MrsYemisiAdewale Agronomist, National Horticultural Research (NIHORT) Idi-Ishin, Ibadan. UIR-F Oladele, O.A.

4. INTRODUCTION • Inclusion of growth promoters in poultry feed is old & common worldwide (Waibei et al., 1954; Libby & Schaible, 1955). • Usually antibiotics due to abundance of pathogenic microorganisms in the environment. • Microorganisms subclinical infections sub-optimal performance. UIR-F Oladele, O.A.

5. The use of antibiotics as growth promoters in poultry has faced intense criticism for public health reasons: i. Antibiotic resistant strains of pathogens have evolved thereby challenging effective therapy in both poultry and humans (Campylobacter, Enterococcus, Salmonella spps and E. coli – Luoet al., 2003; Graham et al., 2007). European Union,1999 – banned, most antibiotics (Casewellet al., 2003). U.S., 2004 – banned, Enrofloxacin in food animals (Graham et al., 2007). ii. Antibiotic residues – poultry meat & eggs made available for human consumption in Nigeria (Dipeoluet al., 2000; 2002; 2004). UIR-F Oladele, O.A.

6. Poultry producers have therefore been searching for alternatives (Javandel et al., 2008). • Non-conventional growth promoters: - improve nutrient digestibility, - control pathogenic micro-organisms, - facilitate favourable intestinal microbial balance and - enhance nutrient absorption through increased absorptive capacity (Al-Harthi, 2002; El-Deek et al., 2003). UIR-F Oladele, O.A.

7. Introduction – Garlic (Allium sativum) • A well known spice and herbal medicine for the prevention and treatment of a variety of diseases (Adimoradi et al., 2006). • A member of the onion family Alliceae having antimicrobial, antioxidant and antihypertensive properties (Konjufca et al., 1997; Sivam, 2001). • Allicin (diallyl-thiosulfinate) is the major organosulfur cpd – biologically active (Raham, 2007). UIR-F Oladele, O.A.

8. In poultry: - improve broiler growth - decreased mortality rate. The use of GP in poultry production is of particular importance in the tropics: - high environmental temperature, - high relative humidity and - poor biosecurity are optimal conditions for disease agents to thrive. UIR-F Oladele, O.A.

9. Justification: • Increasing pressure to reduce/eliminate the use of antibiotics as growth promoters in poultry feed. • Prevalence of antibiotic-resistant bacteria pathogens of poultry has made control a challenge sub-optimal production. • Thus, further depletion of the already inadequate protein supply in Nigeria (FAO, 2010)., thereby hampering food security (MDG). • Search for alternative growth promoters preferably from organic sources for sustainability. UIR-F Oladele, O.A.

10. Preliminary Studies Preliminary studies have shown: • Increased body weight and higher Newcastle disease vaccinal immune response. • Increased villi length & width as well as cryptal depth In commercial broilers fed garlic inclusion (Oladele & Bakare, 2011; Oladele et al., 2012). (The garlic meal used has been patented (Patent No. NG/P/2012/285 of 19 July, 2012). UIR-F Oladele, O.A.

11. Aim: To evaluate the potentials of garlic as a growth promoter in chickens via the assessment of production performance and markers of immune status and disease tolerance. UIR-F Oladele, O.A.

12. Specific Objectives: • To determine quantitatively, the active components of garlic varieties available in Nigeria. • To determine the effect of garlic feed inclusion on: a. performance characteristics of commercial layers. b. immunity (humoral and cellular), haematology and serum biochemistry of commercial chickens. c. disease susceptibility/tolerance of commercial chickens. d. the organoleptic properties of poultry meat. iii) To conduct a cost-benefit analysis of garlic feed inclusion in commercial chicken production. iv) To partner with poultry producers and assess the acceptability of this product. UIR-F Oladele, O.A.

13. Expected outcome: This project is expected to: • provide scientific explanations for some of the claims on garlic. • provide information to poultry producers on the potentials of garlic as a growth promoter with consequent improvement in health and productivity of poultry. • reduce inadvertent use of antibiotics in poultry rearing as well as production of safer meat and egg for human consumption. • stimulate the adoption of the use of garlic as an organic growth promoter by producers with consequent reduction in the emergence of antibiotic-resistant bacteria. UIR-F Oladele, O.A.

14. Experimental Design and Methods Phase 1 1. Quantitative assessment of the active components of garlic varieties available in Nigeria Available garlic varieties in Nigeria will be sourced and their active components i.e. allicin and alliin will be quantitatively determined using the high-performance liquidchromatography and/or the method of Mironet al., 2002. UIR-F Oladele, O.A.

15. 2.Rearing of Commercial Layers • 500 day-old pullet chicks will be purchased and reared in a designated poultry house at the Teaching & Research Farm. • These chicks will be randomly separated into four groups (A, B, C and D) each consisting of two replicates designated as 1 and 2 from day-old and administered all necessary prophylaxis i.e. vaccines and multivitamins. • Appropriate mash will be served with the inclusion of previously prepared garlic meal (Patent No. NG/P/2012/285) viz: 0.125% - group A, 0.25% - group C, 0.5% - group C and 0% - group D (control). UIR-F Oladele, O.A.

16. 3. Assessment of performance characteristics of commercial layers on garlic feed inclusion • Performance characteristics to be assessed include morbidity, mortality, onset of lay, % production, egg quality (size, weight, shell colour and thickness, yolk colour and size, albumen and yolk viscosity) and spent hen value. • Shelf-life of eggs laid will also be evaluated. UIR-F Oladele, O.A.

17. 4. Assessment of immunity, haematology and serum biochemistry of commercial layers on garlic feed inclusion • Cell mediated immunity will be assayed using lymphocyte proliferation ELISA kit (BrdUcolorimetric, Roche Applied Science, UK). • Humoralimmunity (Newcastle disease and infectious bursal disease vaccinalresponse) will be assayed using the ELISA technique. • Haematology(PCV, RBC counts, total and differential WBC counts) and serum biochemistry (protein, enzymes and lipid levels) will be determined using standard procedures (Campbell, 1997). UIR-F Oladele, O.A.

18. 5. Determination of organoleptic properties of poultry meat from commercial layers on garlic feed inclusion • Sensory evaluation of poultry meat will be conducted in a sensory testing room equipped with positive air pressure, individual stations and proper light (ISO, 1988). • Coded samples of freshly prepared poultry meat will be served in separate, well-lit cubicles to 40 panelists at room temperature. • A descriptive 9-point hedonic scale rating will be used to assess the meat for appearance/colour, aroma, chewiness, juiciness, toughness and overall acceptability. • 1 representing ‘like extremely’ and 9 ‘dislike extremely’ (Osman and Aldosari, 2006). UIR-F Oladele, O.A.

19. 6. Cost-benefit analysis of garlic feed inclusion in commercial layer production • The cost of rearing these chickens will be determined and compared across the different groups considering inputs, disease control, mortalities, etc. • This will be compared with gains from egg production and spent layers across the groups to determine profitability. UIR-F Oladele, O.A.

20. Phase 2 • Determination of disease susceptibility/ tolerance of commercial broilers on garlic feed inclusion Three experiments to determine disease susceptibility/tolerance to three classes of pathogens (virus, bacteria and protozoan) will be conducted separately. a) Experimental infection with Infectious bursal disease (IBD) virus • 200 day-old broilers will be purchased and reared in an isolated facility. • Random separation into 6 groups (A, B, C, D, E and F) from day-old and administered all necessary prophylaxis i.e. vaccines (except IBD) and multivitamins. • Garlic inclusion, IBD vaccine and IBD virus will be administered as follows: UIR-F Oladele, O.A.

21. Group A: Garlic + IBD vaccine Group B: Garlic + IBD virus Group C: Garlic + IBD vaccine + IBD virus Group D: IBD vaccine + IBD virus Group E: IBD virus only Group F: IBD vaccine only • Garlic inclusion will be served from day-old at a level pre-determined from Phase 1 as most desirable. • IBD vaccination will be at the recommended age and route. • Experimental infection with IBD virus will be at 5 weeks of age via intraocular route. • LD50would have been pre-determined using 30 four week-old broiler chicks. • Clinical signs, mortality and pathology will be monitored and recorded until 10 days post-infection. UIR-F Oladele, O.A.

22. b) Experimental infection with Salmonella gallinarum • 180 commercial broilers will be purchased. Thirty of them will be separated for the determination of LD50 of the bacterial inoculum. • 150 chicks will be reared in five different groups of 30 each and administered garlic inclusion and S. gallinarum as follows: • Group A: 0.125% garlic + S. gallinarum • Group B: 0.25% garlic + S. gallinarum • Group C: 0.5% garlic + S. gallinarum • Group D: 0% garlic + S. gallinarum • Group E: Garlic inclusion only (at the rate most desirable from Phase 1). • Experimental infection will be orally at 5-6 weeks of age. • Clinical signs, mortality and pathology will be monitored and recorded until 14 days post-infection. UIR-F Oladele, O.A.

23. c) Experimental infection with oocysts of Eimeriaspp As in 1c above. 2. Determination of organoleptic properties of meat from commercial broilers on garlic feed inclusion • Uninfected broilers from control groups will be used for this study. • Same procedure as Phase 1.5. will be adopted. UIR-F Oladele, O.A.

24. Possible limitations a) incessant power outage. b) delays in supply of reagents and equipment. UIR-F Oladele, O.A.

25. Summary of Plan of Work Year 1 a) Construction and renovation of poultry experimental pens. • Quantitative assessment of the active components of garlic varieties available in Nigeria. • Rearing of commercial layers. • Assessment of performance characteristics of commercial layers on garlic feed inclusion. • Assessment of immunity, haematology and serum biochemistry of commercial layers on garlic feed inclusion. f) Cost-benefit analysis of garlic feed inclusion in commercial layer production. UIR-F Oladele, O.A.

26. Year 2 a) Year 1 d and f to be continued. b) Determination of disease susceptibility/tolerance of commercial broilers on garlic feed inclusion. • Experimental infection with infectious bursal disease virus. • Experimental infection with Salmonella galllinarum. • Experimental infection with oocysts of Eimeria spp. c) Determination of organoleptic properties of meat from commercial layers and broilers on garlic feed inclusion. UIR-F Oladele, O.A.

27. Year 3 • Year 2 b and c to be continued. • Consultations with poultry producers through the Poultry Association of Nigeria to encourage the use of garlic as growth promoter. b) Project round-off and final report will be written. UIR-F Oladele, O.A.

28. UIR-F Oladele, O.A.

29. UIR-F Oladele, O.A.

30. = N9,366,650.00 UIR-F Oladele, O.A.

31. Budget Justification • There is sparsity of equipment and reagents in the Poultry Diseases Laboratory at the Dept. of Veterinary Medicine at present such that so much is needed for this project. • Construction of a small poultry house is necessary for the Layers project. • Conversion of an old kennel at Vet. Medicine to poultry experimental house for infectivity studies. • An allowance is necessary for visits to farms and meeting with PAN members for advocacy. • Allowance was made in budget for publications since about five publications is expected from this project. UIR-F Oladele, O.A.

32. References Adimoradi, M., Navidshad, B., Saif, J., Royan, M. 2006. Effect of dietary garlic meal on histological structure of small intestine of Broiler chickens. Poult. Sci., 43: 378 – 383. Al-Harthi MA. Efficacy of vegetable diets with antibiotics and different types of spices or their mixtures on performance, economic efficiency and carcass traits of broilers. J. Agr. Sci. Mansoura University 2002; 27: 3531 – 3545. Campbell, TW: Avian Hematology and Cytology. Ames, IA, Iowa State University Press, 1997. Casewell, M., Friis, C., Marco, E., McMullin, P., Phillips, I. 2003. The European-ban on growth promoting antibiotics and emerging consequences for human and animal health. J. Antimicrob. Chemother. 52:159-161. Dipeolu, MA., Akpan, NJ. and Olutayo, A. 2000. Residues of Tetracycline antibiotic in turkey and egg of chickens sold for human consumption. Poultry Science Journal. 1(1): 4-11. Dipeolu, MA., Eruvbetine, D. and Sowunmi, KS. 2002. Tetracycline residue deposition in egg of layers fed antibiotics and enzyme supplemented feed. Proceedings of the 27thAnnual Conference of the Nigerian Society for Animal Production. 29: 292- 294. Dipeolu, MA., Adebayo, AJ. and Oke, MO. 2004. Residues of Streptomycin antibiotic in commercial layers in Abeokuta and Ibadan metropolis. Nigerian Journal of Animal production, 31(1): 130-134. UIR-F Oladele, O.A.

33. Graham, J.P., Boland, J.J. and Silbergeld, E. 2007. Growth promoting antibiotics in food animal production: An economic analysis. Public Health Reports, 122: 79-87. ISO (International Standard Organisation). 1988. Sensory Analysis-General Guidance for the Design of Test Rooms. ISO 8589, 1-9 . Javandel F, Navidshad B, Seifdavati J, Pourrahimi GH, Baniyaghoubi S. The favourite dosage of garlic meal as a feed additive in broiler chicken ratios. Pak J BiolSci2008; 11(13): 1746-1749. KonjufcaVH, Pest GM and Bakalli RI 1997. Modulation of cholesterol levels in Broiler meat by dietary garlic and copper. Poultry Science 76, 1264 – 1271. Libby, DA, Schaible, PJ. 1955. Observations on growth responses to antibiotics and arsenic acids in poultry feeds. Science 121 : 733. Luo, N., Sahin, O., Lin, J., Michel, L.O., Zang, Q. 2003. In vivo selection of Campylobacter isolates with high levels of fluoroquinolones resistance associated with gyrA mutations and the function of the cmeABC efflux pump. Antimicrob. Agents Chemother. 47: 390-394. UIR-F Oladele, O.A.

34. Miron, T., Shin, I., Feigenblat, G., Weiner, L., Mirelman, D., Wilchek, M., Rabinkov, A. (2002). A spectrophotometric assay for allicin, alliin, and alliinase (alliinlyase) with a chromogenicthiol: reaction of 4-mercaptopyridine with thiosulfinates. Anal Biochem. 307(1):76-83. OladeleOmolade and BakareHauwa (2011). Effects of garlic (Alliumsativum) on growth performance and vaccinal immune response in commercial broilers. Conference Proceedings: 5th Pan Commonwealth Veterinary Association. Journal of Commonwealth Veterinary Association.Special issue. Vol. 27(2), 243-247. www.commonwealthvetassoc.org/ Oladele OA,Emikpe BO, Hauwa B. Effects of dietary garlic (Alliumsativum) supplementation on body weight and gut morphometry of commercial broilers. Int J Morph 2012,30(1), 238-240. http://www.scielo.cl/revistas/ijmorphol Osman, BM, Aldosari, MN. 2006. Sensory evaluation of Awassi and Nadji lambs meat. Scientific Journal of faisal University (Basic and Applied Sciences). Vol. 7(2): 1427H. Raham, M.S. 2007. Allicin and other functional active components of garlic: Health benefitsand bioavalability. International Journal of food properties. Vol. 10: 245-268. Sivam GP 2001. Protection against Helicobacter pylori and other bacterial infections by garlic. Journal of Nutrition 131, 1106S – 1108S. . UIR-F Oladele, O.A.

35. Tollba, AAH and Hasan, MSH. 2003.Using some natural additives to improve physiological and productive performance of broiler chicks under high temperature conditions. Black cumin (Nigella sativa) or garlic (Alliumsativum). Poultry Science, 23:327-340. 2003. Waibel, P.E., Abbott, OJ., Baurmann, CA. and Bird, HR. 1954. Disappearance of the growthresponse of chicks to antibiotics in an “old” environment. Poultry Science. 33: 1141. UIR-F Oladele, O.A.

36. Abstract Pictures Sample of Product (garlic meal) Garlic meal (200g) to be mixed with 160kg poultry feed. UIR-F Oladele, O.A.

37. Abstract Pictures Two chicken layers (Nera Black) on garlic feed inclusion showing lustrous plumage and brightly coloured combs and wattles in spite of being reared on old litter. UIR-F Oladele, O.A.

38. Abstract Pictures Closer picture of the Nera Black layers on garlic feed inclusion immediately after beak trimming. UIR-F Oladele, O.A.

39. Abstract Pictures Isa Brown layer chicken on garlic feed inclusion showing lustrous plumage and brightly coloured combs and wattles. UIR-F Oladele, O.A.