Livestock Science 142 (2011) 305309

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Livestock Science
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Performance, carcass characteristics, and immunity in broiler chickens fed dietary neem (Azadirachta indica) as alternative for an antibiotic growth promoter
N. Landy a,, Gh. Ghalamkari b, M. Toghyani b
a b

Young Researchers Club, Khorasgan Branch, Islamic Azad University, Isfahan, Iran Department of Animal Science, Khorasgan Branch, Islamic Azad University, Isfahan, Iran

a r t i c l e

i n f o

a b s t r a c t
This experiment was conducted to examine the effect of neem (Azadirachta indica) as an antibiotic growth promoter substitute on growth performance, carcass traits, and humoral immune responses in broiler chickens. A total of 192 one-d-old mixed sex broiler chicks (Ross 308) were weighted and randomly allocated to four treatment groups, each with 4 replicate pens of 12 chicks. The dietary treatments consisted of the basal diet (control), control + 4.5 mg flavophospholipol/kg, or control + 7 or 12 g neem fruit powder/kg. Body weights of broilers were determined at d 1, 14, 28, and 42, feed intake was determined at the same periods, and feed conversion ratio was calculated accordingly. At d 42, two birds per replicate were slaughtered for determination of carcass and organ weights. Antibody titers against Newcastle, influenza viruses, and sheep red blood cells (SRBC) were determined. Dietary supplementation of flavophospholipol increased final body weight of broilers at 42 d of age compared with those fed diets supplemented with neem (Pb 0.05). Birds fed 7 g neem/kg in the diet had the highest feed conversion ratio at 42 d, compared with other treatments. Daily feed intake, internal organ weights, and carcass traits were not influenced by the dietary treatments at d 42. Supplementation with 7 g neem/ kg led to greater antibody titers against SRBC compared with 12 g neem/kg (Pb 0.05) and influenza virus compared with the control diet and the diet supplemented with flavophospholipol (Pb 0.05). In conclusion, the results indicate that supplementing broiler diet with 7 g neem/kg could induce favorable influences on immune responses of broilers without any adverse effect on growth performance. 2011 Elsevier B.V. All rights reserved.

Article history: Received 4 March 2011 Received in revised form 1 August 2011 Accepted 31 August 2011 Keywords: Broiler Azadirachta indica Performance Immunity Carcass characteristics

1. Introduction Antibiotic growth promoter has been successfully used at subtherapeutic doses in poultry production to promote growth and protect health of the birds (Engberg et al., 2000; Eyssen and Desomer, 1963; Harms et al., 1986; Miles et al., 1984; Rosen, 1996). Antibiotic growth promoters were supposed to increase growth rate as a result of improved gut health, resulting in better nutrient utilization and decreased feed conversion ratio (Visek, 1978). However, there is the fear that the continuous
Corresponding author. Tel.: + 98 913 207 0979; fax: + 98 311 5354038. E-mail address: n_landy1984@yahoo.com (N. Landy). 1871-1413/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2011.08.017

subtherapeutic use of in-feed antibiotics could lead to the development of antibiotic resistant bacteria, which can be harmful to humans (Nasir and Grashorn, 2006). Thus, efforts have been made in different parts of the world to ban the inclusion of all types of antibiotic growth promoters in animal feeds. Because of the ban on the use of antibiotics in European Union, and the growing pressure on livestock producers in other parts of the world, antibiotic free alternative substances and strategies for animal growth promotion and disease prevention have been investigated over the years. Phytogenic and herbal products have received increased attention in recent years because they have been accepted by consumers as natural additives (Toghyani et al., 2010).

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N. Landy et al. / Livestock Science 142 (2011) 305309 Table 1 The ingredient and calculated composition of basal starter, grower, and finisher diets. Item Ingredient, g/kg Corn Soybean meal Soybean oil Dicalcium phosphate CaCO3 NaCl Trace mineral premixa Vitamin premixb DL-methionine L-lysine Calculated composition Metabolizable energy (kcal/kg) Crude protein (g/kg) Calcium (g/kg) Available phosphorus (g/kg) Methionine + cysteine (g/kg) Lysine (g/kg) Threonine (g/kg) Tryptophan (g/kg) Starter 537.3 400 20 19.3 10.5 3.5 2.5 2.5 3.1 1.3 2870 221 8.6 4.9 10.1 13.3 8.3 3.2 Grower 533 396 35 17.1 8.7 3 2.5 2.5 2 2980 220 7.5 4.4 8.9 11.9 8.3 3.2 Finisher 561.5 370 35 15.6 8.5 3 2.5 2.5 1.4 3000 210 7 4.1 8 11.3 6.3 3

Neem (Azadirachta indica) is a tropical evergreen tree native to Indian sub-continent. Neem possesses limonoids, protolimonoids, tetranortriterpenoids, pentanortri-terpenoids, hexanortriterpenoids, and some nonterpenoid (Koul et al., 2006). Most of the plant parts such as fruits, seeds, leaves, bark, and roots contain compounds with proven antiseptic, antiviral, antipyretic, anti-inflammatory, antiulcer, and antifungal properties (Brahmachari, 2004). Also, NRC (1992), reported that, neem contains the chemical, azadirachtin, which has positive effects on pests, and deformental effect on viruses, mites, fungal pathogens, plant parasitic nematodes, intestinal worms, bacteria, mollusks, and protozoan parasites such as coccidian species. Furthermore, Sadekar et al. (1998) and Ahsan et al. (1999) observed the beneficial influence of neem leaves on antibody titers against new castle disease virus (NCDV) antigen in broiler chickens. Despite these findings, there has been a dearth of information on the possible immunopotentiating effects of neem dry fruit powder on broiler chickens. The present study was designed to compare the efficacy of different levels of neem fruit powder an antibiotic growth promoter on growth performance, carcass characteristics, and some immune responses in broiler chickens when used as supplements in the diet. 2. Materials and methods 2.1. Animals and dietary treatments One hundred ninety two, 1-d-old broiler chickens of mixed sexes (Ross-308) were weighted and randomly assigned to each of the 4 treatment groups, each with 4 replicate pens of 12 chicks. A commercial antibiotic growth promoter was supplemented to basal diet with no additives. The dietary treatments were basal diet (control), control+4.5 mg flavophospholipol/ kg, or control+7 or 12 g neem fruit powder/kg diet. Table 1 lists the basal diet formulated to meet or exceed the nutrient requirements of broilers (Aviagen, 2002). The birds were fed a starter diet from 0 to 14 d, a grower diet from 14 to 28 d, and finisher diet from 28 to 42 d. All the dietary treatments were added to the basal diets at the expense of corn. Chicks were raised on floor pens (120 120 80 cm) for 6 weeks and had free access to feed and water throughout the entire experimental period. The lighting program consisted of a period of 23 h light and 1 h of darkness. The ambient temperature in experimental house was maintained at 32 C during the first week and gradually decreased by 3 C in the second and third weeks, and finally fixed at 22 C thereafter. 2.2. Performance and carcass components Body weights of broilers were determined at 1, 14, 28, and 42 d of age. Daily body weight gain (DWG) and daily feed intake (DFI) were recorded in different periods and feed conversion ratio (feed intake/weight gain) was calculated. Mortality was recorded as it occurred. At 42 d of age, 2 birds per replicate were randomly chosen, based on the average weight of the group and slaughtered through cutting carotid arteries and partial slicing of the neck by a manual neck cutter. Carcass yield was calculated by dividing eviscerated weight by live weight. Empty proventriculus, empty gizzard, empty small intestine, abdominal

a Provided the following per kg of diet: Mg, 56 mg; Fe, 20 mg; Cu, 10 mg; Zn, 50 mg; Co, 125 mg; I, 0.8 mg. b Provided the following per kg of diet: vitamin A, 10,000 IU; vitamin D3, 2000 IU; vitamin E, 5 IU; vitamin K, 2 mg; riboflavin, 4.20 mg; vitamin B12, 0.01 mg; pantothenic acid, 5 mg; nicotinic acid, 20 mg; folic acid, 0.5 mg; choline, 3 mg.

fat pad, and heart were also removed, weighted, and calculated as a percentage of live weight. Small intestine and ceca length were measured. 2.3. Immunity The commercially available oil-adjuvant injectable emulsion against Newcastle Disease virus (NDV) and Avian Influenza virus (AIV) were used (H9N2 subtype) for vaccinating broiler chicks, and they were injected subcutaneously with 0.2 mL per chick at 9 d of age. Also, chicks were orally vaccinated against Newcastle Disease (Lasota) at 21 d of age. Antibody titers against NDV, AIV, and sheep red blood cells (SRBC), and heterophil to lymphocyte (H/L) and albumin to globulin (A/G) ratios were measured as immune responses. At 25 d of the experiment, 2 birds per pen were inoculated via the brachial vein with 1 mL of 1% SRBC suspension. At d 6 post-SRBC, blood samples were taken and plasma was collected. Antibody titers against SRBC were measured by the microtiter procedure described by Wegmann and Smithies (1966). Titers were expressed as the log2 of the reciprocal of the highest dilution giving visible hemagglutination. At 28 d of age, 2 male broilers from each pen were randomly selected, and blood samples were taken by puncture of the brachial vein for analysis of antibody titers against NDV and AIV. Serum antibody titers against NDV and AIV were measured by the hemagglutination inhibition test (HI), and HI antibodies were then converted to log2. At d 42, 2 broilers per pen were selected and their blood samples were collected using syringes containing heparin to avoid blood clot formation. Blood smears were prepared using MayGreenwaldGiemsa stain (Lucas and Jamroz, 1961). One hundred leukocytes per samples were counted

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by heterophil to lymphocyte separation under an optical microscope (Nikon, Tokyo, Japan) with 100 oil immersion lens, then heterophil to lymphocyte (H/L) ratio was calculated and recorded (Gross and Siegel, 1983). To determine albumin to globulin ratio, blood samples (2 broilers per pen) were collected at 42 d of age, and after serum separation, albumin and protein concentrations were determined using spectrophotometer and the kit (Pars Azmoon Company; Tehran, Iran). Globulin concentration in serum was computed by subtracting albumin concentration from proteins. 2.4. Statistical analysis The data were subjected to analysis of variance procedures appropriate for a completely randomized design using the General Linear Model procedures of SAS (SAS Inst. Inc., Cary, NC). Means were compared using Tukey test. Statements of statistical significance are based on Pb 0.05. 3. Results and discussion 3.1. Performance and carcass traits Data on performance indices are summarized in Table 2. The body weight obtained in our broiler chickens was lower than the breed standard at different ages. This project was performed in Khorasgan Azad university research farm, which is located around Isfahan city, with an altitude of 1680 m. According to Julian (2007) the partial pressure of oxygen drops approximately 7 mm Hg for each 1000 m increase in altitude, equivalent to a drop of 2.5% in the air oxygen. Beker et al. (2003) showed that in the broilers raised under low pressure atmospheric O2, growth rate is retarded due to a depression in feed intake. Thus, besides feeding a mash diet, to a large extent, the relatively high altitude could account for the decreased growth rate of broilers at different ages. Because of foregoing problems, our chicks did not reach the target weight
Table 2 Effect of experimental diets on performance indices of broilers at different ages. Variables Dietary treatments Control Body weight (g) 14 d 262 28 d 1004 42 d 2109ab Daily feed intake (g/d) 014 d 24 1428 d 93 2842 d 166 042 d 91 Feed:gain (g:g) 014 d 1428 d 2842 d 042 d
a,b 1

for Ross 308 and had lower weights at different ages. Broilers receiving antibiotic had higher body weights compared to 7 g neem/kg at 42 d of age (P b 0.05). Treatments failed to induce any marked effect on daily feed intake. Broilers receiving flavophospholipol or basal diet had lower feed conversion ratio (FCR) compared to broilers receiving 7 g neem/kg during finisher period and entire experimental period (P b 0.05) but FCR of broilers in other periods was not affected. No differences because of treatment effects were observed on mortality. Antibiotics may control and limit the growth and colonization of a variety of pathogenic and nonpathogenic species of bacteria in chick gut (Ferket, 2004). A more balanced biota population in the gastrointestinal tract of poultry could lead to a greater efficiency in digestibility and utilization of food, resulting in an enhanced growth and improved FCR (Bedford, 2000). Similar to our results, Tipu et al. (2002) reported an enhancement in body weight gain of broilers offered diets containing Salinomycin Sodium (Sacox) in comparison with broilers fed diets containing neem fruit and no differences in feed intake between treatment groups. Unfortunately, no other reports are available on the effects of neem fruit on bird growth performance. Table 3 shows carcass, abdominal fat, and relative organ weights as a percentage of live weight at slaughter, and absolute small intestine and cecum lengths. Carcass yield, abdominal fat, gizzard, proventriculus, and small intestine weights, or small intestine and ceca lengths were not markedly affected by dietary treatments. These results are consistent with those reported by Hernandez and Madrid (2004) who did not find any differences among the control treatment and those containing antibiotic or mixtures of plant extracts on organ weight of 42-d-old broilers. In addition, in another study, carcass traits of broilers fed diet supplemented with Echinacea purpurea L. or antibiotic were not affected by dietary treatments (Landy et al., 2011). 3.2. Immune responses The effect of experimental diets on humoral immune responses is presented in Table 4. The treatments had no effect on antibody titer against NDV. Use of antibiotic, flavophospholipol, failed to have any effect on antibody titer against AIV, and SRBC in comparison with control groups. On the other hand, antibody titer against AIV increased in the group treated with 7 g neem/kg diet compared with those on the control and

SEM1 12 g neem fruit/kg 255 1032 2060b 8 28 45

Flavophospholipol 7 g neem fruit/kg 275 1085 2163a 261 1034 2014b

Table 3 Effect of experimental diets on carcass yield and internal relative organ weight of broilers at 42 d. Relative organ weight Dietary treatments 12 g Control Flavophospholipol 7 g neem neem fruit/kg fruit/kg Carcass (%) 76.2 Abdominal fat (%) 2.03 Gizzard (%) 3.42 Proventriculus (%) 0.71 Small intestine (%) 5.41 Small intestine 195.0 (cm) Cecum (cm) 39.4
a

SEMa

25 94 163 93

24 91 160 89

24 92 161 89

0 2 1 1

1.41 1.88 2.09b 1.84b

1.39 1.74 2.11b 1.83b

1.45 1.76 2.34a 1.99a

1.48 1.74 2.19ab 1.85b

0.03 0.04 0.05 0.03

75.7 1.88 3.02 0.60 4.73 195.6 41.6

74.2 2.20 2.92 0.56 4.37 183.7 41.4

75.5 2.14 3.10 0.49 4.40 182.1 40.0

0.7 0.11 0.16 0.02 0.30 4.8 1.3

Values in the same row not sharing a common superscript differ (P b 0.05). Standard error of mean.

Standard error of mean.

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Table 4 Effect of experimental diets on antibody titers against Newcastle and Influenza viruses at d 28 and sheep red blood cells (SRBC) at d 31, and heterophil to lymphocyte and albumin to globulin ratios at d 42. Variables Dietary treatments Control Flavophospholipol 7g neem fruit/kg 7.87 6.25a 8.0a 0.57 12 g neem fruit/kg 7.85 5.37ab 5.7c 0.56 0.65 0.50 0.9 0.00 SEM1

an index of response to a stressor (Gross and Siegel, 1983). According to the H/L ratio, the neem fruit supplementation at 7 or 12 g/kg diet tended to decrease the H/L ratio of broiler chickens. The H/L ratio is recognized as a measure of stress in birds (Davison et al., 1983; Gross and Siegel, 1983; Maxwell, 1993) and has become a valuable tool in stress research. 4. Conclusion In conclusion, the results indicate that supplementing broiler diet with 7 g neem/kg could indicate favorable influences on immune responses of broilers without any adverse effects on growth performance. Acknowledgment This project was supported by the Young Researchers Clubs, Islamic Azad University, Khorasgan Branch, Isfahan, Iran. References
Ahsan, U.H., Meraj, K.A., Rasool, S., 1999. Effect of supplementing Allium sativum (garlic) and Azadirachta indica (neem) leaves in broilers feed on their blood cholesterol, triglycerides and antibody titer. Int. J. Agri. Biol. 1, 125127. Aviagen, 2002. Ross Broiler Management Manual. Aviagen Ltd, Midlothian, UK. Bedford, M., 2000. Removal of antibiotic growth promoters from poultry diets. World's Poult. Sci. J. 56, 347365. Beker, A., Vanhooser, S.L., Swartzlander, J.H., Teeter, R.G., 2003. Graded atmospheric oxygen level effects on performance and ascites incidence in broilers. Poult. Sci. 82, 15501553. Belay, T., Deyhim, F., Teeter, R.G., 1992. Effect of virginiamycin supplementation on growth and humoral mediated immunity of broilers. Poult. Sci. 71 (Suppl), 137 (Abstr.). Brahmachari, G., 2004. Neem an omnipotent plant. Chem. Biochem. 5, 408421. Cebra, J.J., 1999. Influences of microbiota on intestinal immune system development. Am. J. Clin. Nutr. 69 (Suppl), 1051S1064S. Dafwang, I.I., Cook, M.E., Sunde, M.L., Bird, H.R., 1985. Bursal intestinal and spleen weights and antibody response of chicks fed subtherapuetic levels of dietary antibiotics. Poult. Sci. 64, 634639. Davison, T.F., Rowell, L.G., Rea, J., 1983. Effects of dietary corticosterone on peripheral blood lymphocyte and granulocytes populations in immature domestic fowl. Res. Vet. Sci. 34, 236329. Engberg, R.M., Hedemann, M.S., Leser, T.D., Jensen, B.B., 2000. Effect of zinc bacitracin and salinomycin on intestinal microflora and performance of broilers. Poult. Sci. 79, 13111319. Eyssen, H., DeSomer, P., 1963. The mode of action of antibiotics in stimulating growth of chicks. J. Exp. Med. 117, 127137. Ferket, P.R., 2004. Alternatives to antibiotics in poultry production. In: Lyons, T.P., Jacques, K.A. (Eds.), Nutritional Biotechnology in the Feed and Food Industries. E-publishing Inc., Nottingham, UK, pp. 5767. Gross, W.B., Siegel, P.S., 1983. Evaluation of heterophil to lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27, 972979. Harms, R.H., Ruiz, N., Miles, R.D., 1986. Influence of virginiamycin on broilers fed four levels of energy. Poult. Sci. 65, 19841986. Hernandez, F., Madrid, J., 2004. Influence of two plant extracts on broilers performance, digestibility and digestive organ size. Poult. Sci. 83, 169174. Julian, R.J., 2007. The response of heart and pulmonary arteries to hypoxia, pressure and volume: a short review. Poult. Sci. 86, 10061011. Koul, A., Ghara, A.R., Gangar, S.C., 2006. Chemomodulatory effects of Azadirachta indica on the hepatic status of skin tumor bearing mice. J. Int. Sci. 20, 169177. Landy, N., Ghalamkari, G.h., Toghyani, M., Moattar, F., 2011. The effects of Echinacea purpurea L. (purple coneflower) as an antibiotic growth promoter substitution on performance, carcass characteristics and humoral immune response in broiler chickens. J . Med. Plants. Res. 5, 23322338. Lu, L., Walker, W.A., 2001. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am. J. Clin. Nutr. 73 (Suppl), 1124S1130S.

New castle (log2) Influenza (log2) SRBC (log2) Heterophil to lymphocyte ratio Albumin to globulin ratio
a,b

7.50 3.42c 7.0abc 0.64

6.87 4.37bc 7.5ab 0.62

0.65b

1.01a

0.70b

0.71b

0.01

Values in the same row not sharing a common superscript differ significantly (Pb 0.05). 1 Standard error of mean.

flavophospholipol diets at 28 d of age (Pb 0.05). The highest SRBC antibody titer was observed in the group receiving 7 g neem/kg diet, but it was not different from broilers fed the control or flavophospholipol diet. The SRBC antibody titer observed in birds fed the diet containing 7 g neem/kg diet significantly was greater than those fed 12 g neem/kg diet (Pb 0.05). In other trial with rats, Sairam et al. (1997) reported that, injection of NIM-76, a volatile fraction from neem, resulted in an increase in polymorphonuclear leukocyte, and NIM-76 acted through cell-mediated mechanisms by activating macrophages and lymphocytes. Sadekar et al. (1998) showed that dry leaves of neem might potentiate the humoral immune response to NDV. Also, Ray et al. (1996) reported that, mice treated with 100 mg leaf extract of neem/kg diet had greater IgM and IgG levels, and anti-ovalbumin antibody titers compared to the control group. Ahsan et al. (1999) showed that neem leaves might potentiate the immune response to Infectious Bursal Disease and NDV in broiler chickens. The highest albumin to globulin ratio obtained in broilers fed diets containing antibiotic. Use of flavophospholipol failed to have any effect on antibody titer against NDV, AIV and SRBC in comparison with control group. Also, H/L ratio was not affected by flavophospholipol treatment. Dafwang et al. (1985) reported a slight response of broiler antibodies against SRBC to diets supplemented with oxytetracycline, lincomycin, penicillin, bambermycins or tylan. Belay et al. (1992) reported no effect of dietary virginiamycin on the total IgG, IgM antibody titers in 7 week old broiler chickens. Therefore, the influence of enteric conditioners and their effects on gut microflora could be mainly limited to the mucosal immune complement and not the systemic portion of the immune system. Although the contribution of gut microflora to the development and physiological status of the humoral and cellular mucosal immune system is well understood (Cebra, 1999; Lu and Walker, 2001), the effects on the systemic immune complement may be less dominant. Treatments failed to induce any effect on H/L ratio, though it tended to decrease in broilers fed diets containing 7 or 12 g neem/kg. In birds, the heterophils are phagocytic cells whose main role is to provide protection against invading microorganisms. Heterophils increase and lymphocytes decrease when birds are stressed, so that the ratio between them is

N. Landy et al. / Livestock Science 142 (2011) 305309 Lucas, A.M., Jamroz, C., 1961. Atlas of Avian Hematology. Agriculture Monograph 25. USDA, Washington, DC. Maxwell, M.H., 1993. Avian blood leukocyte responses to stress. World's Poult. Sci. J. 49, 3443. Miles, R.D., Janky, D.M., Harms, R.H., 1984. Virginiamycin and broiler performance. Poult. Sci. 63, 12181221. Nasir, Z., Grashorn, M.A., 2006. Use of Black cumin (Nigella sativa) as alternative to antibiotics in poultry diets. 9th Tagung Schweine-und Geflgelernhrung, Halle (Saale), Germany, pp. 210213. NRC, 1992. Neem: A Tree for Solving Global Problems. Natl. Acad. Press, Washington, DC. Ray, A., Banerjee, B.D., Sen, P., 1996. Modulation of humoral and cell-mediated immune responses by Azadirachta indica (neem) in mice. Indian J. Exp. Biol. 34, 698701. Rosen, G.D., 1996. Pronutrient antibiotic replacement standards discussed. Feedstuffs 75, 1113. Sadekar, R.D., Kolte, A.Y., Barmase, B.S., Desi, V.F., 1998. Immunopotentiating effects of Azadirachta indica (neem) dry leaves powder in broiler, naturally infected with IBD virus. Ind. J. Exp. Biol. 36, 11511153.

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Sairam, M., Sharma, S.K., Ilavazhagan, G., Kumar, D., Selvamurthy, W., 1997. Immunomodulatory effects of NIM-76, a volatile fraction from neem oil. J. Ethnopharmacol. 55, 133139. Tipu, M.A., Pasha, T.N., Zulfiqar, A., 2002. Comparative efficacy of salinomycin sodium and Neem fruit (Azadirachta indica) as feed additive anticoccidials in broilers. Int. J. Poult. Sci. 1, 9193. Toghyani, M., Toghyani, M., Gheisari, A.A., Ghalamkari, G.h., Mohammadrezaei, M., 2010. Growth performance, serum biochemistry, and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and peppermint (Mentha piperita). Livest. Sci. 129, 173178. Visek, W.J., 1978. The mode of growth promotion by antibiotics. J. Anim. Sci. 46, 14471469. Wegmann, T.G., Smithies, O., 1966. A simple hemagglutination system requiring small amounts of red blood cells and antibodies. Transfusion 6, 6773.