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1. Livestock Science 147 (2012) 113–118 Contents lists available at SciVerse ScienceDirect Livestock Science journal homepage: www.elsevier.com/locate/livsci Role of in-feed clove supplementation on growth performance, intestinal microbiology, and morphology in broiler chicken P.S. Agostinia, D. Sol? a-Oriola, M. Nofrarı ´asb, A.C. Barroetaa, J. Gasaa, E.G. Manzanillac,n aGrup de Nutricio ´, Maneig i Benestar Animal, Departament de Ci? encia Animal i dels Aliments, Facultat de Veterinaria, Universitat Aut? onoma de Barcelona, 08193 Bellaterra, Barcelona, Spain bCentre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, 08193 Bellaterra, Barcelona, Spain cDepartment of Animal Science, University of California, Davis, CA 95616, United States a r t i c l e i n f o a b s t r a c t The objective of the present work was to evaluate the effect of different dietary inclusion levels of clove on growth performance, digestibility, intestinal microbiology, and morphology of broiler chickens. In a first experiment, 800 one-d-old male Ross 308 chicks were randomly allocated to 40 replicate pens with 20 chicks per pen. Five inclusion levels of clove were tested: 0, 100, 200, 1000, and 2500 mg/kg. Treatments were fed from 0 to 35 d of age. Feed to gain ratio was modified with clove inclusion showing a cubic effect (Po0.05). Levels of 100 and 200 mg/kg showed the lower feed to gain ratio. At 21 and 35 d of age, 2 broiler chickens per pen were selected from diets containing 0 and 100 mg/kg of clove and samples from ileal content and tissue were collected to evaluate microbiology and intestinal morphology. Enterobacteriaceae counts were not affected by clove supplementation at 21 d of age, but the number of Lactobacillus was increased compared to the control diet (9.62 vs. 9.18 log cfu/g digesta; Po0.05). Moreover, clove inclusion increased the number of intraepithelial lympho- cytes (1.55 vs. 2.22 lymphocytes/100 enterocytes; Po0.01) and lamina propria cell density (5.98 vs. 6.84 cells/1000 mm2; Po0.05) in the intestinal epithelium of 21 d-old broiler chickens. In a second experiment, 150 d-old male Ross 308 broiler chickens were randomly allocated to 30 cages with 5 broiler chickens per pen. From 19 to 21 d of age, feed intake and total excreta from each cage were recorded and sampled daily and the effect of the same 5 levels of clove on DM and OM digestibility was assessed but, no differences were observed. Clove at doses of 100–200 mg/kg seem to improve feed efficiency. Interesting changes in intestinal microbiota and epithelium were observed but further studies are required to clarify the mode of action of clove in broiler chickens. Article history: Received 22 April 2011 Received in revised form 12 April 2012 Accepted 19 April 2012 Keywords: Broiler chickens Clove E. coli Immune response Lactobacillus & 2012 Elsevier B.V. All rights reserved. 1. Introduction decades. However, concerns about the development of antimicrobial resistance from animal to human bacteria have led to a ban in the use of AGP in some regions as the European Union since 2006. The ban of the use of AGP in animal feed is known to have increased the therapeutic use of antibiotics in some cases (Wierup, 2001) which is not a good solution either and effective alternatives are needed. The poultry industry is already using alternative pro- ducts that help replace AGP ensuring optimal growth in broiler chickens. It has been shown that under optimal The use of antibiotics as growth promoters (AGP) in animal feeds has allowed great growth performance and feed efficiency in the intensive poultry industry in the last nCorresponding author. Tel.: þ34 93 581 1556; fax: þ34 93 581 1494. E-mail address: egmanzanilla@gmail.com (E.G. Manzanilla). 1871-1413/$-see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2012.04.010

2. 114 P.S. Agostini et al. / Livestock Science 147 (2012) 113–118 production conditions, it is possible to reach competitive growth results without the use of AGP (Engster et al., 2002). However, poultry rearing is not always done under optimal conditions and more research on alternatives to AGP is required. Different substances have been studied as alter- natives to AGP in broiler chicken production such as probiotics and prebiotics (Patterson and Burkholder, 2003), enzymes (Rosin et al., 2007), acidifiers (Ricke, 2003), immu- nomodulating agents (Dalloul et al., 2006), and essential oils (EO) or plant extracts (Herna ´ndez et al., 2004). In recent years, EO have received special attention as alternatives to AGP but more studies are needed to optimize their use (Brenes and Roura, 2010). Although their mode of action has not yet been fully elucidated, some hypotheses have been proposed. The main hypoth- esis tested was the antimicrobial effect; however, histo- logical changes in the gastrointestinal tract, modulation of the immune response (Middleton and Kandaswami, 1992), stimulation of enzyme Srinivasan, 2004), or anti-oxidant effects (Aruoma et al., 1996) can be other proposed mechanisms of action. Phenolic compounds, such as carvacrol, thymol, or eugenol, are the most commonly used EO as additives in animal nutrition because of their antimicrobial activity. Eugenol is the main compound (72–90%) in Syzigium aromaticum (clove). Clove, and its essential oil, is one of the plant extracts that has been found effective in poultry to improve growth performance (Ertas et al., 2005; Isabel and Santos, 2009; Najafi and Torki, 2010) and to control some intestinal pathogens (Mitsch et al., 2004; Ordonez et al., 2008), and is currently used, alone or in combina- tion with other extracts, in commercial production. It is hypothesized that changes in digestibility, intestinal microbiota, and/or in intestinal mucosa are involved in the mechanism of action of clove. Therefore, the present study was conducted to evaluate the effect of increasing dietary levels of clove on growth performance and digestibility of broiler chickens, and the effects of cur- rently used doses on intestinal microbiology and histolo- gical characteristics. Table 1 Ingredients and chemical composition of the experimental diets. Basal diet (g kg?1) Item Starter Grower Ingredients, as-fed basis Corn Barley Soybean meal, 48% CP Wheat Extruded soybeans Soybean oil Lard L-Lysine DL-Methionine L-Threonine Calcium carbonate Calcium phosphate Sodium Bicarbonate Salt Vitamin-mineral premixa Additivesb Composition (% DM) AMEc(kJ/kg), calculated Ash, analyzed Crude protein, analyzed Ether extract, analyzed Lysine, calculated Methionineþcystine, calculated Threonine, calculated Calcium, calculated Phosphorus, calculated Linoleic acid, calculated 488.9 – 278.2 120.0 50.0 20.8 – 3.33 3.35 0.12 12.34 13.68 1.11 3.00 4.00 1.20 – 147.6 258.6 500.0 – 15.0 43.5 3.79 2.79 0.29 13.99 4.33 0.94 2.50 4.75 2.00 activity (Platel and 12.87 5.98 20.87 5.23 1.269 0.957 0.825 0.850 0.691 – 13.39 5.19 20.40 7.63 1.153 0.868 0.742 0.850 0.493 1.866 aVitamin and mineral premix per kg of feed: vitamin A: 12,000 IU as retinyl acetate; vitamin D: 2400 IU as cholecalciferol; vitamin E: 30 mg as all-rac-alpha-tocopheryl acetate; vitamin B1: 2.2 mg as thiamine hydrochloride; riboflavin: 8 mg; vitamin B12: 11 mg as cyanocobalamin; vitamin B6: 5 mg as pyridoxine HCl; vitamin K: 3 mg as menadione nicotinamide bisulfite; pantotheni acid: 25 mg as calcium-d-pantothe- nate; niacin: 65 mg as nicotinic acid; biotin: 150 mg as d-(þ)-biotin; folic acid: 1.5 mg; Fe: 80 mg as FeSO4?H2O; Cu: 8 mg as CuSO4?5H2O; Zn: 40 mg as ZnSO4; Mn: 60 mg as MnO; I: 0.33 mg as Ca(IO3); Se: 0.15 mg as Na2SeO3; and ethoxyquin: 150 mg. bStarter diet: Maxiban (0.60 g/kg in a 50% premix added at 1.20 g/kg), Lilly S.A., Madrid, Spain. Grower diet: Nutral-fit Broiler (phytase, 0.75 g/kg) and Nutralzyme Aviar-T (beta-glucanase and xylanase, 0.75 g/kg), Nutral S.A., Colmenar Viejo, Madrid, Spain; and Elancoban (0.5 g/kg), Lilly S.A., Madrid, Spain. cAME¼apparent metabolizable energy. 2. Materials and methods 2.1. Diets and clove product were prepared for each phase: 0, 100, 200, 1000, or 2500 mg/kg of the clove product. Thus, estimated con- centrations of clove and eugenol in the supplemented diets were 30, 60, 300, and 750 mg, and 25, 50, 250, and 625 mg/kg, respectively. The feeding program consisted of a starter diet until 21 d of age and a grower diet from 21 to 42 d of age. Diets were pelleted and the starter feed was offered as crumbles. Diets were formulated to meet or exceed broiler chickens nutrient requirements (FEDNA, 2008). Composition of the experi- mental diets is shown in Table 1. Narasin/nicarbasin (Maxiban, Lilly S.A., Madrid, Spain) and monensin (Elancoban, Lilly S.A., Madrid, Spain) were included in the starter and grower diet, respectively, as coccidiostats. The grower diet also contained phytase (Nutral-fit Broiler, Nutral S.A., Colmenar Viejo, Madrid, Spain) and a beta-glucanase/ xylanase combination (Nutralzyme Aviar-T, Nutral S.A., Colmenar Viejo, Madrid, Spain). Diets were supplemented with the experimental pro- duct containing 30% (wt/wt) clove oil (85% eugenol) in an inert mineral carrier. A total of 5 experimental treatments 2.2. Animals and experimental design Two experiments were conducted. All experimental procedures involving broiler chickens in this study were conducted at the Animal Nutrition Unit of the Universitat Aut? onoma de Barcelona, after approval by the Ethical Committee on Animal Experimentation. All broiler chick- ens used were 1 d-old male Ross 308 chicks vaccinated for infectious bronchitis and infectious bursal disease. Feed and water were provided ad libitum and temperature was

3. 115 P.S. Agostini et al. / Livestock Science 147 (2012) 113–118 controlled throughout the experiments following the Ross 308 broiler manual (/http://en.aviagen.com/ross-308S). In experiment 1, a total of 800 chicks were allocated to 40 floor pens of 20 chicks each and the pens were randomly assigned to the experimental treatments (8 replicates per treatment). The same experimental treatments were pro- vided continuously from 1 d until 35 d of age (starter–grower phase). From d 35 to 42 (finishing phase), all broiler chickens were fed the control diet. Broiler chickens were slaughtered under commercial conditions at 42 d of age. Broiler chickens were weighed and feed intake per pen was recorded on d 1, 21, 35, and 42 of the experiment, and feed to gain ratio (F:G) was calculated. At 21 and 35 d of age, individual broiler chickens were weighed, and 2 broiler chickens per pen, closest to the mean, were selected and killed by carbon dioxide. The gastrointestinal tract was immediately removed from broiler chickens on the control and 100 mg/kg of clove, and the small intestine was then separated. The ileum was defined as the region comprised between Meckel’s diverti- culum and the ileocecal junction. The content of the distal half of the ileum was collected into plastic tubes containing ethanol, and a tissue sample of the ileum was collected, opened longitudinally, and fixed by immersion in 10% (vol/ vol) buffered formalin for the histological study. Experiment 2 was designed to assess the digestibility of the same experimental diets used in experiment 1. A total of 150 broiler chickens were allocated to 30 meta- bolism cages (5 per cage) and the cages were randomly assigned to 5 experimental treatments (6 replicates per treatment). Fresh feed and water were provided daily. From d 19 to 21, feed intake and total excreta from each cage were recorded and sampled daily. Samples were stored at ?18 1C until laboratory analyses. Intravillus lamina propria cell density was determined by counting total visibly stained nuclei in 10 fields (total area of 4000 mm2) from each section using a grid ocular (Olympus, Microplanet). Cell density was expressed as number of total stained cells per 1000 mm2. All the morphometric analyses were conducted by the same person, who was blinded to the treatments. 2.4. Calculations and statistical analysis The apparent total tract digestibility coefficients of DM and organic matter (OM) were calculated based on amount of DM and OM excreted in feces, and relative to the amount of nutrient consumed using the following formula: 100?[(Total amount of DM or OM excreted in feces/Total amount of DM or OM ingested in feed)?100]. Growth performance traits and nutrient digestibility values were assessed by ANOVA. When ANOVA was significant values were assessed for linear, quadratic, and cubic orthogonal contrasts. Microbial counts and intestinal morphometric characteristics were assessed by t-test. The alpha level used for the determination of significance was 0.05. All statistical analyses were per- formed using SAS (Version 9.2; SAS Inst. Inc., Cary, NC). 3. Results 3.1. Growth performance Growth performance results are presented in Table 2. Initial BW (39.470.70 g/chick) did not differ between groups prior to the assignment of the experimental diets. On d 21 post-hatch, treatment showed a cubic trend on BW (Po0.07). No other differences were found on BW or ADFI. Treatment with clove product showed cubic effects on F:G on d 0 to 21 and 21 to 35 (Po0.05 and Po0.01, respectively) being the lowest values found for doses of 100 and 200 mg/kg. When treatments were removed from the diets in finishing phase (35–42 d) and all broiler chickens were fed control diet, no differences were found on growth performance. No differences were found on digestibility of DM and OM among treatments. 2.3. Analytical methods Feed and fecal samples were analyzed for dry matter (DM), ash, crude protein (CP), and ether extract contents following AOAC (1995) procedures. Samples of 400 mg of digesta preserved in ethanol were precipitated by centrifu- gation (13,000g for 5 min). The DNA in the precipitate was extracted and purified using a commercial kit (QIAamp DNA Stool Mini Kit, Qiagen, West Sussex, UK). Lactobacillus and enterobacteria were quantified using real-time PCR follow- ing the methods described by Castillo et al. (2006). Ileum samples were dehydrated and embedded in paraffin wax, sectioned at 4 mm, and stained with hema- toxylin and eosin. Morphometric measurements were performed with a light microscope (BHS, Olympus, Bar- celona, Spain). Villus height (VH), crypt depth (CD), intraepithelial lymphocyte (IEL) number in the villi, and intravillus lamina propria cell density were measured. Measurements were taken in 10 well-oriented villi and crypts from each intestinal section of each broiler chicken. On the basis of cellular morphology, differences between the nuclei of enterocytes, and lymphocytes were clearly distinguishable at 400? magnification. The VH and CD were measured using a linear ocular micrometer (Olym- pus, Microplanet, Barcelona, Spain). The same villus and crypt columns were used to determine the number of IEL expressed as absolute values and per 100 enterocytes. 3.2. Intestinal microbial counts The log colony forming unit values of enterobacteria and lactobacillus and the enterobacteria to and lactoba- cillus ratio (E:L) determined in the ileal contents at 21 and 35 d of experiment are presented in Table 3. At d 21, no differences were observed for enterobacteria between the broiler chickens fed the control diet and those fed the diet supplemented with 100 mg/kg of clove product. However, the inclusion of 100 mg/kg of clove product increased the number of lactobacillus decreased E:L (Po0.05). No treatment differences were observed at d 35 between groups. (Po0.05), resulting in a 3.3. Ileal morphology Morphometric measurements made in the ileum are presented in Table 4. No differences were found in VH, CD,

4. 116 P.S. Agostini et al. / Livestock Science 147 (2012) 113–118 Table 2 Effects of dietary clove product inclusion on growth performance and digestibility of broiler chickensa. SEMc P-valued Clove product (mg/kg) Itemb Quad Cubic 0 100 200 1000 2500 Linear LW d 0 d 21 d 35 d 42 ADFI d 0 to 21 d 21 to 35 d 35 to 42 d 0 to 42 F:G d 0 to 21 d 21 to 35 d 35 to 42 d 0 to 42 Digestibility (%) DM OM 39.4 919 1940 2505 39.4 942 1997 2593 39.4 932 2016 2593 39.5 916 1975 2583 39.6 916 1966 2551 0.3 14 28 45 0.07 0.44 0.07 58.8 148 198 110 58.7 147 196 110 56.0 147 201 108 57.6 149 199 109 58.2 150 197 110 0.8 2 5 2 o0.05 o0.01 o0.05 0.93 0.13 0.66 1.39 2.06 2.43 1.92 1.33 1.96 2.29 1.82 1.30 1.87 2.55 1.84 1.43 1.99 2.25 1.85 1.40 1.98 2.40 1.88 0.03 0.03 0.14 0.03 75.7 78.6 76.0 78.8 75.0 77.9 75.6 78.5 75.2 78.1 0.7 0.6 aClove product was removed from the diets from 35 to 42 d (finishing phase) and all broiler chickens were fed the control diet (0 mg/kg). bLW: live weight (g/broiler chicken); ADFI: average daily feed intake (g/broiler chicken/d); F:G: feed to gain; DM: dry matter; and OM: organic matter. cSEM: standard error of the mean (n¼8 and 6 for growth performance and digestibility, respectively). dData was assessed for linear, quadratic (Quad), and cubic effects by orthogonal contrasts when one-way ANOVA was significant (Po0.05). Table 3 Effects of clove product inclusion on in the ileal microbiota of broiler chickens. Table 4 Effects of dietary clove supplementation in epithelium of the ileum of broiler chickens. Itema SEMb Itema SEMb Clove product (mg/kg) P-value Clove product (mg/kg) P-value 0 100 0 100 d 21 Enterobacteriaceae Lactobacillus E:L d 35 Enterobacteriaceae Lactobacillus E:L d 21 VH, mm CD, mm VH:CD IEL, total IEL, 100 enterocytes LPC LPL LPC:LPL d 35 VH, mm CD, mm VH:CD IEL, total IEL, 100 enterocytes LPC LPL LPC:LPL 10.73 9.18 1.55 10.70 9.62 1.07 0.06 0.13 0.13 0.72 787.1 160.3 4.96 12.21 1.55 5.98 1.53 0.26 760.5 162.7 4.72 17.12 2.22 6.84 1.46 0.21 18.4 5.2 0.21 0.96 0.09 0.13 0.10 0.01 0.51 0.76 0.59 0.03 o0.05 o0.05 o0.01 o0.01 0.71 0.15 10.36 11.64 0.89 10.64 11.75 0.91 0.12 0.11 0.01 0.12 0.30 0.31 aEnterobacteriaceae (E) and Lactobacillus (L) are expressed as log cfu/g of digesta. bSEM: standard error of the mean (n¼8). 1,025.7 239.7 955.4 252.3 3.86 32.51 3.53 9.81 2.94 0.29 43.7 14.6 0.34 1.53 0.21 0.20 0.15 0.01 0.44 0.67 0.35 0.09 0.09 0.41 0.94 0.79 4.53 26.63 2.73 9.46 2.96 0.29 and VH:CD at d 21 and at 35 post-hatch by the inclusion of 100 mg/kg of clove product in the diet. However, cell numbers were affected by the clove inclusion at d 21 and tended to be affected at d 35. At d 21, broiler chickens fed the clove supplemented diets had higher number of IEL, both absolute counts (Po0.05) and per 100 enterocytes (Po0.01), and higher cellular density in the lamina propria (Po0.01). At 35 d the number of IEL, absolute (P¼0.09) and per 100 enterocytes (P¼0.90), tended to be greater for broiler chickens fed the clove supplemented diet than for the control diet. No effects were observed between diets for the density of lymphocytes in the lamina propria, or in the ratio of total cells to lymphocytes in the lamina propria at d 21 and 35. aVH: villous height; CD: crypt depth; IEL: intraepithelial lympho- cytes total and per 100 enterocytes; LPC: celular density of the lamina propria measured as cells per 1,000 mm2; and LPL: lymphocyte density of the lamina propria measured as lymphocytes per 1,000 mm2. bSEM: standard error of the mean (n¼8). 4. Discussion Clove oil has been found effective in improving broiler chicken growth performance in previous experiments (Ertas et al., 2005; Isabel and Santos, 2009; Najafi and

5. 117 P.S. Agostini et al. / Livestock Science 147 (2012) 113–118 Torki, 2010). However, there is some variability in the results depending on the dose. Thus, one of the objectives in this study was to assess the effect of different inclusion levels of clove oil on broiler chicken growth performance and digestibility. The improvement found on growth performance was not linearly related to the inclusion levels of clove, and the greater performance values were obtained at 100 and 200 mg/kg of clove oil inclusion. Similarly, Ertas et al. (2005) found better growth results in broiler chickens fed 100 or 200 mg/kg of a blend of EOs (oregano, clove, and anise) than using a 400 mg/kg dose. In the present study, the product also showed to be safe in doses as high as 750 mg/kg of clove, not showing any negative effects of clove reported by Kreydiyyeh et al. (2000) in rats. It is also interesting to see that growth performance improvement disappeared when clove oil was removed from the diets in finishing phase (35–42 d) and all broiler chickens were fed the control diet. Many plant extracts that can improve poultry growth performance are referred to as antimicrobial products. Dorman and Deans (2000) and Friedman et al. (2002) found that cinnamaldehyde, thymol, and carvacrol are very active molecules against a wide range of bacterial species in vitro. In those experiments, eugenol had good antimicrobial activity against some bacterial species such as Listeria monocytogenes and Campylobacter jejuni but had medium to low antimicrobial activity against enter- obacteria. However, Michiels et al. (2009) tested these extracts on pig intestinal microbiota and found similar effects of eugenol on coliforms compared to cinnamalde- hyde, thymol, and carvacrol, and lower effects on lactic acid bacteria. Combinations of cinnamaldehyde, thymol, and carvacrol have been found to decrease E. coli and lactobacilli in vivo in broiler chickens (Jamroz et al., 2003; Tiihonen et al., 2010), but there have been no data on the in vivo effect of clove oil or eugenol in broiler chicken intestinal microbiota. In this study, microbiological counts and intestinal morphometry were studied in broiler chickens, and their growth performance was improved when fed 100 mg/kg of clove product. Lactobacilli counts and E:L were increased for broiler chickens treated with clove. This change in the ratio could be related to a positive selection of eugenol towards lactic acid bacteria as shown by Michiels et al. (2009). However, the dose that improve growth performance in the present study, 100 mg clove or 30 mg active compound/kg, was much lower than the minimum inhibitory concentrations found for clove or eugenol against most bacteria (200–500 mg/ kg, Dorman and Deans, 2000). Therefore, no direct anti- microbial effects should be expected in vivo. In clove, it is also common to find b-caryophyllene, cinnamaldehyde, or a-humulene but in concentrations lower than 1% (Chaieb et al., 2007), which also rules out a blend effect that could have had greater antibacterial activity than its major components, as proposed by Mourey and Canillac (2002). Changes in immune response or in intestinal physiol- ogy could be proposed as mechanisms of action of clove, indirectly affecting microbial populations. In the present study, we observed an increase in the number of IEL and density of cells in the lamina propria. Whether a change on intestinal cell number is related to an improvement or an impairment of immune function is difficult to ascer- tain. A greater presence of lymphocytes in the epithelium is normally associated with an improvement of the immune response because these cells are involved in the identification of antigens and the modulation of the epithelial response (Matsumoto et al., 1999). However, infiltration of cells in the lamina propria is mainly related to an elevated inflammatory response (Jiang et al., 2000), which would be negative for growth performance indices. Nofrarı ´as et al. (2006) found that increased infiltration of lymphocytes in the lamina propria of ileum and colon was associated to greater lactobacilli counts in piglets fed a diet with a mixture of EO (carvacrol, cinnamaldehyde, and capsicum oleoresin) compared to control. In any case, effects of intestinal microbiota on immunity are well known (Haghighi et al., 2005) and the increase in IEL and density of cells in the lamina propria observed in our study could be related to the increase in the number of lactobacilli in the gut. 5. Conclusions This study showed that clove oil improves growth performance of broiler chickens, but it would depend on the dose in a non-linear way with the optimum dose being approximately 30–60 mg clove/kg feed. Changes on intestinal microbiota and epithelium may be involved in the mechanism of action of plant extracts. Future experi- ments should be designed to describe changes in the whole range of intestinal microbial populations and to clarify the causality chain among microbiota, immune system, and other possible changes in the intestinal ecosystem. Conflict of interest statement All authors declare that there is no conflict of interest of any financial or other relationship with other people or organizations that may inappropriately influence the author’s work or that might be construed to influence the results or interpretation of their manuscript. 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