SYMPOSIUM ARTICLES Effects of a bioactive olive pomace extract from Olea europaea on growth performance, gut function, and intestinal microbiota in broiler chickens J. Herrero-Encinas,∗ M. Blanch,† J. J. Pastor,† A. Mereu,† ,1 I. R. Ipharraguerre,† ,2 and D. Menoyo∗ ,3 ∗ Departamento de Producción Agraria, Universidad Politécnica de Madrid, ETS Ingeniería Agronómica Alimentaria y de Biosistemas, 28040 Madrid, Barcelona, Spain; and † Lucta S.A., Innovation Division, UAB Research Park, Edifici Eureka, 08193 Bellaterra, Barcelona, Spain ABSTRACT The present study aims to investigate served in birds fed the Control compared to Monensin the effects of supplementing broiler diets with a bioac- and OE groups. Performance of birds fed OE or Mo- tive olive pomace extract (OE) from Olea europaea on nensin was similar throughout the trial. The apparent growth performance, digestibility, gut microbiota, bile ileal digestibility of crude protein was higher in birds fed acid composition, and immune response. To this end, Monensin than Control treatment (P < 0.05). No signif- three hundred and six 1-day-old broiler chickens (Ross icant changes on bacterial composition at a family level 308) were housed in floor pens (6 pens/treatment, with were observed in the caeca of birds fed the experimen- 17 birds/pen). Animals were fed with a standard non- tal diets. Moreover, no significant differences on plasma medicated starter diet for 21 D, and from 22 to 42 D of and intestinal bile acid composition were observed age with their respective experimental diet: a negative among treatments. Birds fed the OE showed a signifi- control with no additives (Control), a positive control cant decrease of IL-8 expression in the ileum (P < 0.05). with 100 ppm of monensin (Monensin) and the basal Additionally, the expression of TGF-β 4, and Bu-1 was diet supplemented with 750 ppm of an OE (Lucta S.A., significantly upregulated (P < 0.01) in broilers fed the Spain). Feed intake and growth rate were monitored OE and Monensin diets compared to those fed the Con- weekly throughout the trial. From 21 to 42 D of age, trol. In conclusion, the inclusion of 750 ppm of a bioac- no significant differences in feed intake were observed tive olive pomace extract from Olea europaea in broiler among dietary treatments; however, lower average daily chicken diets improved animal growth likely as result of gain and higher feed conversion ratio (P < 0.05) was ob- its anti-inflammatory properties. Key words: broiler chicken, olive extract, performance, gut function, immune response 2020 Poultry Science 99:2–10 http://dx.doi.org/10.3382/ps/pez467 INTRODUCTION of byproducts such as olive pomace and cake in the feed has no adverse effects in broiler performance (El Agricultural eco-innovation is based on circular econ- Hachemi et al., 2007; Sayehban et al., 2016). Moreover, omy and cradle to cradle concepts. This innovation con- recent interest is being generated in the purification cept aims at “zero waste” economy where new products of the bioactive compounds (polyphenols, oleuropeo- and applications are created from raw material wastes side, flavonoid, and simple phenolics) from botanicals (Mirabella et al., 2014). Spain is the largest olive oil such as the olive by-products to enhance animal health (Olea europaea L.) producer worldwide with around 1.3 and performance (Liehr et al., 2017; Leskovec et al., million tons that generates wastes such as olive pomace 2018). In a global strategy to reduce the use of drugs and leaves totaling annually more than 4 and 0.2 million in animal production, plant extracts rich in bioactive tons, respectively (International Olive Council, 2017). compounds with anti-microbial, antioxidant, and anti- One of the ways to take advantage of the olive inflammatory properties are promising alternatives to oil industry wastes is its use in animal feed. It has antibiotics (Niewold, 2007, 2014; Lillehoj et al., 2018). been shown that the inclusion of up to 150 g/kg According to Niewold (2014) the positive effects of an- tibiotics on animal performance are directly related to their anti-inflammatory effects, which attenuate the Received January 18, 2019. Accepted July 30, 2019. intestinal inflammatory insults that take place under 1 Present address: Yara International ASA, P. O. Box 343, Skøyen, normal productive circumstances. Moreover, bioactive N-0213 Oslo, Norway phytochemicals can stimulate innate immunity and 2 Present address: Institute of Human Nutrition and Food Science, might be an alternative to control coccidiosis in poultry Christian-Albrechts-University Kiel, Hermann-Rodewald-Straße 6-8, D-24118 Kiel, Germany (Lillehoj and Lee, 2012). In this regard, it has been 3 Corresponding author: david.menoyo@upm.es recently suggested that olive pomace extracts might © 2019 The Authors. Published by Elsevier on behalf of Poultry Science Association Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 OLIVE POMACE EXTRACT IN BROILER DIETS 3 improve the intestinal health of calves and pigs (Liehr Table 1. Ingredients and chemical composition (%, as fed basis, et al., 2017; Morrison et al., 2017). Also, inclusion of an unless otherwise indicated) of pre-experimental (1 to 21 D) and experimental control diet (22 to 42 D). olive pomace bioactive extract in diets fed to sea bream showed a positive effect on growth performance by im- Pre-experimental diet Control diet proving liver lipid metabolism and the intestinal innate Ingredient immune function (Gisbert et al., 2017). Wheat (10.2% PB) 46.9 35.5 In addition, phenolic compounds extracted from olive Soy bean meal (47% PB) 37.3 30.7 leafs might be beneficial to broilers through their Barley 6.51 20.5 Fat1 5.50 8.00 antimicrobial activity against intestinal pathogenic bac- Celite – 2.00 teria (Sarica and Ürkmez, 2016). Also, phenolic com- Dicalcium phosphate 1.40 1.33 pounds can stimulate or inhibit digestive enzyme activ- Calcium carbonate 1.15 0.98 Vitamin and mineral premix2 0.30 0.30 ities affecting nutrient digestibility in broilers (Brenes Sodium chloride 0.40 0.35 and Roura, 2010; Leskovec et al., 2018). The stimu- L-Lys HCl (78%) 0.14 0.12 lation of other digestive secretions such as bile acids DL-met (99%) 0.26 0.23 L-Thr (98%) 0.05 0.05 by plant bioactives has been reported in rats (Platel Etoxiquin 66% 0.02 0.02 and Srinivasan, 2000). Because of their potential use in Endofeed DC 0.01 0.01 poultry feeds there is a need to increase our knowl- Phyzyme XP 5000 0.01 0.01 edge on olive byproducts extracts and their effects Calculated values Dry matter 88.3 89.0 on gut microbiota, nutrient digestibility, and bile acid AMEn (Kcal/Kg) 2,950 3,050 metabolism. Crude protein 21.9 19.2 The present study aimed to investigate the effects Ether extract 7.11 9.60 Crude fiber 3.03 2.90 of supplementing broiler diets with an olive pomace ex- Neutral detergent fiber 10.4 11.0 tract from Olea europaea on animal performance, nutri- Starch 32.0 32.3 ent digestibility, bile acid composition, gut microbiota, Sugars 4.75 4.20 Ca 1.00 0.90 and immune function. Total P 0.61 0.55 Digestible P 0.36 0.34 Na 0.17 0.15 Ash 6.40 7.80 MATERIALS AND METHODS 1 Animal fat and soybean oil blend. 2 Provided the following (per kilogram of diet): vitamin A (transretinyl Housing and Experimental Animals acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,000 IU; vitamin E (all-rac-tocopherol acetate), 20 IU; vitamin K (bisulfate menadione com- A feeding trial was carried out at the Polytech- plex), 3 mg; riboflavin, 5 mg; pantothenic acid (D-calcium pantothenate), nic University of Madrid (UPM) experimental facilities 10 mg; nicotinic acid, 30 mg; pyridoxine (pyridoxine·HCl), 3 mg; thi- amine (thiamine-mononitrate), 1 mg; vitamin B12 (cyanocobalamine), (Agricultural Production Department, Madrid). All the 12 µg; D-biotin, 0.15 mg; choline (choline chloride), 300 mg; folic acid, experimental procedures used were approved by the An- 0.5 mg; Se (Na2 SeO3 ), 0.1 mg; I (KI), 2.0 mg; Cu (CuSO4 ·H2 O), imal Ethic Committee of the Universidad Politécnica de 10 mg; Fe (FeSO4 ·7H2 0), 30 mg; Zn (ZnO), 100 mg; Mn (MnSO4 ·H2 O), 100 mg; and ethoxyquin, 110 mg. Madrid, according with principles of care of animals in experimentation (Boletin Oficial del Estado, 53/2013, ble form for the first 3 wk (Table 1). After that, 252 BOE, 2013). animals with 21 D of age (84 birds/treatment) and sim- A total of three hundred and six 1-day-oldmixed ilar body weight (BW) were used in the feeding trial (14 sex broiler chickens (Ross 308) were obtained from a birds/pen × 6 pens/treatment × 3 treatments). The commercial hatchery (Avimosa Group, Toledo, Spain). design was completely randomized with 3 treatments, a Chicks were assigned to 18 floor pens (1.1 m × 1.1 m) negative control with no additives (Control), a positive with 17 birds per pen (initial live weight 40.6 ± 0.7 g). control with 100 ppm of monensin (Monensin; Elanco Pens were bedded with wood shaving and provided with Valquimia S.A.) and the basal diet supplemented with a hopper feeder and a bell drinker. Environmental con- 750 ppm of an olive extract (OE, Lucta S. A.; Spain) ditions such as temperature, humidity, ventilation, and which consisted of an olive pomace extract standard- illumination were automatically controlled during the ized to contain a minimum of 10% total triterpenes experiment, depending on the age of the birds (33°C and 2% polyphenols. The theoretical concentration during the first week of age and then was reduced 2°C of OE (750 ppm, with 2% of polypenols and 10% of each week until reaching 23°C at 6 wk of age). Regard- triterpenes) was similar to the concentration found ing light program, chicks received 23 h light and 1 h in the experimental diet (722 ± 23 ppm, with 2.4 ± dark for the first 7 D of life and then 18 h light and 0.25% of polypenols and 12.9 ± 0.54% of triterpenes). 6 h dark until the end of the experiment. This analysis developed with ultra high performance liquid chromatography-mass spectroscopy (UPLC-MS) Experimental Design and Diets technique indicated that extract mixed procedure and stability of the product fitted with the expected values. All birds were raised with a standard non-medicated The experimental diets were formulated to have sim- starter diet based on wheat and soybean meal in crum- ilar nutritive value (Table 1) according to FEDNA 4 HERRERO-ENCINAS ET AL. (2010) and manufactured at IRTA (Mas de Bover, gen analyzer (LECO, St. Joseph, MI, EE.UU). The GE Constantí, Spain). Celite was added in the feed at 2% as was analyzed by adiabatic bomb calorimeter (PARR, acid insoluble ash marker for apparent ileal digestibility 1356 model; Parr Instrument Company, Moline, IL, (AID) determination. Animals were fed ad libitum the EE.UU). Acid insoluble ash of diets, and ileal contents experimental diets as pellets with 3 mm diameter from were determined as indicated by De Coca-Sinova et al. 21 to 42 D of age. (2011). Productive Traits and Sampling Bile Acids Analysis Body weight and feed consumption were determined Bile acids analysis was performed by UPLC-MS chro- by pen at 21, 28, 35, and 42 D of age, to calculate matography in an AQUITY I-Class (Waters Corp., Mil- the average daily gain (ADG), average daily feed in- ford, MA) connected to a Xevo-G2 QTof MS detector. take (ADFI), and feed conversion ratio (FCR). Mortal- Separation was run on an ACQUITY UPLC BEH C18 ity was recorded and weighed as produced. At the end 1.7 mm column (2.1 × 100 mm, Waters Corp.) using of the experiment at 42 D of age, birds were slaughtered water and acetonitrile as mobile phases, both contain- by asphyxiation in CO2 atmosphere. To obtain enough ing a 0.1% of formic acid. Mass spectroscopy detec- ileal content to determine nutrient digestibility and to tion was performed in full-scan negative mode (100 to sample all animals in the fed state birds were deprived 1,200 Da). Concentration of bile acid was determined of food for 2 h, and then were refed under ad libitum based on standard curves with QuanLynx software conditions for 1.30 h to achieve as homogeneous feed (Waters Corp.). Bile acids were extracted using the intake as possible. Two birds per pen were randomly following methodologies and using chenodeoxycholic selected and sampled to run microbial, gene expression, acid-d4 (CDCA-d4) as internal standard: lyophilized blood and bile acid analysis. To determine the relative ileal digesta samples were homogenized in absence of abundance of bacterial families approximately 1 g of solvent on a TissueLyzer II (QIAGEN, Hilden, Ger- caecal content was sampled, immediately placed in dry many) and 20 mg of homogenate were extracted with ice and stored at −80°C. For gene expression analy- 840 µL of H2 O: ACN (1:1) including internal stan- sis around 200 mg of ileal (approximately 4 cm from dard, respectively. After homogenization, the mixture the Meckel diverticulum) mucosal scrapings were sam- was centrifuged (15,000 g × 10 min, 4°C) and the su- pled in RNA later (Invitrogen, Carlsbad, CA) following pernatant diluted in H2 O: ACN (1:50) for UPLC-MS the manufacturer’s instructions, and further stored at analysis. Plasma proteins were precipitated by addition −80°C. Blood samples were collected from the heart us- of 200 µL of ACN with 5 µL internal standard to 50 µL ing sterile syringes and needles. To obtain the plasma of plasma. After centrifugation, supernatants were di- blood was collected into tubes containing EDTA and rectly analyzed by UPLC analysis. aprotinin (BD Vacutainer), held in ice for 30 min, cen- trifuged at 2,000 × g for 10 min and stored at −80°C Gut Microbiota Analysis to later analyze bile acids. Finally, 5 g of ileal content were collected and stored at −80°C to analyze bile acid The bacterial DNA extraction and sequence analy- concentrations. sis were performed using the methodology described by Andreano et al. (2017). Briefly, bacterial DNA was iso- Nutrient Apparent Ileal Digestibility lated of caecal content samples to obtain the micro- biome profile by massive sequencing of the 16S rRNA The ileal digesta from the remaining birds (10 to 12 gene regions. Amplicons of the V1 to V2 16S rRNA re- broiler per pen) was collected as indicated in Mandalawi gion were amplified by PCR with F27 forward and R338 et al. (2014). Samples were pooled, homogenized, frozen reverse primers with the sequences and conditions in- at −20°C, and freeze-dried. Then, the dried samples dicated in Andreano et al. (2017). For each amplicon, were ground using a mortar and pestle to pass through a quality and concentration were analyzed using Agilent 0.5 mm screen and maintained in airtight containers at Bioanalyzer 2100 (Agilent Technologies, Santa Clara, room temperature until determination of nutrient AID. CA). Samples were massive sequenced with the Ion 318 The AID of dry matter (DM), organic matter (OM), Chip Kit v2 (Life Technologies) under manufacturer’s crude protein (CP), ether extract (EE), and gross en- conditions on an Ion Torrent Personal Genome Machine ergy (GE) was estimated using the indigestible marker (PGM). method (De Coca-Sinova et al., 2011). Gene Expression Analysis Ileal Digesta Analysis Total RNA was extracted from approximately 50 mg The ileal content of broilers were analyzed following of ileal mucosal scraping with TRIzol reagent (Invitro- the standard methods of AOAC (2000) for DM and OM gen, Carlsbad, CA, USA), disrupted with a mixer mill (934.01), EE (920.39), total ash in muffle (942.05), and MM-400 (Retsch, Stuttgart, Germany) and isolated by CP by combustion method (968.06) using FP-528 nitro- using the GenElute Mammalian Total RNA Miniprep OLIVE POMACE EXTRACT IN BROILER DIETS 5 Kit (Sigma-Aldrich Corporation, St. Louis, MO, USA). Table 2. Effect of experimental diets on broiler chickens growth To prevent genomic DNA contamination an “in col- performance from 21 to 42 D of age.1 umn” DNase step was performed by using the RNAse- Item2 Control Monensin OE SEM3 P-value Free DNase Set (Quiagen, Australia). Extracted RNA 21 to 28 D yield and quality were measured by spectrophotometry ADG (g/bird) 103 103 100 2.69 0.77 (Epoch, BioTek, Winoosky, VT, USA) combined with ADFI (g/bird) 149 146 146 3.06 0.59 the Take3 Micro-Volume Plate (BioTek, Santa Barbara, FCR (g/g) 1.46 1.42 1.46 0.014 0.12 CA, USA) by absorbance at wavelengths of 260 and 28 to 35 D ADG (g/bird) 105 107 108 2.46 0.60 280 nm. ADFI (g/bird) 185 182 184 4.16 0.87 Reverse transcription of around 2,400 ng of extracted FCR (g/g) 1.77a 1.71b 1.71b 0.018 0.039 RNA was performed with the SuperScript VILO Mas- 35 to 42 D ter Mix (Invitrogen, Carlsbad, CA, USA). The quan- ADG (g/bird) 77.8b 103a 97.2a 4.18 0.002 ADFI (g/bird) 193 202 205 4.68 0.22 titative real-time PCR (qRT-PCR) analysis was per- FCR (g/g) 2.50a 1.98b 2.12b 0.083 0.001 formed in a 7300 Real Time PCR System (Applied 21 to 42 D Biosystems, Foster City, CA, USA) with already tested ADG (g/bird) 95.1b 104a 102a 2.03 0.018 and published primer conditions. Following primers ADFI (g/bird) 176 177 178 3.37 0.89 and PCR conditions were obtained from the litera- FCR (g/g) 1.91a 1.70b 1.76b 0.029 <0.001 ture: chicken β Actin (housekeeping) (Wang et al., a,b Means within a column and main effect not sharing a common 2009); ubiquitin (UB) (housekeeping) (De Boever et al., superscript are significantly different by Tukey’s test (P ≤ 0.05). 1 Control, negative control with no additives; Monensin, basal diet 2008); liver X receptor (LXR), carbohydrate responsive supplemented with 100 ppm of monensin; OE, basal diet supplemented element-binding protein (ChREBP), sterol regulatory with 750 ppm of OE. 2 element-binding protein 1 (SREBP1), (Proszkowiec- ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio. Weglarz et al., 2009); apical sodium dependent bile 3 SEM, standard error of means (n = 6 replicates with 14 birds each). acid transporter (ASBT) (Mcquaid, 2012); fatty acid binding protein 2 (FABP2) and 6 (FABP6), inter- leukin 6 (IL-6), claudin 1 (Claudin1), transforming OTUs was performed using RDP classifier (Wang et al., growth factor beta 4 (TGF-β 4), toll like receptor 4 2007). Representative sequences were aligned by Py- (TLR4) and 2β (TLR-2β ) (Chen et al., 2015); chicken Nast as default in quantitative insights into microbial B-cell marker chB6 (Bu-1), marker of active avian ecology (QIIME) pipeline (Casparoso et al., 2010) and T lymphocytes (CD3γδ ) (Bar-Shira et al., 2003); in- 0.005% of total OTUs were discarded. Shannon index terleukin 8 (former CXCLi2) (Rasoli et al., 2015); was assessed to analyze alpha diversity and the non- interferon-gamma (IFN-γ ) (Rothwell et al., 2004). parametric Kruskal–Wallis test was applied to evaluate Primers of chicken interleukin 2 (IL-2) (Forward: 5 - the statistical significance (P < 0.05). CAAGATTCATCTCGAGCTCTACACA-3 , Reverse: Differences in gene expression resulting from the 5 -CCCAGGTAACACTGCAGAGTTTG-3 ) were de- comparison of chickens fed Monensin and OE treat- signed from the GenBank sequence with accession num- ments relative to the control group were determined ber AF000631.1 using Primer Express v.2 software (Ap- using a mixed-model in which a gene-specific effect plied Biosystems, Foster City, CA, USA). Samples were and a sample-specific effect were treated as random analyzed in triplicate using the right amount of each variables and treatment was considered fixed (Steibel primer, ultra-purified water, and SYBR Green Master et al., 2009). For genes displaying efficiencies different Mix (Applied Biosystems, Foster City, CA, USA). from 2 (E = 2), cycle threshold (Ct) values were ad- justed according to the model described by Steibel et al. (2009). The standard error was used to recalculate the Statistical Analysis lower and upper 95% confidence intervals for each fold change. ANOVA was performed to analyze dietary effects on performance, AID, and bile acids by using the mixed- RESULTS model procedure of SAS (release 9.2; SAS Institute), with diet as the fixed effect and pen as the experimental Daily Gain, Feed Intake, and Feed unit. Data was analyzed by Shapiro–Wilk and Levene’s Conversion test to determine normality and variance homogene- ity. Tables show the average values of each treatment Growth performance data are shown in Table 2. From and the standard error of the mean (SEM). When the 28 to 35 D of age, broilers fed the OE and Monensin di- ANOVA was significant, differences among means were ets showed better FCR than those fed the Control diet separated by Tukey’s test at α = 0.05. (P < 0.05). During the last period, from 35 to 42 D of To de-multiplex, quality-filter, and analyze the mi- age, no significant differences were observed in ADFI. crobial raw sequencing reads QIIME 1.9.1 was utilized However, animals fed with Control showed lower ADG (Caporaso et al., 2010). Then, for taxonomy analyses, (P < 0.01) than those fed Monensin and OE diets re- reads were clustered into operational taxonomic units sulting in a worse FCR (P < 0.001) for Control diet. (OTUs) and taxonomic assignment of representative In the global period, from 21 to 42 D of age, a lower 6 HERRERO-ENCINAS ET AL. Table 3. Effect of experimental diets on the apparent ileal di- 100 gestibility (AID, %) of nutrients in broilers at 42 D of age.1 90 Proteobacteria (Gamma) Item Control Monensin OE SEM2 P-value Enterobacteriaceae 80 Digestibility Firmicutes (Clostriciales) Dry matter 65.4 68.6 66.2 0.99 0.095 70 Peptostreptococcaceae Relative Abundance (%) Gross energy 69.5 72.5 70.2 1.04 0.13 Ether extract 83.0 84.2 82.1 1.54 0.62 60 Firmicutes (Clostriciales) Organic matter 68.5 71.7 69.2 1.06 0.12 Clostridiaceae Crude protein 72.1b 77.8a 73.8a,b 1.41 0.035 50 a,b Means within a column and main effect not sharing a common 40 Firmicutes (Turicibacterales) superscript are significantly different by Tukey’s test (P ≤ 0.05). Turicibacteraceae 1 Control, negative control with no additives; Monensin, basal diet 30 supplemented with 100 ppm of monensin; OE, basal diet supplemented Firmicutes (Lactobacillales) with 750 ppm of OE. 20 Streptococcaceae 2 SEM, standard error of means (n = 6). 10 Firmicutes (Lactobacillales) Table 4. Effect of experimental diets on bile acid composition in Lactobacillaceae ileal contents and plasma of broilers at 42 D of age.1 0 Control Monensin OE Item2 Control Monensin OE SEM3 P-value4 Figure 1. Effects of feeding broiler chicken diets supplemented with Ileal content (µmol/g of sample) 100 ppm of monensin or 750 ppm of OE on the relative abundance of TCA 0.094 0.15 0.11 0.042 0.93 bacteria families in caecal content (n = 6). Control, negative control TCDCA 1.21 3.84 1.27 0.99 0.14 with no additives; Monensin, basal diet supplemented with 100 ppm AVCA 0.17 0.31 0.23 0.055 0.19 of monensin; OE, basal diet supplemented with 750 ppm of OE. CA 0.56 1.39 1.01 0.27 0.094 CDCA 4.61 9.63 8.07 2.14 0.12 Σ Conjugated 1.30 3.98 1.37 1.01 0.14 4.5 Σ Unconjugated 5.35 11.3 9.31 2.24 0.097 Total BA 6.65b 15.3a 10.7a,b 2.89 0.055 4.0 Plasma (nmol/mL of plasma) 3.5 TCA 0.41 0.72 0.77 0.19 0.37 Shannon Index TCDCA 14.2 13.6 14.8 2.59 0.97 3.0 TLCA 0.29 0.29 0.29 0.022 0.95 CDCA 0.31 0.36 0.64 0.21 0.24 2.5 Σ Conjugated 14.9 14.6 15.9 2.75 0.96 2.0 Σ Unconjugated 0.31 0.36 0.64 0.21 0.24 Total BA 15.2 14.9 16.5 2.9 0.72 1.5 a,b Means within a column and main effect not sharing a common 1.0 superscript are significantly different by Tukey’s test (P ≤ 0.05). 1 Control, negative control with no additives; Monensin, basal diet 0.5 supplemented with 100 ppm of monensin; OE, basal diet supplemented 0.0 with 750 ppm of OE. 2 TCA, Taurocholic acid; TCDCA, Tauroursodeoxycholic acid; TLCA, Control Monensin OE Taurolithocholic acid; AVCA, Avicholic acid; CA, Cholic acid; CDCA, chenodeoxycholic acid. Figure 2. Effects of feeding broiler chicken diets supplemented with 3 100 ppm of monensin or 750 ppm of OE on the relative diversity of SEM, standard error of means (n = 6). 4 caecal microbiota (Shannon index) (n = 6). Control, negative control P-values are from square root data transformation analysis. with no additives; Monensin, basal diet supplemented with 100 ppm ADG (P < 0.05) was observed in birds fed Control com- of monensin; OE, basal diet supplemented with 750 ppm of OE. pared to Monensin and OE groups. Furthermore, no significant differences in ADFI were reported among plasma (Table 4). However, the sum of the total bile treatments and hence broilers in Control diet showed acids tended (P = 0.055) to be higher in birds fed the worse FCR than animals fed the Monensin and OE Monensin. The predominant bile acid in ileal digesta (P < 0.001). was CDCA while tauroursodeoxycholic acid (TCDCA) was the predominant in plasma. Apparent Ileal Digestibility Caecal Microbiota No significant differences on GE, OM, and EE AID were observed among treatments (Table 3). However, Firmicutes was the predominant phylum in the caeca birds fed Monensin showed higher CP AID (P < 0.05) (Figure 1). No significant differences among diets were than those fed Control diet, and OE group showed in- observed in relative abundance of the main bacte- termediate values. rial families. Numerically, the most abundant fami- lies were Clostridiciaceae in Control (41.7%) and Mo- Bile Acids nensin (51.1%) treatments, and Lactobacillaceae in OE (65.5%) treatment. Additionally, the diversity of caecal No differences were observed in conjugated or un- microbiota measured by the Shannon index was similar conjugated bile acid concentration in ileal digesta and among treatments (Figure 2). OLIVE POMACE EXTRACT IN BROILER DIETS 7 2.4 *** *** ** 2.0 * 1.6 Fold change 1.2 0.8 0.4 * 0.0 R T SR P P1 γ 2 6 -2 -6 Cl -8 δ TG 1 β4 TL 1 2β R4 N 3γ BP BP n - B SB LX IL IL IL BU di F- R- EB IF TL RE CD FA FA au A Ch Monensin OE Figure 3. Effects of feeding broiler chicken diets supplemented with 100 ppm of monensin (Monensin) or 750 ppm of OE (OE) on the expression of selected genes in the ileum. Gene expression values are fold change relative to the mRNA levels in the control diet (C) set to be 1.0 (horizontal axis). Bars indicate the 95% confidence interval (Fold change up—Fold change low) (n = 12; *: P < 0.05; **: P < 0.01; ***: P < 0.001). Gene Expression cal origin but also plant location, harvesting conditions, processing, and storage can affect the extract composi- Results of gene expression in the ileum are shown tion and activity (Huyghebaert et al., 2011). Therefore, in Figure 3. The expression of LXR, ASBT, REBP, the different concentration of the bioactive substances SREBP1, IFNγ , FABP2, FABP6, IL-2, IL-6, Claudin1, in the extracts (i.e., percentage of total triterpenes and TLR-2β , TLR4, and CD3γδ in the ileum was not af- polyphenols) and/or the route of administration (feed fected by dietary treatments. However, the expression vs. water) might be behind the different results on per- of IL-8 was significantly down-regulated in birds fed the formance among olive byproduct extract studies. OE compared to those fed Control diet (P < 0.05). In Plant extracts might improve broiler growth and feed addition, the expression of TGF-β 4 was significantly efficiency by increasing nutrient digestibility as they up-regulated in birds fed Monensin (P < 0.05) and can affect digestive enzyme function, intestinal mor- OE treatments (P < 0.01) compared to C. The expres- phology gastrointestinal tract motility, or bile acid se- sion of Bu-1 showed a similar pattern with a signifi- cretion (Lee et al., 2003; Brenes and Roura, 2010; cant up-regulation in Monensin (P < 0.001) and OE Bozkurt et al., 2016; Leskovec et al., 2018). In the (P < 0.001). present study no significant differences were observed on the AID of DM, EE, GE, and OM among dietary DISCUSSION treatments. This is in agreement with the study of Leskovec et al. (2018) who reported no significant dif- In the present study supplementing broiler diets with ferences in nutrient apparent total tract digestibility co- 750 ppm of OE during the grower-finisher period did efficients among broilers fed the olive leaf extract and not affect ADFI but improved ADG and FCR com- the no supplemented control. However, in the present pared to a non-supplemented control. This is in agree- study birds fed the Monenisn supplemented diet showed ment with Sarica and Ürmez (2016) who reported also a better protein AID than the control but similar to higher final BW and better FCR in broilers fed olive those fed the OE. Recent studies support that the pos- leaf extracts. By contrast Leskovec et al. (2018) and itive effects of Monensin on broiler growth and FCR King et al. (2014) were unable to detect significant dif- might partially be explained by a rise in intestinal diges- ferences in productive parameters after supplementing tive enzyme activities including chymotrypsin (Bozkurt the feed and water with olive leaf and pomace extracts, et al., 2016). Other studies have also reported improve- respectively. However, the specified examples were per- ment on broiler FCR with Monensin due to an increase formed with extracts from olive leaves (Leskovec et al., of conjugated bile acids (TCA and TCDCA) and hence 2018) or olive pulp (King et al., 2014) rich in polyphe- better dietary fat digestibility (Guban et al., 2006). nols, while in the present study extract contained a Moreover, studies performed in rats have shown the higher prevalence of triterpenes over the polyphenolic ability of plant extracts rich in polyphenols to affect fraction. Contradictory results on broiler performance lipid metabolism and bile acid composition (Fotschki with other plant extracts rich in bioactive compounds et al., 2017). In the present study no significant differ- have also been described (Leskovec et al., 2018) and ences were observed on intestinal or plasma bile acid it has been proven that not only the different botani- composition among treatments, in agreement with a 8 HERRERO-ENCINAS ET AL. lack of effect on dietary fat digestibility. Moreover, the counts in ileal samples of birds fed olive leaf extracts or ileal expression of genes encoding proteins involved in a control diet with no additives. Again, different ex- bile acid and lipid metabolism such as LXR, ASBT, tracts origin (leaf vs. pomace) and composition (oleu- ChREBP, SREBP1, FABP2, and FABP6 was not af- ropein vs. triterpenes) might potentially explain these fected by dietary treatments. However, in the present discrepancies. On the other hand, monensin inclusion in study total bile acid concentration tended to be higher chicken diets has been described to decrease Lactobacil- in birds fed the Monensin supplemented diet. Besides lus species (Guban et al., 2006; Danzeisen et al., 2011). their role as physiological detergents that facilitate the No evidence were found that could justify these contra- absorption of lipids bile acids act as “hormone-like” dictory results; however, the limited number of repli- molecules involved in several signaling pathways in- cates in the present study, or factors such as diet, broiler cluding the regulation of lipid, glucose, and energy breed, the environment of the experimental farm, or metabolism (Li and Chiang, 2015). The composition of technical methodologies might be behind discrepant re- the bile acids pool is a balance between the primary (un- sults among microbiome studies in poultry (Danzeisen conjugated) bile acids synthesized in the liver and their et al., 2011). Further analysis to confirm the lack of ef- modifications to secondary (conjugated) bile acids in fect of the OE on bacteria at the genus or species level the intestine generated by the microbiome (Li and Chi- is needed. ang, 2015). After the transport of bile acids back to the Plant extracts may exert their beneficial effects on liver via portal blood the synthesis of new primary bile growth performance among others because of their an- acids takes place according to the physiological needs. tioxidant and/or immunomodulatory effects (Lillehoj This link between gut microbiota and hepatic bile acid et al., 2018). Previous studies conducted with ex- synthesis might be useful to detect functional changes in tracts of similar composition to the one used in this the microbiome or potential changes in microbial pop- study showed better animal performance related to ulations impacting the animal energy metabolism. Re- its anti-inflammatory function rather than the antiox- sults from this study suggest a better performance in idant one (Gisbert et al., 2017; Liehr et al., 2017; birds fed the Monensin supplemented diets because of Tedó et al., 2018). This made us explore only the a better protein digestibility and higher ileal total bile potential reduction of the inflammatory status of acid concentrations. By contrast, our results show that the animals. Some plant extracts have shown im- the positive effects of OE on performance seem not to munomodulatory effects in studies in which poultry and be due to improvement in nutrient digestibility or bile pigs were experimentally infected (Zeng et al., 2015; acid pool modifications. Sugiharto, 2016). These effects include increase in lym- Gastrointestinal microbiome plays an important role phocyte proliferation, serum level antibodies, decrease for gut health and nutrition in poultry production (Xiao of pro-inflammatory, and increase of anti-inflammatory et al., 2016). Phytogenics, probiotics, prebiotics, or ex- cytokines (Zeng et al., 2015; Sugiharto, 2016). In a re- ogenous enzymes are commonly used to modulate gut cent study, Liehr et al. (2017) reported a beneficial microbiome (Dibner and Richards, 2005; Oakley et al., effect of an OE extract in pigs challenged with LPS 2014). In this context, bioactive compounds of olive po- on immune response by reducing the pro-inflammatory mace such as oleuropein and hydroxytyrosol are good IL-1β in plasma leading also to a better animal per- candidates to modulate the composition of gut micro- formance. Despite inflammation is normally associated biota and enhance gut integrity (Sarica and Ürmez, with pathogen outbreaks there might be also a moder- 2016). It is known that triterpenes offered through the ate intestinal inflammation in response to the high en- diet are only partially absorbed in the upper digestive ergy diets used under normal productive circumstances tract (Yin et al., 2012) and are also present along the (Niewold, 2014). Moreover, as suggested by Niewold GIT tract (Lozano-Mena et al., 2016). Therefore, coat- (2014) plant extracts, like antibiotics, might increase ing technologies are not required in this kind of extracts broiler performance because of their anti-inflammatory to reach the gastrointestinal tract and perform its ac- effects at intestinal level. In the present study, the ex- tivity. In the present study Firmicutes was the most pression of intestinal IL-2, IL-6, and IFNγ was not af- abundant phylum in the broiler caeca, this is in agree- fected by dietary means. However, birds fed the OE ment with other studies in broilers (Danzeisen et al., showed a significant decrease of IL-8 expression in the 2011). No significant differences were observed on cae- ileum. This chemokine plays an important role in the cal microbiota diversity or relative abundance of the chicken inflammatory response recruiting heterophil at main bacterial families among experimental treatments. a local and systemic level in response to bacterial pres- This is in agreement to results obtained by Liehr et al. ence (Kogut, 2002). Moreover, a significant upregula- (2017) who observed no differences in composition and tion of the anti-inflammatory cytokine TGF-β 4 and diversity of gut microbiome, at the phylum level, in the B cell marker Bu-1 was observed in the ileum of pigs fed diets supplemented with an olive oil bioactive birds fed the EO and Monensin diets compared to extract similar than the one used in this study. How- those fed the Control diet. Therefore, it is plausive ever, our results partially agree with Sarica and Ürmez that the better performance observed in birds fed the (2016) who reported no changes in total aerobic bacte- Monensin and EO diets in our study might be related ria but significant differences on E. coli and Lactobacilli to a lower intestinal inflammatory response explained OLIVE POMACE EXTRACT IN BROILER DIETS 9 by the immunosuppressant effect on IL-8 and the insoluble ash for evaluation of nutrient retention in broiler diets. increase anti-inflammatory expression of TGF-β 4 un- Span. J. Agric. Res. 9:761–768. Dibner, J. J., and J. D. Richards. 2005. Antibiotic growth promoters der non-acute inflammatory conditions. in agriculture: history and mode of action. Poult. Sci. 84:634–643. In conclusion, the present study shows that the inclu- El Hachemi, A., K. E. El, Mecherfi, K. Benzineb, D. Saidi, and O. sion of 750 ppm of an olive pomace extract containing a Kheroua. 2007. Supplementation of olive mill wastes in broiler minimum of 10% total triterpenes and 2% polyphenols chicken feeding. Afr. J. Biotechnol. 6:1848–1853. FEDNA (Fundación Española Desarrollo Nutrición Animal). 2010. positively affects growth in broiler chickens. Among the Normas FEDNA para la Formulación de Piensos Compuestos. possible mechanism of action studied so far in this work 3rd ed. In C. De Blas, G. G. Mateos, and P. G. Rebollar, ed. improvement in performance is likely related to the ex- Fund. Esp. Desarro. Nutri. Anim., Madrid, Spain. tract anti-inflammatory properties. However, other pos- Fotschki, B., J. Juśkiewicz, A. Jurgoński, N. Rigby, M. Sójka, K. Kołodziejczyk, A. Mackie, and Z. Zduńczyk. 2017. Raspberry po- sible mechanisms such as antioxidant capacity remain mace alters cecal microbial activity and reduces secondary bile to be explored. acids in rats fed a high-fat diet. J. Nutr. Biochem. 46:13–20. Gisbert, E., K. B. Andree, J. C. Quintela, J. A. Calduch-Giner, I. R. Ipharraguerre, and J. Pérez-Sánchez. 2017. Olive oil bioac- ACKNOWLEDGMENTS tive compounds increase body weight, and improve gut health and integrity in gilthead sea bream (Sparus aurata). Br. J. Nutr. The authors gratefully acknowledge the support of 117:351–363. Lucta S. A., Comunidad de Madrid (Spain) and Euro- Guban, J., D. R. Korver, G. E. Allison, and G. W. Tannock. 2006. pean Structural and Investment Funds for Javier Her- Relationship of dietary antimicrobial drug administration with broiler performance, decreased population levels of Lactobacillus rero (Project MEDGAN-CM S2013/ABI2913). salivarius, and reduced bile salt deconjugation in the ileum of broiler chickens. Poult. Sci. 85:2186–2194. Huyghebaert, G., R. Ducatelle., and F. Van Immerseel. 2011. An up- date on alternatives to antimicrobial growth promoters for broil- REFERENCES ers. Vet. J. 187:182–188. International Olive Council. 2017. Accessed Dec. 2017. http://www. Andreano, S. D., A. S. Bonastre, O. Francino, A. C. Martí, C. Lecchi, internationaloliveoil.org. G. Grilli, D. Giovanardi, and F. Ceciliani. 2017. Gastrointestinal King, A. J., J. K. Griffin, and F. Roslan. 2014. In vivo and in vitro microbial population of turkey (Meleagris gallopavo) affected by addition of dried olive extract in poultry. J. Agric. Food Chem. hemorrhagic enteritis virus. Poult. Sci. 96:3550–3558. 62:7915–7919. AOAC International. 2000. Official Methods of Analysis of AOAC Kogut, M. H. 2002. Dynamics of protective avian inflammatory re- International. 17th ed. AOAC Int., Gaithersburg, MD. sponse: the role of an IL-8 like cytokine in the recruitment of Bar-Shira, E., D. Sklan, and A. Friedman. 2003. Establishment of heterophils to the site of organ invasion by Salmonella enteri- immune competence in the avian GALT during the immediate tidis. Comp. Immunol. Microbiol. Infect. Dis. 25:159–172. post-hatch period. Dev. Comp. Immunol. 27:147–157. Lee, K. W., H. Everts, H. J. Kappert, M. Frehner, R. Losa, and Boletín Oficial del Estado. 2013. Real Decreto por el que se estable- A. C. Beynen. 2003. Effects of dietary essential oil components cen las normas básicas aplicables para la protección de los ani- on growth performance, digestive enzymes and lipid metabolism males utilizados en experimentación y otros fines científicos, in- in female broiler chickens. Br. Poult. Sci. 44:450–457. cluyendo la docencia. Boletín Oficial del Estado. 34:11370–11421. Leskovec, J., A. Levart., S. Žgur, D. Jordan, T. Pirman, J. Salobir, Bozkurt, M., G. Ege, N. Aysul, H. Akşit, A. E. Tüzün, K. and V. Rezar. 2018. Effects of olive leaf and marigold extracts on Küçükyılmaz, A. E. Borum, M. Uygun, D. Akşit, S. Aypak, the utilization of nutrients and on bone mineralization using two E. Şimşek, K. Seyrek, B. Koçer, E. Bintaş, and A. Orojpour. different oil sources in broilers. J. Poult. Sci. 55:17–27. 2016. Effect of anticoccidial monensin with oregano essential oil Li, T., and J. Y. L. Chiang. 2015. Bile acids as metabolic regulators. on broilers experimentally challenged with mixed Eimeria spp. Curr. Opin. Gastroenterol. 31:159–165. Poult. Sci. 95:1858–1868. Liehr, M., A. Mereu, J. J. Pastor, J. C. Quintela, S. Staats, G. Brenes, A., and E. Roura. 2010. Essential oils in poultry nutri- Rimbach, and I. R. Ipharraguerre. 2017. Olive oil bioactives tion: main effects and modes of action. Anim. Feed. Sci. Technol. protect pigs against experimentally-induced chronic inflamma- 158:1–14. tion independently of alterations in gut microbiota. PLoS One Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. 12:e0174239. Bushman, E. K. Costello, N. Fierer, A. G. Peña, J. K. Goodrich, Lillehoj, H. S., and K. W. Lee. 2012. Immune modulation of innate J. I. Gordon, G. A. Huttley, S. T. Kelley, D. Knights, J. E. Koenig, immunity as alternatives-to-antibiotics strategies to mitigate the R. E. Ley, C. A. Lozupone, D. McDonald, B. D. Muegge, M. use of drugs in poultry production. Poult. Sci. 91:1286–1291. Pirrung, J. Reeder, J. R. Sevinsky, P. J. Turnbaugh, W. A. Wal- Lillehoj, H., Y. Liu, S. Calsamiglia, M. E. Fernandez-Miyakawa, F. ters, J. Widmann, T. Yatsunenko, J. Zaneveld, and R. Knight. Chi, R. L. Cravens, S. Oh, and C. G. Gay. 2018. Phytochemicals 2010. QIIME allows analysis of high-throughput community se- as antibiotic alternatives to promote growth and enhance host quencing data. Nat. Methods 7:335–336. health. Vet. Res. 49:76. Chen, J., G. Tellez, J. D. Richards, and J. Escobar. 2015. Identi- Lozano-Mena, G., M. Sánchez-Gonzalez, A. Parra, M. E. Juan, and fication of potential biomarkers for gut barrier failure in broiler J. M. Planas. 2016. Identification of gut-derived metabolites of chickens. Front. Vet. Sci. 2:14. maslinic acid, a bioactive compound from Olea europaea L. Mol. Danzeisen, J. L., H. B. Kim, R. E. Isaacson, Z. J. Tu, and T. J. Nutr. Food Res. 60:2053–2064. Johnson. 2011. Modulations of the chicken cecal microbiome and Mandalawi, H. A., M. V. Kimiaeitalab, V. Obregon, D. Menoyo, and metagenome in response to anticoccidial and growth promoter G. G. Mateos. 2014. Influence of source and level of glycerin in treatment. PLoS One 6:e27949. the diet on growth performance, liver characteristics, and nutrient De Boever, S., C. Vangestel, P. De Backer, S. Croubels, and S. U. Sys. digestibility in broilers from hatching to 21 days of age. Poult. Sci. 2008. Identification and validation of housekeeping genes as inter- 93:2855–2863. nal control for gene expression in an intravenous LPS inflamma- Mcquaid, R. 2012. Characterization of the ileal lipid binding protein tion model in chickens. Vet. Immunol. Immunopathol. 122:312– (FABP6) in tissues involved in bile acid and steroid metabolism 317. in poultry. PhD Diss. McGrill Univ., Montreal, Canada. De Coca-Sinova, A., G. G. Mateos, J. M. González-Alvarado, C. Mirabella, N., V. Castellani, and S. Sala. 2014. Current options for Centeno, R. Lázaro, and E. Jiménez-Moreno. 2011. Comparative the valorization of food manufacturing waste: a review. J. Clean. study of two analytical procedures for the determination of acid Prod. 65:28–41. 10 HERRERO-ENCINAS ET AL. Morrison, S. Y., J. J. Pastor, J. C. Quintela, J. J. Holst, B. Hart- timicrobial feed additives in broiler diets. Eurp. Poult. Sci. 80:1– mann, J. K. Drackley, and I. R. Ipharraguerre. 2017. Short 13. communication: promotion of GLP-2 secretion in dairy calves Sayehban, P., A. Seidavi, M. Dadashbeiki, A. Ghorbani, W. A. G. with a bioactive extract from Olea europea. J. Dairy Sci. Araújo, and L. F. T. Albino. 2016. Effects of different levels of 100:1940–1945. two types of olive pulp with or without exogenous enzyme sup- Niewold, T. A. 2007. The non-antibiotic anti-inflammatory effect plementation on broiler performance and economic parameters. of antimicrobial growth promoters, the real mode of action? A Rev. Bras. Cienc. Avic. 18:489–500. hypothesis. Poult. Sci. 86:605–609. Steibel, J. P., R. Poletto, P. M. Coussens, and G. J. Rosa. 2009. A Niewold, T. A. 2014. Why anti-inflammatory compounds are the powerful and flexible linear mixed model framework for the analy- solution for the problem with in feed antibiotics. Qual. Assur. sis of relative quantification RT-PCR data. Genomics 94:146–152. Saf. Crop. Foods. 6:119–122. Sugiharto, S. 2016. Role of nutraceuticals in gut health and Oakley, B. B., H. S. Lillehoj, M. H. Kogut, W. K. Kim, J. J. growth performance of poultry. J. Saudi Soc. Agric. Sci. 15:99– Maurer, A. Pedroso, M. D. Lee, S. R. Collett, T. J. Johnson, and 111. N. A. Cox. 2014. The chicken gastrointestinal microbiome. FEMS Tedó, G., A. Martínez, C. Muller, M. Reina, M. Blanch, and Microbiol. Lett. 360:100–112. J. J. Pastor. 2018. Targeting gut inflammation in pigs with the Platel, K., and K. Srinivasan. 2000. Stimulatory influence of select use of olive fruits bioactives positively influences pig perfor- spices on bile secretion in rats. Nutr. Res. 20:1493–1503. mance. Proc. 14th Internat. Symp. Digest. Physiol. Pigs. 9:262. Proszkowiec-Weglarz, M., M. P. Richards, B. D. Humphrey, (Abstr.). R. W. Rosebrough, and J. P. McMurtry. 2009. AMP-activated Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007. Naive protein kinase and carbohydrate response element binding pro- Bayesian classifier for rapid assignment of rRNA sequences into tein: a study of two potential regulatory factors in the hepatic the new bacterial taxonomy. Appl. Environ. Microbiol. 73:5261– lipogenic program of broiler chickens. Comp. Biochem. Physiol. 5267. B, Biochem. Mol. Biol. 154:68–79. Wang, P. H., Y. H. Ko, H. J. Chin, C. Hsu, S. T. Ding, and C. Y. Rasoli, M., S. K. Yeap, S. W. Tan, K. Roohani, Y. W. Kristeen-Teo, Chen. 2009. The effect of feed restriction on expression of hepatic N. B. Alitheen, Y. A. Rahaman, I. Aini, M. H. Bejo, P. Kaiser, and lipogenic genes in broiler chickens and the function of SREBP1. A. R. Omar. 2015. Differential modulation of immune response Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 153:327–331. and cytokine profiles in the bursae and spleen of chickens infected Xiao, Y., Y. Xiang, W. Zhou, J. Chen, K. Li, and H. Yang. 2016. Mi- with very virulent infectious bursal disease virus. BMC Vet. Res. crobial community mapping in intestinal tract of broiler chicken. 11:75. Poult. Sci. 96:1387–1393. Rothwell, L., J. R. Young, R. Zoorob, C. A. Whittaker, P. Hesketh, Yin, M. C., M. C. Lin, M. C. Mong, and C. Y. Lin. 2012. Bioavailabil- A. Archer, A. L. Smith, and P. Kaiser. 2004. Cloning and char- ity, distribution, and antioxidative effects of selected triterpenes acterization of chicken IL-10 and its role in the immune response in mice. J. Agric. Food Chem. 60:7697–77101. to Eimeria maxima. J. Immunol. 173:2675–2682. Zeng, Z., S. Zhang, H. Wang, and X. Piao. 2015. Essential oil and Sarica, S., and D. Ürkmez. 2016. The use of grape seed-, olive aromatic plants as feed additives in non-ruminant nutrition. J. leaf- and pomegranate peel-extracts as alternative natural an- Anim. Sci. Biotechnol. 6:1–10.