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The effect of lavender (Lavandula angustifolia) essential oil as a drinking water supplement on the production performance, blood biochemical parameters, and ileal microflora in broiler chickens
The effect of lavender (Lavandula angustifolia) essential oil as a drinking water supplement on the production performance, blood biochemical parameters, and ileal microflora in broiler chickens Michalina Adaszy´ nska-Skwirzy´ nska1 and Danuta Szczerbi´ nska Department of Poultry and Ornamental Bird Breeding, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology, Szczecin 71-270, Poland periment. The analyses reveal that the addition of LEO has a positive effect on body weight in the second period of rearing (d 22–24). Treatment broilers (LEO1–42 and LEO22–42 ) weighed on average 6.35% more compared to the control (P < 0.01). LEO addition positively affected weight gains and feed conversion ratio (P < 0.01) in the second period of rearing (d 22–24). No differences were found between the groups feed intake, water intake, survival rate, and blood biochemical parameters (P > 0.05). The addition of LEO to drinking water had a positive impact on the gut microflora of the ileum: the numbers of pathogenic microorganisms decreased (Escherichia coli and coliform) while the number of probiotic bacteria increased (P < 0.01).
ABSTRACT The aim of this study was to evaluate growth performance, selected biochemical blood parameters, and the microbiota of ileal digesta in broiler chickens provided with drinking water containing an addition of natural lavender essential oil (LEO). The experiment was carried out on a commercial farm using n = 300 unsexed Ross-308 broiler chickens. One-day-old chicks were randomly assigned to three groups of 100 chickens each (five replications, 20 individuals each). The control group broilers were provided with drinking water without the addition of LEO. Groups LEO1–42 and LEO22–42 had access to water containing 0.4 ml/L LEO (for 6 h/day) from days 1 to 42 (LEO1–42 ) and 22 to 42 (LEO22–42 ). Body weight, feed intake, water intake, and mortality were recorded throughout the ex-
Key words: broiler, lavender essential oil, performance, blood biochemistry, intestinal microflora 2018 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pey385 (e.g., myrcene, α-pinene, caryophyllene), alcohols (e.g., linalool, α-terpineol, borneol), ketones (e.g., camphor, carvone, eucarvone), esters (e.g., linalool acetate, lavandulyl acetate, geranyl acetate), aldehydes (e.g., neral), oxides (e.g., caryophyllene oxide), and ethers (e.g., eucalyptol; Lis-Balchin, 2002). In addition to these compounds, coumarins and organic acids are found in LEO ´ (Prusinowska and Smigielski, 2014). Recent studies, analyzing LEO chemical constituents an attempt to describe their action mechanisms on rats and human models, confirm many beneficial properties of this oil. According to the literature, LEO has antibacterial, antifungal, antioxidant, analgesic, anti-inflammatory, and antispasmodic properties (Yang et al., 2010; Prusi´ nowska and Smigielski, 2014; Carrasco et al., 2016; Giovannini et al., 2016). Studies on animal and human models alike have shown that lavender has potential immunostimulatory, anxiolytic, sedative, hypnotic, analgesic, and anticonvulsant effects, and may also improve one’s mood (Ghelardini et al., 1999; Sasannejad et al., ´ 2012; Prusinowska and Smigielski, 2014). The literature lacks reports on the possibility of using LEO in poultry production as a supplement in the diet of broiler chickens in order to stimulate production
INTRODUCTION Essential oils (EO) are classified as phytobiotics, i.e., natural substances having a protective function against harmful pathogenic microorganisms (Zeng et al., 2015). Lavender essential oil (LEO) is a by-product of lavender flowers used in human aromatherapy, pharmacy, perfumery, cosmetics, and the food industry (Prusi´ nowska and Smigielski, 2014; Kirimer et al., 2017). Lavender flowers (Lavandulae flos) contain up to 3% of EO, whose qualitative and quantitative composition is variable and depends on the genotype, climatic conditions, mode of reproduction, and morphological char´ acteristics of the plant (Prusinowska and Smigielski, 2014). LEO is a volatile, biologically active substance with a characteristic scent. Chemically, it is a multicomponent mixture of terpenoid compounds (monoterpenes, sesquiterpenes, and their oxygen derivatives), characterized based on isoprenoid units (Carrasco et al., 2016). LEO compounds include hydrocarbons C 2018 Poultry Science Association Inc. Received October 4, 2016. Accepted August 2, 2018. 1 Corresponding author: [email protected]
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performance. Therefore, this study was undertaken with the purpose of evaluating the applicability of LEO in broiler production. The effects of LEO offered as a drinking water supplement on production performance, selected biochemical blood parameters, and ileal microbiota in broiler chickens were analyzed.
iment was carried out with the approval of the Local Ethics Committee for the Experiments on Animals in Szczecin (permit no. 19/2015 dated 2015 May 22).
MATERIALS AND METHODS
In order to determine the levels of selected biochemical parameters, on d 42 of age blood was collected from the left brachial vein of 10 randomly selected birds from each group (five replications of two individuals each). The tests were carried out on the day of sampling. Blood samples were centrifuged at 15,000 × G for 5 min in order to obtain the serum, which was then stored for analysis under controlled refrigerated conditions (+5◦ C ±3◦ C). The temperature of serum samples during the analysis was maintained at +18◦ C ±1◦ C. Blood biochemical parameters were measured using the VetTest 8008 (Idexx Laboratories, Inc., Westbrook, USA) chemistry analyzer with dry-slide technology using the Idexx Laboratories methodology. Blood parameters analyzed in this study included: glucose, cholesterol, triglycerides, and uric acid. Additionally, at 42 d of age, five randomly selected broilers from each group (five replications of one individual each), with BW similar to the group’s average, were slaughtered, and ileal digesta samples were collected for microbiological analyses aimed at preselected groups of microorganisms (Clostridium spp., Escherichia coli, coliform, aerobic, anaerobic, and lactic acid bacteria). The intestine was spread individually on the autopsy table in the following orientation: the duodenum (from the gizzard outlet to the end of the pancreatic loop), small intestine (from the site where the duodenum emerges from the gizzard to the beginning of the ceca), ceca, and large intestine (from the colon to the rectum). Then, the duodenum, ceca, and large intestine were removed. The remaining small intestine was composed of two parts, the jejunum (from the pancreatic loop to Meckel’s diverticulum) and the ileum divided by Meckel’s diverticulum. The ileum was isolated at the intestine fragment between the Meckel diverticulum and the ileocecal junction. Three points were designated in this area: the middle of the ileum, and half way from the center to each end (with three repetitions for the final calculations). The ileum was opened with scissors, ileal content (1 g/each point) was collected by gentle fingers-stripping of the surrounding ileal segment. The samples were collected using small, sterile plastic tubes (volume 12 ml) and were kept in an ice box. Serial dilutions were performed within 1 h of collection. The initial suspension and decimal dilutions were performed according to the European Standard PNEN ISO 6887-1:2000 (10−1 dilution as the initial dilution up to 10−9 as the final dilution) under a laminar flow safety cabinet (2nd class; BioBase, South Gongye Road, Jinan, China). Buffered peptone water (Merck, Darmstadt, Germany) was used as a diluent. The initial
Bird Trial The experiment was performed in a commercial poul˙ owko, Poland) using 300 unsexed Rosstry farm (Zab´ 308 broilers. The chickens were raised in the intensive system. The experimental period was 42 d. Chicks were purchased from a commercial hatchery (Park Dro´ lowo, Poland). On arrival, 1-d-old biarski Sp. z o.o., Smi birds were weighed and then randomly assigned to three treatments groups with five replicate pens containing 20 birds each. Throughout the experiment, the control group broilers were provided with drinking water without an addition of LEO. Groups LEO1–42 and LEO22–42 were provided with water (for 6 h/day—the mean time of water intake (WI) with LEO throughout the production cycle) with an addition of 0.4 ml/L oil, from 1 to 42 d of age (LEO1–42 ) and from 22 to 42 d of age (LEO22–42 ). The birds were provided with water in 5 L manual reservoir tank reversible drinkers (in each pen). Once all LEO-infused water was drunk, the reservoir tanks were replaced with new ones containing clean water without the addition of LEO. The remaining water in each pen was measured by volume. Over the entire duration of the experiment, broilers had unlimited access to water. Broilers were housed in the same shed for 42 d, on wheat-straw bedding, with a stocking density of 14 individuals per m2 . All groups were managed under uniform, standard environmental conditions. The following lighting program was applied: (d 1–6) 24L:0D, (d 7–10) 22L:2D, (d 11–32) 20L:4D, and (d 33–42) 23L:1D (this lightening program is adopted from the standards of genetic science—Ross Aviagen Brand and the chick ´ lowo, Poland). At the initial supplier—Poultry Park Smi stage of rearing (d 1–2), mean temperature in the shed was 32◦ C–33◦ C and decreased in subsequent periods as follows: 30◦ C (d 3), 28◦ C–30◦ C (d 4–7), 26◦ C–28◦ C (d 8–14), 24◦ C–26◦ C (d 15–21), about 22◦ C (d 22–42). Average humidity ranged from 50% at the beginning to 70% at the final stage of the cycle, increasing gradually from week to week. Broilers were fed ad libitum with the following feeds: starter (1–12 d of age), grower I (13–22 d of age), grower II (23–32 d of age), and finisher (33–42 d of age). The composition and nutritional value of the applied premixes is shown in Table 1. In this study, the body weight (BW) of the broilers (in 1, 21, and 42 d of age), daily feed intake (FI), WI, and the number of deaths were recorded. The resulting data were used to calculate body weight gain (BWG), feed conversion ratio (FCR), and survival rate. The exper-
Blood Biochemistry and Enumeration of Bacteria
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT
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Table 1. Ingredient and nutrient compositions of basal diets (%). Item
Starter (0–12 d)
Grower I (13–22 d)
Grower II (23–32 d)
Finisher (33–42 d)
Ingredient Wheat, 11.6% Soybean meal, 46% Maize, 8% Soya oil Canola meal, 32.5% Potato protein, 73% Limestone Monocalcium phosphate Vitamin and mineral premix1 Poultry fat L-Lys-HCl DL-Met Salt NaHCO3 Thr Choline chloride Phytase premix2 Total Calculated analysis ME (kcal/kg) Lys Met Ca P Na
37.00 29.05 25.93 2.68 – 1.50 1.27 0.92 0.53 – 0.39 0.26 0.24 0.14 0.07 – 0,02 100
38.00 24.94 27.39 2.78 2.50 1.0 0.85 0.43 0.53 0.50 0.40 0.17 0.27 0.13 0.06 0.03 0.02 100
33.00 27.16 28.74 3.79 2.50 0.50 0.78 0.30 0.60 1.50 0.41 0.18 0.28 0.15 0.07 0.02 0.02 100
40.00 24.0 25.32 3.66 3.01 – 0.67 0.11 0.59 1.50 0.43 0.17 0.28 0.14 0.07 0.03 0.02 100
2800.0 1.26 0.56 0.86 0.56 0.15
2865.0 1.16 0.46 0.67 0.47 0.15
2984.1 1.15 0.45 0.71 0.42 0.15
3020.0 1.07 0.44 0.65 0.39 0.15
Analyzed nutrient composition3 Crude protein Crude fiber Crude fat Crude ash
21.06 2.93 4.34 5.42
19.58 3.04 4.60 4.67
19.56 3.36 7.18 4.28
18.52 3.38 7.36 4.00
1 Vitamin-mineral premix contained the following per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 5,000 IU; vitamin E, 50 mg; vitamin B1, 3 mg; vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 15 μ g; nicotinic acid, 60 mg; pantothenic acid, 14.7 mg; folic acid, 1.5 mg; iron, 63 mg; copper, 15 mg; cobalt, 1.0 mg; zinc, 100 mg; iodine, 1.0 mg; selenium, 0.3 mg, antioxidant (BHA). 2 Phytase premix was prepared by dilution with calcium carbonate to contain 1.000 FTU (phytase units)/g (Optiphos, Huvepharma AD, Sofia, Bulgaria). 3 Based on a DM content of 87.5%.
suspension was thoroughly mixed. Prior to further dilutions, larger particles were always allowed to settle (up to 15 min). Subsequently (in less than 45 min), the following individual procedures were performed: ISO 4833:2013-13 for the general enumeration of microorganisms (Escherichia coli, coliform, total aerobic and anaerobic bacteria) using the pour plate technique ISO 20128:2006 for the enumeration of lactic acid bacteria, PN-ISO 15213:2005 and PN-EN ISO 7937:2005 for detection of Clostridium spp. (including Clostridium perfringens). Selective agar media were used for enumeration of target bacterial groups: total aerobes (Nutrient Agar—Merck, Darmstadt, Germany); total anaerobes (Wilkens-Chalgren Agar—HiMedia, Mumbai, India); lactic acid bacteria (MRS agar—Merck, Darmstadt, Germany); Escherichia coli and total coliform bacteria (MacConkey Agar—Merck, Darmstadt, Germany) and Clostridium spp. (TSC Agar—HiMedia, Mumbai, India). Samples incubated under anaerobic conditions at 37◦ C for 72 ± 3 h were used for the determination of the total numbers of Clostridium spp., lactic acid bacteria, and total anaerobic bacteria, whereas samples incubated under aerobic conditions at 37◦ C for
72 h were used for the determination of the total numbers of Escherichia coli and coliform. Total aerobic bacteria were determined after incubation for 72 h at 30◦ C. Finally, the number of bacterial colonies was calculated (PN-EN ISO 7218:2008). The numbers of colony forming units (CFU) were expressed as log10 CFU per gram.
Essential Oil Commercially available lavender (Lavandula angustifolia) flower EO (Avicenna Oil, Wroclaw, Poland) was analyzed for chemical composition. Chromatography was performed using the Agilent 6890 N Network gas chromatograph (Agilent Technologies, Palo Alto, USA) with the 5973 Network mass selective detector, equipped with the 7683 Series Injector automatic liquid sampler. Chromatography operating conditions were adjusted experimentally for the optimum analyte separation. A GC capillary column HP-5MSI (30 m length, 0.25 mm inner diameter) was used. The operating temperatures were as follows: injector 250◦ C, ion source 230◦ C, and quadrupole 150◦ C. The split-mode (10:1) injection of 3-μl samples was applied. Analysis was
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performed in scan mode in the m/z range 40–500. The column temperature program was: 60◦ C (hold 3 min), 10◦ C/min to 300◦ C (hold 13 min). In order to confirm the compliance of the LEO chemical composition with the requirements of ISO 3515:2002, we performed a quantitative and qualitative analysis. The identification of the main compounds of the LEO was carried out based on mass spectra and retention indices. In order to confirm the identification, the retention indices of the compounds were calculated and compared with the literature (Babushok et al., 2011). In order to determine n-alkanes retention indexes, we used the C7– C30 standard (Supelco, Bellefonte, USA) analyzed under the same chromatographic conditions. The relative content of each compound was measured by the peak area share in the total ion current of all compounds present in the analyte.
Statistical Analysis The obtained results of BW, BWG, FCR, FI, and WI were subjected to statistical analysis, calculating the means and mean standard deviation and performing the two-way analysis of variance (ANOVA), assessing intergroup differences using post-hoc Tuckey’s test at P < 0.01. The results of selected biochemical parameters of blood serum and the numbers of microorganisms in the ileum content were developed by statistically calculating the mean values, the mean standard deviation, and performing the one-way ANOVA. The significance of differences between the means was assessed using post-hoc Tukey’s test. The test probability at P < 0.05 was considered statistically significant. Survival rate in the examined groups was compared with Fisher’s precise test. Statistical analysis was performed using the PQStat v. 1.6.2 package (PQStat Sofware, Poland). In this study, H0 hypothesis assumed that an addition of 0.4 ml/L LEO to the drinking water of chickens has no effect on BW, BWG, FCR, FI, WI, survival rate, selected biochemical parameters of blood, and the number of microorganisms in the ileum. An alternative hypothesis—H1 — assumes the occurrence of a significant effect on the considered variables.
RESULTS Chemical Composition of Essential Oil The composition of LEO is presented in Table 2. In total, 26 substances have been identified, mainly linalool acetate (46.25%) and linalool (35.17%). The following compounds were found in lower concentrations (<5%): caryophyllene, α-pinene, borneol, neryl acetate, cis-β -ocimene, p-cymene, lavandulyl acetate, and 4terpineol. Chromatography showed that the relative percentage content of each class of compounds in LEO was as follows: monoterpene hydrocarbons (8.17%),
Table 2. Main components (%) detected chromatography-mass spectrometry in LEO.
Compound
RT1 (min)
RI2
α -pinene camphene β -pinene β -myrcene hexyl acetate 3-carene p-cymene eucalyptol trans-β -ocimene cis-β -ocimene linalool oxide linalool camphor borneol terpinen-4-ol p-menth-1-en-8-ol α -terpineole linalool acetate bornyl acetate lavandulol acetate carvacrol nerol acetate geraniol acetate copaene caryophyllene β -farnesene
5.14 5.50 6.22 6.64 7.14 7.34 7.59 7.71 8.00 8.30 9.03 10.05 11.04 11.76 12.07 12.54 13.74 14.34 14.47 15.17 15.41 17.13 17.36 17.88 18.49 19.63
935 947 974 985 985 1008 1020 1030 1036 1045 1073 1102 1044 1065 1178 1184 1191 1253 1283 1285 1301 1361 1375 1381 1420 1454
RI3
Ref
936 950 977 985 989 1011 1024 1031 1038 1048 1075 1099 1043 1068 1177 1183 1190 1256 1284 1289 1300 1362 1376 1380 1420 1456
by
Gas
Percentage4 (%)
SEM
1.90 0.60 0.59 0.89 0.70 0.25 1.14 0.85 0.10 0.10 0.10 35.17 0.46 1.94 0.54 0.52 0.10 46.25 0.10 0.81 0.10 1.54 0.16 0.17 2.63 0.13
0.04 0.01 0.11 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.15 0.00 0.01 0.01 0.01 0.01 0.13 0.01 0.01 0.00 0.04 0.01 0.01 0.03 0.01
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Retention time. Retention index. 3 Reference average retention index values (Babushok et al., 2011). 4 Results are mean. 2
oxygenated monoterpenes (87.79%), and sesquiterpene hydrocarbons (2.93%).
Performance Parameters Table 3 presents the production performance of broilers. From d 22–42, broilers treated with LEO exhibited significantly (P < 0.01) higher BW as compared to the control group. An addition of LEO to drinking water improved chickens slaughter BW, irrespective of how long it had been applied for. Upon completion of the rearing cycle, the highest BWG (P < 0.01) was recorded in groups LEO1–42 (2.88 kg) and LEO22–42 (2.84 kg), and the lowest in the control group (2.68 kg). No significant differences between the groups were found in relation to FI (P > 0.05). The average daily FI remained at a similar level (0.11 kg/d). No significant differences between the groups were observed in WI (P > 0.05). Total WI over the entire duration of rearing ranged from 9.25 L/bird, in group LEO22–42 , to 9.39 L/bird, in the control. Average daily WI (L/bird) over the entire experiment ranged between 0.2203, in group LEO1–42 , and 0.2236, in the control. LEO application had a positive effect on FCR in the second period (d 22–42) and for the entire duration of rearing (d 1–42; P < 0.01). The highest FCR (kg/kg) was found in the control group, which did not have LEO added in the water. Survival rates were similar (P > 0.05) between the groups, from 96%, in the control, to 99%, in LEO22–42 .
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT Table 3. Effect of LEO on the performance parameters of broiler chickens from 1 to 42 d of age.1 Measurement per period (P3 , d) BW (kg/bird) 1 21 42 BWG (kg/bird) 1–21 22–42 1–42 FI (kg/day per bird) 1–21 22–42 1–42 FCR (kg/kg) 1–21 22–42 1–42 WI (L/day per bird) 1–21 22–42 1–42 P-value4 BW BWG FI FCR WI
Group (G)2 Control
LEO1–42
LEO22–42
SEM
0.04 1.17 2.72a
0.04 1.17 2.93b
0.04 1.15 2.89b
0.003 0.016 0.020
1.12 1.56a 2.68a
1.12 1.76b 2.88b
1.10 1.74b 2.84b
0.016 0.022 0.019
0.06 0.15 0.11
0.06 0.15 0.11
0.06 0.15 0.11
0.065 0.010 0.020
1.18 2.03a 1.74a
1.16 1.79b 1.57b
1.19 1.81b 1.61b
0.016 0.007 0.001
0.13 0.32 0.22
0.13 0.31 0.22
0.13 0.31 0.22
0.011 0.010 0.016
Period (P) < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
Group (G) < 0.01 < 0.01 0.99 < 0.01 0.98
Period × group < 0.01 < 0.01 0.98 < 0.01 0.97
Values in rows with different letters differ significantly (P < 0.01). Results are means of 5 replicates per treatment. 2 Group (G): control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. 3 Period (P), BW: body weight, BWG: body weight gain, FI: feed intake, FCR: feed conversion ratio, WI: water intake. 4 Significance level of treatment factor: P = period (growing period), G = group, (P × G) = interaction between the period of growing and groups of treatments. a,b 1
Table 4. Level of biochemical blood parameters of broiler chickens at d 42.1,2 Group3
Cholesterol (mmol/L) Glucose (mmol/L) Triglyceride (mmol/L) Uric acid (mmol/L)
Control
LEO1–42
LEO22–42
SEM
P-value
3.67 13.50 0.88 0.31
3.83 13.24 0.91 0.32
3.57 12.83 0.86 0.25
0.07 0.14 0.04 0.02
0.344 0.133 0.840 0.308
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Results are means of 5 replicates per treatment. Reference average of blood parameters values (mmol/L): cholesterol = 2.96–6.80; glucose = 6.1–17.1; triglyceride = 0.15–1.1; uric acid = 0.19–0.46 (Mazurkiewicz et al., 2005). 3 Control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. 2
Blood Biochemistry and Intestinal Microflora Table 4 summarizes the blood biochemical parameters in the chickens after 42 d of the experiment. The addition of LEO had no effect on the biochemical parameters of chicken blood serum (P > 0.05). The results of quantitative analysis of the ileal microbial flora
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in 42-d-old chicks are shown in Table 5. The numbers of anaerobic and mesophilic aerobic bacteria were the highest and similar in all groups (control, LEO1–42 , and LEO22–42 ). Differences were found in Escherichia coli, coliform, and lactic acid bacteria counts in LEO1–42 and LEO22–42 , as compared with the control group (P < 0.01). No anaerobic Clostridium sp. bacteria have been detected in any of the samples.
DISCUSSION LEO is a mixture of volatile organic compounds. GS-MS, applied in this study, is a tool for screening volatile secondary plant metabolites. In this study, oxygenated monoterpenes, which included linalool and linalool acetate, represented the highest proportion among the components. It should be emphasized that linalool is a compound of proven antimicrobial and antioxidant activity (Carrasco et al., 2016). LEO should be in accordance to the requirements of the pharmacopoeia or ISO 3515:2002 (the main components of the oil should be linalool: 20.0%–45.0%, and linalool acetate: 25.0%–47.0%). According to the European Pharmacopoeia (Eur Ph 8.0, 2014), LEO should contain the following components: eucalyptol (<2.5%), camphor (<1.2%), linalool (20.0%–45.0%), linalool acetate (25.0%–47.0%), 4-terpineol (0.1%–8.0%), lavandulyl acetate (>0.1%), α-terpineol (<2.0%). The composition of oil used in the present study was consistent with the guidelines of the pharmacopoeia and ISO 3515:2002. In this study, it has been shown that an addition of LEO had a significant impact on the final BW of broilers. Chickens provided with LEO-infused drinking water weighed on average 6.35% more (on d 42 of the experiment; P < 0.01) as compared to the control group. The application of LEO in the second period of rearing (d 22–42) had a positive effect on BWG and FCR (P < 0.01). In this study, no differences in WI were found in any of the examined periods of bird rearing (P > 0.05). The use of LEO did not affect WI, probably due to the good tolerance of broiler chickens to its smell (flowery-herbal) and taste (slightly sweet). A decrease in WI, in turn, was observed in studies by Khosravinia (2013, 2016), where EO from Satureja khuzistanica, whose main chemical component was carvacrol (92.2%), was added to drinking water. The reduction in WI could be connected to a change in its taste ( more bitter) and intensive spicy smell. It should be noted that reduced WI may have a negative impact on kidney function (Khosravinia et al., 2013). Other authors also found that an addition of bitter EO, whose main components were carvacrol, thymol, and cinnamaldehyde, to water and feed had an unfavorable effect on WI and FI (Lee et al., 2004; Windisch et al., 2008). In contrast, Alali et al. (2013) observed that an addition of a mixture of carvacrol, thymol, eucalyptol, lemon oil, and citric acid had no effect on WI. In this study, a favorable effect of the addition of LEO to water for broiler chickens on production performance was found,
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Table 5. Bacterial quantification (log10 CFU) in ileum content at d 42.1 Group2 Microorganisms (CFU/g)
Control
LEO1–42
LEO22–42
SEM
P-value
Total anaerobic bacteria Total aerobic bacteria Escherichia coli Coliform Clostridium spp. Probiotic bacteria
7.42 5.72 4.28a 3.29a nd 5.23a
7.64 5.30 3.22b 2.15b nd 6.36b
7.96 5.30 3.21b 2.56b nd 6.09b
0.14 0.15 0.08 0.15 – 0.14
0.47 0.16 < 0.01 < 0.01 – < 0.01
Values in rows with different letters differ significantly (P < 0.01). Results are means of 5 repicates per treatment. Control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. nd: none determined. a,b 1 2
but no differences between the period of supplying LEO (d 1–42 and d 22–42); hence, it may be assumed that supplying LEO in the second period of rearing is more profitable for economic reasons (d 22–42). Other authors, who experimented on broiler chickens, found a positive effect of EO in respect to BW and FCR over the entire rearing cycle (Alali et al., 2013; Khattak et al., 2014). Khattak et al. (2014) reported that the application of a mixture of EO extracted from basil, cumin, bay leaf, lemon, sage, and thyme in the feed of broiler chickens increased BW and improved FCR (P < 0.05). Botsoglu et al. (2002) and Cross et al. (2007) did not observed any improvements in the performance parameters resulting from feed with an addition of EO in an amount of 50–1000 mg/kg of feed. In contrast, a negative impact of summer savory (Satureja hortensis) oil was found by Andi (2015), who experimented with concentrations ranging from 0.1 to 0.5 ml/L in drinking water. The addition of oil caused a decrease in the BW and FI of chickens. The author suggests that the likely cause of a decrease in FI and WI by the birds was the bitterspicy taste of the EO (Andi, 2015). A deterioration of production performance resulting from the use of EO in the feed was observed by Kirkpinar et al., 2011. Differences in the efficiency of the application of EO may result from incorrect concentrations (too low or too high doses, which may have toxic effects), improper chemical composition (some components of EO have toxic more specifically nephron—and neurotoxic effects, e.g., tujon and anteol derivatives), too short duration, and method of application as well as the different taste and smell of EO. The experiment revealed that in all groups the mortality was low throughout the study (<5%). There were no differences between treatment groups (P > 0.05). Betancourt et al. (2014) have shown that EO from three chemotypes of oregano (Origanum vulgare) reduced the mortality of birds by 59% on average. Conversely, Alali et al. (2013) and Vuki´c-Vranjeˇs et al. (2013) reported the lack of effects of a mixture of EO added to water and feed on the mortality of chickens. Opinions on the argument that the mortality of chickens may be reduced by the use of EO are varied, since researchers use EO of various chemical compositions, and, in consequence,
of different biological activity and impact on the avian body. Moreover, it should be emphasized that most experiments have been carried out in ideal hygienic conditions, with smaller stocking densities; hence, it is difficult to achieve a significant improvement of survival rates. Under production conditions, however, densities of chickens and the loads of the sheds are much higher, which worsens the hygienic conditions of management, creating a background that allows as to see clearly the beneficial effects of EO on the reduction of mortality. This study revealed that an addition of LEO to water had no effect on the selected blood serum biochemical parameters (cholesterol, glucose, triglyceride, uric acid). Conversely, B¨ol¨ ukba¸sı et al. (2008) found lower levels of cholesterol and triglycerides, as compared with the control group, in the blood of laying hens receiving feed supplemented with a mixture of EO from thyme, sage, and rosemary. The authors (B¨ ol¨ ukba¸sı et al., 2008) report that selected EO and their components (borneol, cineole, citral, fenchone, geraniol, carvacrol, menthol) have an inhibitory effect on HMG-CoA reductase, the enzyme responsible for the synthesis of cholesterol in the liver, thereby reducing its content in the blood of birds. Other researchers (Najafi and Torki, 2010; Khattak et al., 2014; Ghazanfari et al., 2015; Kim et al., 2016) reported no effects of the EO used in the rearing of chickens in terms of blood biochemical parameters. Intestinal microflora is an important factor for avian health and poultry production yields. They are, however, also crucial for consumer safety, since gut contents may contaminate the carcass with a variety of pathogens, such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Clostridium perfringens, Campylobacter, and Salmonella (Choi et al., 2015). The qualitative and quantitative composition of intestinal microflora in poultry is affected by many factors, e.g., environmental stress, housing conditions farm microclimate, age of birds, and the composition of feed. Homeostasis of gastrointestinal microflora can be influenced by, among others, dietary supplementation with certain active substances, such as EO (Roberts et al., 2015). This study proves that LEO added to the water of broiler chickens had a bacteriostatic effect,
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT
reducing CFU of Escherichia coli and coliform bacteria, additionally increasing the numbers of probiotic bacteria (P < 0.01). This result indicates that LEO may represent a good dietary additive to poultry feed, which is particularly important due to the increasing resistance of E. Coli to antimicrobials (Mainali et al., 2013). Another in vivo experiment, which was aimed at investigating the effect of different selected EO on bacterial colonization in various sections of avian intestines, confirmed the reduction of the numbers of Escherichia coli, Clostridium perfringens, and the groups Enterococcus, Salmonella and Staphylococcus (Cross et al., 2007; Tiihonen et al., 2010; Kirkpinar et al., 2011; Erhan et al., 2012; Hong et al., 2012; Vuki´c-Vranjeˇs et al., 2013). Moreover, an experiment revealed that pennyroyal EO (Mentha pulegium L.) added to feed in amounts of 0.25% and 0.5% may positively affect the numbers of lactic acid bacteria (Erhan et al., 2012). Isolated cases have also reported on the inhibitory effects against the proliferation of Lactobacillus (Tiihonen et al, 2010; Hong et al., 2012). It is important to select appropriate biologically active substances, which will reduce the numbers of enteric pathogens without affecting on lactic acid bacteria (Choct, 2009). The underdeveloped gastrointestinal tract in the first week chicks after hatch and its microflora are often unable to fight successfully pathogenic organisms, since bacteria such as Escherichia coli, Salmonella sp., or Clostridium sp. proliferate much more quickly than natural gut microbiota strains. It should also be noted that the activity of the given EO depends on the dose and the part of the gastrointestinal tract under study. Besides reducing the populations of certain pathogenic microbial species, the effects of EO may also be associated with acidification of the gut contents (Gopi et al., 2014). EO may also affect intestinal morphology. Reducing the number of pathogenic bacteria in the gut can improve the capacity of epithelial cells in terms of intestinal villi regeneration, and thereby increase the absorption of nutrients (Choi et al., 2015; Zeng et al., 2015). Data on this, however, are inconsistent and involve only the increased or constant length of the villi in the jejunum and colon of broilers fed with an EO-supplemented diet (Vuki´c-Vranjeˇs et al., 2013; Khattak et al., 2014).
CONCLUSION Based on the conducted study, a favorable effect of adding LEO to water intended for broiler chickens has been found. LEO stimulated the growth and proper functioning of the broiler chickens’ bodies, which resulted in their improved health and increased production parameters. Throughout the production cycle (1– 42 d of age), broiler chickens that received LEO in an amount of 0.4 mL/L in water were characterized by on average 6.35% higher BW (P < 0.01) and, on average 8.62% lower FCR (P < 0.01), as compared with the control group. The study has confirmed that LEO has a high potentially positive effect on the intesti-
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nal microflora. LEO is characterized by the appropriate chemical composition (high concentration of linalool and linalool acetate); it shows antimicrobial activity towards intestinal pathogens (Escherichia coli, coliform bacteria), without inhibiting the number of lactic fermentation bacteria, and it even promotes the growth of beneficial bacteria. There was no effect, however, of the addition of LEO on the biochemical parameters of chicken blood serum. Moreover, no differences were observed between the periods of adding LEO (d 1–42 and d 22–42); thus, it may be assumed that adding LEO in the second period of rearing (d 22–42) is more profitable for economic reasons. The addition of LEO to water from 22 d of age is justified by the influence of the operation of intensive rearing conditions, which intensify in the second rearing period (inter alia deterioration of the quality of litter, as well as an increase—in the quantity of conditionally pathogenic bacteria, the concentrations of ammonia in the air and the giveaway heat from the body to the environment). In order to assess fully the potential use of LEO in chicken fattening, further research into its mechanism of action, dosage, diet compatibility, activity against other pathogens, and possible toxicity is needed.
REFERENCES Alali, W. Q., C. L. Hofacre, G. F. Mathis, and G. Faltys. 2013. Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers. Poult. Sci. 92:836–841. Andi, M. A. 2015. Influence of Saturejahortensis L. essential oil in drinking water on broiler production and some blood biochemical parameters. Int. J. Adv. Biol. Biomed. Res. 3:391–396. Babushok, V. I., P. J. Linstrom, and I. G. Zenkevich. 2011. Retencion indices for frequently reported compounds of plant essential oils. J. Phys. Chem. Ref. Data. 4:431011–431046. Betancourt, L., F. Rodriguez, V. Phandanouvong, C. Ariza-Nieto, M. Hume, D. Nisbet, G. Afanador-T´ellez, A. Martynova Van Kley, and A. Nalian. 2014. Effect of Origanum chemotypes on broiler intestinal bacteria. Poult. Sci. 93:2526–2535. ˝ Kaynar. 2008. The effect of B¨ol¨ ukba¸sı, S ¸ . C., M. K. Erhan, and O. feeding thyme, sage and rosemary oil on laying hen performance, cholesterol and some proteins ratio of egg yolk and Escherichia coli count in feces. Arch. Gefl¨ ugelk. 5:231–237. Botsoglou, N. A., P. Florou-Paner, E. Christaki, D. J. Fletouris, and A. B. Spais. 2002. Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br. Poult. Sci. 43:223– 230. Carrasco, A., R. Martinez-Gutierrez, V. Tomas, and J. Tudela. 2016. Lavandula angustifolia and Lavandula latifolia essential oils from Spain: aromatic profile and bioactivities. Planta Med. 82:163–170. Choct, M. 2009. Managing gut health through nutrition. Br. Poult. Sci. 50:9–15. Choi, K. Y., T. K. Lee, and W. J. Sul. 2015. Metagenomic analysis of chicken gut microbiota for improving metabolism and health of chickens—a review. Asian-Australas. J. Anim. Sci. 28:1217– 1225. Cross, D. E., R. M. McDevitt, K. Hillman, and T. Acamovic. 2007. The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. Br. Poult. Sci. 48:496–506. Erhan, M. K., S. C. Bolukbas, and H. Urusan. 2012. Biological activities of pennyroyal (Mentha pulegium L.) in broilers. Livestock Sci. 146:189–192. European Pharmacopeia 8.0-8.8. (Eur Ph). 2014.
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Ghazanfari, S. I., Z. I. Mohammadi, and M. Adib Moradi. 2015. Effects of coriander essential oil on the performance, blood characteristics, intestinal microbiota and histological of broilers. Braz. J. Poult. Sci. 5:419–426. Ghelardini, C., N. Galeotti, G. Salvatore, and G. Mazzanti. 1999. Local anaesthetic activity of the essential oil of Lavandula angustifolia. Planta Med. 65:700–703. Giovannini, D., A. Gismodni, A. Basso, L. Canuti, R. Braglia, A. Canini, F. Mariani, and G. Cappelli. 2016. Lavandula angustifolia Mill. essential oil exerts antibacterial and anti-Inflammatory effect in macrophage mediated immune response to Staphylococcus aureus. Immunol. Invest. 45:11–28. Gopi, M., K. Karthik, H. V. Manjunathachar, P. Tamilmahan, M. Kesavan, M. Dashprakash, B. L. Balaraju, and M. R. Purushothaman. 2014. Essential oils as a feed additive in poultry nutrition. Adv. Anim.Vet. Sci. 1:1–7. Hong, J. C., T. Steiner, A. Aufy, and T. F. Lien. 2012. Effects of supplemental essential oil on growth performance, lipid metabolites and immunity, intestinal characteristics, microbiota and carcass traits in broilers. Livestock Sci. 144:253–262. ISO 3515:2002. Oil of lavender (Lavandula angustifolia Mill.). ISO 20128:2006. Milk products–enumeration of presumptive Lactobacillus acidophilus on a selective medium–colony-count technique at 37 degrees C. ISO 4833-1:2013-12. Microbiology of the food chain-horizontal method f or the enumeration of microorganisms-part 1: colonycount at 30 degrees C by the pour plate technique. Khosravinia, H. 2016. Mortality, production performance, water intake and organ weight of the heat stressed broiler chicken given savory (Satureja khuzistanica) essential oils through drinking water. J. App. Anim. Res. 1:273–280. Khosravinia, H. 2013. Productive performance, litter characteristics and carcass defects of the broiler chickens given drinking water supplemented with Satureja khuzistanica essential oils. J. Med. Plant Res. 7:1754–1760. Khosravinia, H., S. Ghasemi, and A. E. Rafiei. 2013. Effect of savory (Satureja khuzistanica) essential oils on performance, liver and kidney functions in broiler chicks. J. Anim. Feed Sci. 1:50– 55. Kim, S. J., K. W. Lee, C. W. Kang, and B. K. An. 2016. Growth performance, relative meat and organ weights, cecal microflora, and blood characteristics in broiler chickens fed fiets containing different nutrient density with or without essential oils. AsianAustralas. J. Anim. Sci 4:549–554. Kirkpinar, F., H. B. Unulu, and G. Ozdemir. 2011. Effects of oregano and garlic essential oils on performance, carcase, organ and blood characteristics and intestinal microflora of broilers. Livestock Sci. 137:219–225. Khattak, F., A. Ronchi, P. Castelli, and N. Sparks. 2014. Effects of natural blend of essential oil on growth performance, blood biochemistry, cecal morphology, and carcass quality of broiler chickens. Poult. Sci. 93:132–137. Kirimer, N., S. Mokhtarzadeh, B. Demirci, F. Goger, K. M. Khawar, and F. Demirci. 2017. Phytochemical profiling of volatile components of Lavandula angustifolia Miller propagated under in vitro conditions. Ind. Crops Prod. 96:120–125. Lee, K. W., H. Everts, H. J. Kappert, M. Frehner, H. Wouterse, and A. C. Beynen. 2004. Cinnamanaldehyde, but not thymol, coun-
teracts the carboxymethyl cellulose-induced growth depression in female broiler chickens. Int. J. Poult. Sci. 3:608–612. Lis-Balchin, M. 2002. Lavender. The genus Lavandula, 1st edition. CRC Press, London, Taylor & Francis. Mainali, C., M. McFall, and R. Irwin. 2013. Evaluation of antimicrobial resistance profiles of Escherichia coli isolates of broiler chickens at slaughter in Alberta, Canada. J. Food Prot. 76:2045– 2051. Mazurkiewicz, M. et al. 2005. Chapter 13.2.2. Biochemical diagnostics. Pages 686–697 in Poultry Diseases. (Academic Press; 1st Edition). Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland. Najafi, P., and M. Torki. 2010. Performance, blood metabolites and immunocompetence of broiler chicks fed diets included essential oils of medicinal herbs. J. Anim. Vet. Adv. 7:1164–1168. PN EN ISO 7218:2008. Microbiology of food and animal feeding stuffs – general requirements and guidance for microbiological examinations (ISO 7218:2007/Amd 1:2013) (in Polish). PN EN ISO 7937-2005. Microbiology of food and animal feeding stuffs - horizontal method for the enumeration of Clostridium perfringens - colony-count technique (in Polish). PN ISO 15213-2005. Microbiology of food and animal feeding stuffs - horizontal method for the enumeration of sulfite-reducing bacteria growing under anaerobic conditions (in Polish). PN EN ISO 6887-1-2000. Microbiology of food and animal feeding stuffs - preparation of test samples, initial suspension and decimal dilutions for microbiological examination - part 1: general rules for the preparation of the initial suspension and decimal dilutions (in Polish). ´ Prusinowska, R., and K. Smigielski. 2014. Composition, biological properties and therapeutic effects of lavender (Lavandula angustifolia L.). A Review. Herba Polonica 2:56–66. Roberts, T., J. Wilson, A. Guthrie, J. Cookson, D. Vancraynest, K. Schaeffer, R. Moody, and S. Clark. 2015. New issues and science in broiler chicken intestinal health: emerging technology and alternative interventions. J. Appl. Poult. Res. 1:1–10. Sasannejad, P., M. Saeedi, A. Shoeibi, A. Gorgi, M. Abbasi, and M. Foroughipour. 2012. Effect of essential oil in the treatment of migraine headache: a placebo-controlled clinical trial. Eur. Neurol. 67:288–291. Tiihonen, K., H. Kettunen, M. L. Bento, M. Saarinen, S. Lahtinen, A. C. Ouwehand, H. Schulze, and N. Rautonen. 2010. The effect of feeding essential oils on broiler performance and gut microbiota. Br. Poult. Sci. 3:381–392. ˇ Vuki´c-Vranjeˇs, N., D. Tolimir, R. Vukmirovi´c, V. Colovi´ c, P. Stana´cev, and S. Ikoni´c. 2013. Effect of phytogenic additives on performance, morphology and caecal microflora of broiler chickens. Bio. Anim. Husb. 2:311–319. Windisch, W., K. Schedle, C. Plitzner, and A. Kroismay. 2008. Use of phytogenic products as feed additives for swine and poultry. J. Anim. Sci. 86:E140–E148 Yang, S. A., S. K. Jeon, E. J. Lee, Ch. H. Shim, and I. Lee. 2010. Comparative study of the chemical composition and antioxidant activity of six essential oils and their components. Nat. Prod. Res. 24:140–151. Zeng, Z., S. Zhang, H. Wang, and X. Paio. 2015. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. J. Anim. Sci. Biotechnol. 6:7–15.
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ABSTRACT The aim of this study was to evaluate growth performance, selected biochemical blood parameters, and the microbiota of ileal digesta in broiler chickens provided with drinking water containing an addition of natural lavender essential oil (LEO). The experiment was carried out on a commercial farm using n = 300 unsexed Ross-308 broiler chickens. One-day-old chicks were randomly assigned to three groups of 100 chickens each (five replications, 20 individuals each). The control group broilers were provided with drinking water without the addition of LEO. Groups LEO1–42 and LEO22–42 had access to water containing 0.4 ml/L LEO (for 6 h/day) from days 1 to 42 (LEO1–42 ) and 22 to 42 (LEO22–42 ). Body weight, feed intake, water intake, and mortality were recorded throughout the ex-
Key words: broiler, lavender essential oil, performance, blood biochemistry, intestinal microflora 2018 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pey385 (e.g., myrcene, α-pinene, caryophyllene), alcohols (e.g., linalool, α-terpineol, borneol), ketones (e.g., camphor, carvone, eucarvone), esters (e.g., linalool acetate, lavandulyl acetate, geranyl acetate), aldehydes (e.g., neral), oxides (e.g., caryophyllene oxide), and ethers (e.g., eucalyptol; Lis-Balchin, 2002). In addition to these compounds, coumarins and organic acids are found in LEO ´ (Prusinowska and Smigielski, 2014). Recent studies, analyzing LEO chemical constituents an attempt to describe their action mechanisms on rats and human models, confirm many beneficial properties of this oil. According to the literature, LEO has antibacterial, antifungal, antioxidant, analgesic, anti-inflammatory, and antispasmodic properties (Yang et al., 2010; Prusi´ nowska and Smigielski, 2014; Carrasco et al., 2016; Giovannini et al., 2016). Studies on animal and human models alike have shown that lavender has potential immunostimulatory, anxiolytic, sedative, hypnotic, analgesic, and anticonvulsant effects, and may also improve one’s mood (Ghelardini et al., 1999; Sasannejad et al., ´ 2012; Prusinowska and Smigielski, 2014). The literature lacks reports on the possibility of using LEO in poultry production as a supplement in the diet of broiler chickens in order to stimulate production
INTRODUCTION Essential oils (EO) are classified as phytobiotics, i.e., natural substances having a protective function against harmful pathogenic microorganisms (Zeng et al., 2015). Lavender essential oil (LEO) is a by-product of lavender flowers used in human aromatherapy, pharmacy, perfumery, cosmetics, and the food industry (Prusi´ nowska and Smigielski, 2014; Kirimer et al., 2017). Lavender flowers (Lavandulae flos) contain up to 3% of EO, whose qualitative and quantitative composition is variable and depends on the genotype, climatic conditions, mode of reproduction, and morphological char´ acteristics of the plant (Prusinowska and Smigielski, 2014). LEO is a volatile, biologically active substance with a characteristic scent. Chemically, it is a multicomponent mixture of terpenoid compounds (monoterpenes, sesquiterpenes, and their oxygen derivatives), characterized based on isoprenoid units (Carrasco et al., 2016). LEO compounds include hydrocarbons C 2018 Poultry Science Association Inc. Received October 4, 2016. Accepted August 2, 2018. 1 Corresponding author: [email protected]
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´ ´ ´ ADASZYNSKA-SKWIRZY NSKA AND SZCZERBINSKA
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performance. Therefore, this study was undertaken with the purpose of evaluating the applicability of LEO in broiler production. The effects of LEO offered as a drinking water supplement on production performance, selected biochemical blood parameters, and ileal microbiota in broiler chickens were analyzed.
iment was carried out with the approval of the Local Ethics Committee for the Experiments on Animals in Szczecin (permit no. 19/2015 dated 2015 May 22).
MATERIALS AND METHODS
In order to determine the levels of selected biochemical parameters, on d 42 of age blood was collected from the left brachial vein of 10 randomly selected birds from each group (five replications of two individuals each). The tests were carried out on the day of sampling. Blood samples were centrifuged at 15,000 × G for 5 min in order to obtain the serum, which was then stored for analysis under controlled refrigerated conditions (+5◦ C ±3◦ C). The temperature of serum samples during the analysis was maintained at +18◦ C ±1◦ C. Blood biochemical parameters were measured using the VetTest 8008 (Idexx Laboratories, Inc., Westbrook, USA) chemistry analyzer with dry-slide technology using the Idexx Laboratories methodology. Blood parameters analyzed in this study included: glucose, cholesterol, triglycerides, and uric acid. Additionally, at 42 d of age, five randomly selected broilers from each group (five replications of one individual each), with BW similar to the group’s average, were slaughtered, and ileal digesta samples were collected for microbiological analyses aimed at preselected groups of microorganisms (Clostridium spp., Escherichia coli, coliform, aerobic, anaerobic, and lactic acid bacteria). The intestine was spread individually on the autopsy table in the following orientation: the duodenum (from the gizzard outlet to the end of the pancreatic loop), small intestine (from the site where the duodenum emerges from the gizzard to the beginning of the ceca), ceca, and large intestine (from the colon to the rectum). Then, the duodenum, ceca, and large intestine were removed. The remaining small intestine was composed of two parts, the jejunum (from the pancreatic loop to Meckel’s diverticulum) and the ileum divided by Meckel’s diverticulum. The ileum was isolated at the intestine fragment between the Meckel diverticulum and the ileocecal junction. Three points were designated in this area: the middle of the ileum, and half way from the center to each end (with three repetitions for the final calculations). The ileum was opened with scissors, ileal content (1 g/each point) was collected by gentle fingers-stripping of the surrounding ileal segment. The samples were collected using small, sterile plastic tubes (volume 12 ml) and were kept in an ice box. Serial dilutions were performed within 1 h of collection. The initial suspension and decimal dilutions were performed according to the European Standard PNEN ISO 6887-1:2000 (10−1 dilution as the initial dilution up to 10−9 as the final dilution) under a laminar flow safety cabinet (2nd class; BioBase, South Gongye Road, Jinan, China). Buffered peptone water (Merck, Darmstadt, Germany) was used as a diluent. The initial
Bird Trial The experiment was performed in a commercial poul˙ owko, Poland) using 300 unsexed Rosstry farm (Zab´ 308 broilers. The chickens were raised in the intensive system. The experimental period was 42 d. Chicks were purchased from a commercial hatchery (Park Dro´ lowo, Poland). On arrival, 1-d-old biarski Sp. z o.o., Smi birds were weighed and then randomly assigned to three treatments groups with five replicate pens containing 20 birds each. Throughout the experiment, the control group broilers were provided with drinking water without an addition of LEO. Groups LEO1–42 and LEO22–42 were provided with water (for 6 h/day—the mean time of water intake (WI) with LEO throughout the production cycle) with an addition of 0.4 ml/L oil, from 1 to 42 d of age (LEO1–42 ) and from 22 to 42 d of age (LEO22–42 ). The birds were provided with water in 5 L manual reservoir tank reversible drinkers (in each pen). Once all LEO-infused water was drunk, the reservoir tanks were replaced with new ones containing clean water without the addition of LEO. The remaining water in each pen was measured by volume. Over the entire duration of the experiment, broilers had unlimited access to water. Broilers were housed in the same shed for 42 d, on wheat-straw bedding, with a stocking density of 14 individuals per m2 . All groups were managed under uniform, standard environmental conditions. The following lighting program was applied: (d 1–6) 24L:0D, (d 7–10) 22L:2D, (d 11–32) 20L:4D, and (d 33–42) 23L:1D (this lightening program is adopted from the standards of genetic science—Ross Aviagen Brand and the chick ´ lowo, Poland). At the initial supplier—Poultry Park Smi stage of rearing (d 1–2), mean temperature in the shed was 32◦ C–33◦ C and decreased in subsequent periods as follows: 30◦ C (d 3), 28◦ C–30◦ C (d 4–7), 26◦ C–28◦ C (d 8–14), 24◦ C–26◦ C (d 15–21), about 22◦ C (d 22–42). Average humidity ranged from 50% at the beginning to 70% at the final stage of the cycle, increasing gradually from week to week. Broilers were fed ad libitum with the following feeds: starter (1–12 d of age), grower I (13–22 d of age), grower II (23–32 d of age), and finisher (33–42 d of age). The composition and nutritional value of the applied premixes is shown in Table 1. In this study, the body weight (BW) of the broilers (in 1, 21, and 42 d of age), daily feed intake (FI), WI, and the number of deaths were recorded. The resulting data were used to calculate body weight gain (BWG), feed conversion ratio (FCR), and survival rate. The exper-
Blood Biochemistry and Enumeration of Bacteria
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT
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Table 1. Ingredient and nutrient compositions of basal diets (%). Item
Starter (0–12 d)
Grower I (13–22 d)
Grower II (23–32 d)
Finisher (33–42 d)
Ingredient Wheat, 11.6% Soybean meal, 46% Maize, 8% Soya oil Canola meal, 32.5% Potato protein, 73% Limestone Monocalcium phosphate Vitamin and mineral premix1 Poultry fat L-Lys-HCl DL-Met Salt NaHCO3 Thr Choline chloride Phytase premix2 Total Calculated analysis ME (kcal/kg) Lys Met Ca P Na
37.00 29.05 25.93 2.68 – 1.50 1.27 0.92 0.53 – 0.39 0.26 0.24 0.14 0.07 – 0,02 100
38.00 24.94 27.39 2.78 2.50 1.0 0.85 0.43 0.53 0.50 0.40 0.17 0.27 0.13 0.06 0.03 0.02 100
33.00 27.16 28.74 3.79 2.50 0.50 0.78 0.30 0.60 1.50 0.41 0.18 0.28 0.15 0.07 0.02 0.02 100
40.00 24.0 25.32 3.66 3.01 – 0.67 0.11 0.59 1.50 0.43 0.17 0.28 0.14 0.07 0.03 0.02 100
2800.0 1.26 0.56 0.86 0.56 0.15
2865.0 1.16 0.46 0.67 0.47 0.15
2984.1 1.15 0.45 0.71 0.42 0.15
3020.0 1.07 0.44 0.65 0.39 0.15
Analyzed nutrient composition3 Crude protein Crude fiber Crude fat Crude ash
21.06 2.93 4.34 5.42
19.58 3.04 4.60 4.67
19.56 3.36 7.18 4.28
18.52 3.38 7.36 4.00
1 Vitamin-mineral premix contained the following per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 5,000 IU; vitamin E, 50 mg; vitamin B1, 3 mg; vitamin B2, 10 mg; vitamin B6, 3 mg; vitamin B12, 15 μ g; nicotinic acid, 60 mg; pantothenic acid, 14.7 mg; folic acid, 1.5 mg; iron, 63 mg; copper, 15 mg; cobalt, 1.0 mg; zinc, 100 mg; iodine, 1.0 mg; selenium, 0.3 mg, antioxidant (BHA). 2 Phytase premix was prepared by dilution with calcium carbonate to contain 1.000 FTU (phytase units)/g (Optiphos, Huvepharma AD, Sofia, Bulgaria). 3 Based on a DM content of 87.5%.
suspension was thoroughly mixed. Prior to further dilutions, larger particles were always allowed to settle (up to 15 min). Subsequently (in less than 45 min), the following individual procedures were performed: ISO 4833:2013-13 for the general enumeration of microorganisms (Escherichia coli, coliform, total aerobic and anaerobic bacteria) using the pour plate technique ISO 20128:2006 for the enumeration of lactic acid bacteria, PN-ISO 15213:2005 and PN-EN ISO 7937:2005 for detection of Clostridium spp. (including Clostridium perfringens). Selective agar media were used for enumeration of target bacterial groups: total aerobes (Nutrient Agar—Merck, Darmstadt, Germany); total anaerobes (Wilkens-Chalgren Agar—HiMedia, Mumbai, India); lactic acid bacteria (MRS agar—Merck, Darmstadt, Germany); Escherichia coli and total coliform bacteria (MacConkey Agar—Merck, Darmstadt, Germany) and Clostridium spp. (TSC Agar—HiMedia, Mumbai, India). Samples incubated under anaerobic conditions at 37◦ C for 72 ± 3 h were used for the determination of the total numbers of Clostridium spp., lactic acid bacteria, and total anaerobic bacteria, whereas samples incubated under aerobic conditions at 37◦ C for
72 h were used for the determination of the total numbers of Escherichia coli and coliform. Total aerobic bacteria were determined after incubation for 72 h at 30◦ C. Finally, the number of bacterial colonies was calculated (PN-EN ISO 7218:2008). The numbers of colony forming units (CFU) were expressed as log10 CFU per gram.
Essential Oil Commercially available lavender (Lavandula angustifolia) flower EO (Avicenna Oil, Wroclaw, Poland) was analyzed for chemical composition. Chromatography was performed using the Agilent 6890 N Network gas chromatograph (Agilent Technologies, Palo Alto, USA) with the 5973 Network mass selective detector, equipped with the 7683 Series Injector automatic liquid sampler. Chromatography operating conditions were adjusted experimentally for the optimum analyte separation. A GC capillary column HP-5MSI (30 m length, 0.25 mm inner diameter) was used. The operating temperatures were as follows: injector 250◦ C, ion source 230◦ C, and quadrupole 150◦ C. The split-mode (10:1) injection of 3-μl samples was applied. Analysis was
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performed in scan mode in the m/z range 40–500. The column temperature program was: 60◦ C (hold 3 min), 10◦ C/min to 300◦ C (hold 13 min). In order to confirm the compliance of the LEO chemical composition with the requirements of ISO 3515:2002, we performed a quantitative and qualitative analysis. The identification of the main compounds of the LEO was carried out based on mass spectra and retention indices. In order to confirm the identification, the retention indices of the compounds were calculated and compared with the literature (Babushok et al., 2011). In order to determine n-alkanes retention indexes, we used the C7– C30 standard (Supelco, Bellefonte, USA) analyzed under the same chromatographic conditions. The relative content of each compound was measured by the peak area share in the total ion current of all compounds present in the analyte.
Statistical Analysis The obtained results of BW, BWG, FCR, FI, and WI were subjected to statistical analysis, calculating the means and mean standard deviation and performing the two-way analysis of variance (ANOVA), assessing intergroup differences using post-hoc Tuckey’s test at P < 0.01. The results of selected biochemical parameters of blood serum and the numbers of microorganisms in the ileum content were developed by statistically calculating the mean values, the mean standard deviation, and performing the one-way ANOVA. The significance of differences between the means was assessed using post-hoc Tukey’s test. The test probability at P < 0.05 was considered statistically significant. Survival rate in the examined groups was compared with Fisher’s precise test. Statistical analysis was performed using the PQStat v. 1.6.2 package (PQStat Sofware, Poland). In this study, H0 hypothesis assumed that an addition of 0.4 ml/L LEO to the drinking water of chickens has no effect on BW, BWG, FCR, FI, WI, survival rate, selected biochemical parameters of blood, and the number of microorganisms in the ileum. An alternative hypothesis—H1 — assumes the occurrence of a significant effect on the considered variables.
RESULTS Chemical Composition of Essential Oil The composition of LEO is presented in Table 2. In total, 26 substances have been identified, mainly linalool acetate (46.25%) and linalool (35.17%). The following compounds were found in lower concentrations (<5%): caryophyllene, α-pinene, borneol, neryl acetate, cis-β -ocimene, p-cymene, lavandulyl acetate, and 4terpineol. Chromatography showed that the relative percentage content of each class of compounds in LEO was as follows: monoterpene hydrocarbons (8.17%),
Table 2. Main components (%) detected chromatography-mass spectrometry in LEO.
Compound
RT1 (min)
RI2
α -pinene camphene β -pinene β -myrcene hexyl acetate 3-carene p-cymene eucalyptol trans-β -ocimene cis-β -ocimene linalool oxide linalool camphor borneol terpinen-4-ol p-menth-1-en-8-ol α -terpineole linalool acetate bornyl acetate lavandulol acetate carvacrol nerol acetate geraniol acetate copaene caryophyllene β -farnesene
5.14 5.50 6.22 6.64 7.14 7.34 7.59 7.71 8.00 8.30 9.03 10.05 11.04 11.76 12.07 12.54 13.74 14.34 14.47 15.17 15.41 17.13 17.36 17.88 18.49 19.63
935 947 974 985 985 1008 1020 1030 1036 1045 1073 1102 1044 1065 1178 1184 1191 1253 1283 1285 1301 1361 1375 1381 1420 1454
RI3
Ref
936 950 977 985 989 1011 1024 1031 1038 1048 1075 1099 1043 1068 1177 1183 1190 1256 1284 1289 1300 1362 1376 1380 1420 1456
by
Gas
Percentage4 (%)
SEM
1.90 0.60 0.59 0.89 0.70 0.25 1.14 0.85 0.10 0.10 0.10 35.17 0.46 1.94 0.54 0.52 0.10 46.25 0.10 0.81 0.10 1.54 0.16 0.17 2.63 0.13
0.04 0.01 0.11 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.15 0.00 0.01 0.01 0.01 0.01 0.13 0.01 0.01 0.00 0.04 0.01 0.01 0.03 0.01
1
Retention time. Retention index. 3 Reference average retention index values (Babushok et al., 2011). 4 Results are mean. 2
oxygenated monoterpenes (87.79%), and sesquiterpene hydrocarbons (2.93%).
Performance Parameters Table 3 presents the production performance of broilers. From d 22–42, broilers treated with LEO exhibited significantly (P < 0.01) higher BW as compared to the control group. An addition of LEO to drinking water improved chickens slaughter BW, irrespective of how long it had been applied for. Upon completion of the rearing cycle, the highest BWG (P < 0.01) was recorded in groups LEO1–42 (2.88 kg) and LEO22–42 (2.84 kg), and the lowest in the control group (2.68 kg). No significant differences between the groups were found in relation to FI (P > 0.05). The average daily FI remained at a similar level (0.11 kg/d). No significant differences between the groups were observed in WI (P > 0.05). Total WI over the entire duration of rearing ranged from 9.25 L/bird, in group LEO22–42 , to 9.39 L/bird, in the control. Average daily WI (L/bird) over the entire experiment ranged between 0.2203, in group LEO1–42 , and 0.2236, in the control. LEO application had a positive effect on FCR in the second period (d 22–42) and for the entire duration of rearing (d 1–42; P < 0.01). The highest FCR (kg/kg) was found in the control group, which did not have LEO added in the water. Survival rates were similar (P > 0.05) between the groups, from 96%, in the control, to 99%, in LEO22–42 .
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT Table 3. Effect of LEO on the performance parameters of broiler chickens from 1 to 42 d of age.1 Measurement per period (P3 , d) BW (kg/bird) 1 21 42 BWG (kg/bird) 1–21 22–42 1–42 FI (kg/day per bird) 1–21 22–42 1–42 FCR (kg/kg) 1–21 22–42 1–42 WI (L/day per bird) 1–21 22–42 1–42 P-value4 BW BWG FI FCR WI
Group (G)2 Control
LEO1–42
LEO22–42
SEM
0.04 1.17 2.72a
0.04 1.17 2.93b
0.04 1.15 2.89b
0.003 0.016 0.020
1.12 1.56a 2.68a
1.12 1.76b 2.88b
1.10 1.74b 2.84b
0.016 0.022 0.019
0.06 0.15 0.11
0.06 0.15 0.11
0.06 0.15 0.11
0.065 0.010 0.020
1.18 2.03a 1.74a
1.16 1.79b 1.57b
1.19 1.81b 1.61b
0.016 0.007 0.001
0.13 0.32 0.22
0.13 0.31 0.22
0.13 0.31 0.22
0.011 0.010 0.016
Period (P) < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
Group (G) < 0.01 < 0.01 0.99 < 0.01 0.98
Period × group < 0.01 < 0.01 0.98 < 0.01 0.97
Values in rows with different letters differ significantly (P < 0.01). Results are means of 5 replicates per treatment. 2 Group (G): control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. 3 Period (P), BW: body weight, BWG: body weight gain, FI: feed intake, FCR: feed conversion ratio, WI: water intake. 4 Significance level of treatment factor: P = period (growing period), G = group, (P × G) = interaction between the period of growing and groups of treatments. a,b 1
Table 4. Level of biochemical blood parameters of broiler chickens at d 42.1,2 Group3
Cholesterol (mmol/L) Glucose (mmol/L) Triglyceride (mmol/L) Uric acid (mmol/L)
Control
LEO1–42
LEO22–42
SEM
P-value
3.67 13.50 0.88 0.31
3.83 13.24 0.91 0.32
3.57 12.83 0.86 0.25
0.07 0.14 0.04 0.02
0.344 0.133 0.840 0.308
1
Results are means of 5 replicates per treatment. Reference average of blood parameters values (mmol/L): cholesterol = 2.96–6.80; glucose = 6.1–17.1; triglyceride = 0.15–1.1; uric acid = 0.19–0.46 (Mazurkiewicz et al., 2005). 3 Control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. 2
Blood Biochemistry and Intestinal Microflora Table 4 summarizes the blood biochemical parameters in the chickens after 42 d of the experiment. The addition of LEO had no effect on the biochemical parameters of chicken blood serum (P > 0.05). The results of quantitative analysis of the ileal microbial flora
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in 42-d-old chicks are shown in Table 5. The numbers of anaerobic and mesophilic aerobic bacteria were the highest and similar in all groups (control, LEO1–42 , and LEO22–42 ). Differences were found in Escherichia coli, coliform, and lactic acid bacteria counts in LEO1–42 and LEO22–42 , as compared with the control group (P < 0.01). No anaerobic Clostridium sp. bacteria have been detected in any of the samples.
DISCUSSION LEO is a mixture of volatile organic compounds. GS-MS, applied in this study, is a tool for screening volatile secondary plant metabolites. In this study, oxygenated monoterpenes, which included linalool and linalool acetate, represented the highest proportion among the components. It should be emphasized that linalool is a compound of proven antimicrobial and antioxidant activity (Carrasco et al., 2016). LEO should be in accordance to the requirements of the pharmacopoeia or ISO 3515:2002 (the main components of the oil should be linalool: 20.0%–45.0%, and linalool acetate: 25.0%–47.0%). According to the European Pharmacopoeia (Eur Ph 8.0, 2014), LEO should contain the following components: eucalyptol (<2.5%), camphor (<1.2%), linalool (20.0%–45.0%), linalool acetate (25.0%–47.0%), 4-terpineol (0.1%–8.0%), lavandulyl acetate (>0.1%), α-terpineol (<2.0%). The composition of oil used in the present study was consistent with the guidelines of the pharmacopoeia and ISO 3515:2002. In this study, it has been shown that an addition of LEO had a significant impact on the final BW of broilers. Chickens provided with LEO-infused drinking water weighed on average 6.35% more (on d 42 of the experiment; P < 0.01) as compared to the control group. The application of LEO in the second period of rearing (d 22–42) had a positive effect on BWG and FCR (P < 0.01). In this study, no differences in WI were found in any of the examined periods of bird rearing (P > 0.05). The use of LEO did not affect WI, probably due to the good tolerance of broiler chickens to its smell (flowery-herbal) and taste (slightly sweet). A decrease in WI, in turn, was observed in studies by Khosravinia (2013, 2016), where EO from Satureja khuzistanica, whose main chemical component was carvacrol (92.2%), was added to drinking water. The reduction in WI could be connected to a change in its taste ( more bitter) and intensive spicy smell. It should be noted that reduced WI may have a negative impact on kidney function (Khosravinia et al., 2013). Other authors also found that an addition of bitter EO, whose main components were carvacrol, thymol, and cinnamaldehyde, to water and feed had an unfavorable effect on WI and FI (Lee et al., 2004; Windisch et al., 2008). In contrast, Alali et al. (2013) observed that an addition of a mixture of carvacrol, thymol, eucalyptol, lemon oil, and citric acid had no effect on WI. In this study, a favorable effect of the addition of LEO to water for broiler chickens on production performance was found,
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Table 5. Bacterial quantification (log10 CFU) in ileum content at d 42.1 Group2 Microorganisms (CFU/g)
Control
LEO1–42
LEO22–42
SEM
P-value
Total anaerobic bacteria Total aerobic bacteria Escherichia coli Coliform Clostridium spp. Probiotic bacteria
7.42 5.72 4.28a 3.29a nd 5.23a
7.64 5.30 3.22b 2.15b nd 6.36b
7.96 5.30 3.21b 2.56b nd 6.09b
0.14 0.15 0.08 0.15 – 0.14
0.47 0.16 < 0.01 < 0.01 – < 0.01
Values in rows with different letters differ significantly (P < 0.01). Results are means of 5 repicates per treatment. Control = no additive, LEO1–42 = addition of 0.4 ml/L LEO from d 1 to 42 d of age, LEO22–42 = addition of 0.4 ml/L LEO from d 22 to 42 d of age. nd: none determined. a,b 1 2
but no differences between the period of supplying LEO (d 1–42 and d 22–42); hence, it may be assumed that supplying LEO in the second period of rearing is more profitable for economic reasons (d 22–42). Other authors, who experimented on broiler chickens, found a positive effect of EO in respect to BW and FCR over the entire rearing cycle (Alali et al., 2013; Khattak et al., 2014). Khattak et al. (2014) reported that the application of a mixture of EO extracted from basil, cumin, bay leaf, lemon, sage, and thyme in the feed of broiler chickens increased BW and improved FCR (P < 0.05). Botsoglu et al. (2002) and Cross et al. (2007) did not observed any improvements in the performance parameters resulting from feed with an addition of EO in an amount of 50–1000 mg/kg of feed. In contrast, a negative impact of summer savory (Satureja hortensis) oil was found by Andi (2015), who experimented with concentrations ranging from 0.1 to 0.5 ml/L in drinking water. The addition of oil caused a decrease in the BW and FI of chickens. The author suggests that the likely cause of a decrease in FI and WI by the birds was the bitterspicy taste of the EO (Andi, 2015). A deterioration of production performance resulting from the use of EO in the feed was observed by Kirkpinar et al., 2011. Differences in the efficiency of the application of EO may result from incorrect concentrations (too low or too high doses, which may have toxic effects), improper chemical composition (some components of EO have toxic more specifically nephron—and neurotoxic effects, e.g., tujon and anteol derivatives), too short duration, and method of application as well as the different taste and smell of EO. The experiment revealed that in all groups the mortality was low throughout the study (<5%). There were no differences between treatment groups (P > 0.05). Betancourt et al. (2014) have shown that EO from three chemotypes of oregano (Origanum vulgare) reduced the mortality of birds by 59% on average. Conversely, Alali et al. (2013) and Vuki´c-Vranjeˇs et al. (2013) reported the lack of effects of a mixture of EO added to water and feed on the mortality of chickens. Opinions on the argument that the mortality of chickens may be reduced by the use of EO are varied, since researchers use EO of various chemical compositions, and, in consequence,
of different biological activity and impact on the avian body. Moreover, it should be emphasized that most experiments have been carried out in ideal hygienic conditions, with smaller stocking densities; hence, it is difficult to achieve a significant improvement of survival rates. Under production conditions, however, densities of chickens and the loads of the sheds are much higher, which worsens the hygienic conditions of management, creating a background that allows as to see clearly the beneficial effects of EO on the reduction of mortality. This study revealed that an addition of LEO to water had no effect on the selected blood serum biochemical parameters (cholesterol, glucose, triglyceride, uric acid). Conversely, B¨ol¨ ukba¸sı et al. (2008) found lower levels of cholesterol and triglycerides, as compared with the control group, in the blood of laying hens receiving feed supplemented with a mixture of EO from thyme, sage, and rosemary. The authors (B¨ ol¨ ukba¸sı et al., 2008) report that selected EO and their components (borneol, cineole, citral, fenchone, geraniol, carvacrol, menthol) have an inhibitory effect on HMG-CoA reductase, the enzyme responsible for the synthesis of cholesterol in the liver, thereby reducing its content in the blood of birds. Other researchers (Najafi and Torki, 2010; Khattak et al., 2014; Ghazanfari et al., 2015; Kim et al., 2016) reported no effects of the EO used in the rearing of chickens in terms of blood biochemical parameters. Intestinal microflora is an important factor for avian health and poultry production yields. They are, however, also crucial for consumer safety, since gut contents may contaminate the carcass with a variety of pathogens, such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Clostridium perfringens, Campylobacter, and Salmonella (Choi et al., 2015). The qualitative and quantitative composition of intestinal microflora in poultry is affected by many factors, e.g., environmental stress, housing conditions farm microclimate, age of birds, and the composition of feed. Homeostasis of gastrointestinal microflora can be influenced by, among others, dietary supplementation with certain active substances, such as EO (Roberts et al., 2015). This study proves that LEO added to the water of broiler chickens had a bacteriostatic effect,
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EFFECT OF LAVENDER ESSENTIAL OIL AS A DRINKING WATER SUPPLEMENT
reducing CFU of Escherichia coli and coliform bacteria, additionally increasing the numbers of probiotic bacteria (P < 0.01). This result indicates that LEO may represent a good dietary additive to poultry feed, which is particularly important due to the increasing resistance of E. Coli to antimicrobials (Mainali et al., 2013). Another in vivo experiment, which was aimed at investigating the effect of different selected EO on bacterial colonization in various sections of avian intestines, confirmed the reduction of the numbers of Escherichia coli, Clostridium perfringens, and the groups Enterococcus, Salmonella and Staphylococcus (Cross et al., 2007; Tiihonen et al., 2010; Kirkpinar et al., 2011; Erhan et al., 2012; Hong et al., 2012; Vuki´c-Vranjeˇs et al., 2013). Moreover, an experiment revealed that pennyroyal EO (Mentha pulegium L.) added to feed in amounts of 0.25% and 0.5% may positively affect the numbers of lactic acid bacteria (Erhan et al., 2012). Isolated cases have also reported on the inhibitory effects against the proliferation of Lactobacillus (Tiihonen et al, 2010; Hong et al., 2012). It is important to select appropriate biologically active substances, which will reduce the numbers of enteric pathogens without affecting on lactic acid bacteria (Choct, 2009). The underdeveloped gastrointestinal tract in the first week chicks after hatch and its microflora are often unable to fight successfully pathogenic organisms, since bacteria such as Escherichia coli, Salmonella sp., or Clostridium sp. proliferate much more quickly than natural gut microbiota strains. It should also be noted that the activity of the given EO depends on the dose and the part of the gastrointestinal tract under study. Besides reducing the populations of certain pathogenic microbial species, the effects of EO may also be associated with acidification of the gut contents (Gopi et al., 2014). EO may also affect intestinal morphology. Reducing the number of pathogenic bacteria in the gut can improve the capacity of epithelial cells in terms of intestinal villi regeneration, and thereby increase the absorption of nutrients (Choi et al., 2015; Zeng et al., 2015). Data on this, however, are inconsistent and involve only the increased or constant length of the villi in the jejunum and colon of broilers fed with an EO-supplemented diet (Vuki´c-Vranjeˇs et al., 2013; Khattak et al., 2014).
CONCLUSION Based on the conducted study, a favorable effect of adding LEO to water intended for broiler chickens has been found. LEO stimulated the growth and proper functioning of the broiler chickens’ bodies, which resulted in their improved health and increased production parameters. Throughout the production cycle (1– 42 d of age), broiler chickens that received LEO in an amount of 0.4 mL/L in water were characterized by on average 6.35% higher BW (P < 0.01) and, on average 8.62% lower FCR (P < 0.01), as compared with the control group. The study has confirmed that LEO has a high potentially positive effect on the intesti-
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nal microflora. LEO is characterized by the appropriate chemical composition (high concentration of linalool and linalool acetate); it shows antimicrobial activity towards intestinal pathogens (Escherichia coli, coliform bacteria), without inhibiting the number of lactic fermentation bacteria, and it even promotes the growth of beneficial bacteria. There was no effect, however, of the addition of LEO on the biochemical parameters of chicken blood serum. Moreover, no differences were observed between the periods of adding LEO (d 1–42 and d 22–42); thus, it may be assumed that adding LEO in the second period of rearing (d 22–42) is more profitable for economic reasons. The addition of LEO to water from 22 d of age is justified by the influence of the operation of intensive rearing conditions, which intensify in the second rearing period (inter alia deterioration of the quality of litter, as well as an increase—in the quantity of conditionally pathogenic bacteria, the concentrations of ammonia in the air and the giveaway heat from the body to the environment). In order to assess fully the potential use of LEO in chicken fattening, further research into its mechanism of action, dosage, diet compatibility, activity against other pathogens, and possible toxicity is needed.
REFERENCES Alali, W. Q., C. L. Hofacre, G. F. Mathis, and G. Faltys. 2013. Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers. Poult. Sci. 92:836–841. Andi, M. A. 2015. Influence of Saturejahortensis L. essential oil in drinking water on broiler production and some blood biochemical parameters. Int. J. Adv. Biol. Biomed. Res. 3:391–396. Babushok, V. I., P. J. Linstrom, and I. G. Zenkevich. 2011. Retencion indices for frequently reported compounds of plant essential oils. J. Phys. Chem. Ref. Data. 4:431011–431046. Betancourt, L., F. Rodriguez, V. Phandanouvong, C. Ariza-Nieto, M. Hume, D. Nisbet, G. Afanador-T´ellez, A. Martynova Van Kley, and A. Nalian. 2014. Effect of Origanum chemotypes on broiler intestinal bacteria. Poult. Sci. 93:2526–2535. ˝ Kaynar. 2008. The effect of B¨ol¨ ukba¸sı, S ¸ . C., M. K. Erhan, and O. feeding thyme, sage and rosemary oil on laying hen performance, cholesterol and some proteins ratio of egg yolk and Escherichia coli count in feces. Arch. Gefl¨ ugelk. 5:231–237. Botsoglou, N. A., P. Florou-Paner, E. Christaki, D. J. Fletouris, and A. B. Spais. 2002. Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br. Poult. Sci. 43:223– 230. Carrasco, A., R. Martinez-Gutierrez, V. Tomas, and J. Tudela. 2016. Lavandula angustifolia and Lavandula latifolia essential oils from Spain: aromatic profile and bioactivities. Planta Med. 82:163–170. Choct, M. 2009. Managing gut health through nutrition. Br. Poult. Sci. 50:9–15. Choi, K. Y., T. K. Lee, and W. J. Sul. 2015. Metagenomic analysis of chicken gut microbiota for improving metabolism and health of chickens—a review. Asian-Australas. J. Anim. Sci. 28:1217– 1225. Cross, D. E., R. M. McDevitt, K. Hillman, and T. Acamovic. 2007. The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. Br. Poult. Sci. 48:496–506. Erhan, M. K., S. C. Bolukbas, and H. Urusan. 2012. Biological activities of pennyroyal (Mentha pulegium L.) in broilers. Livestock Sci. 146:189–192. European Pharmacopeia 8.0-8.8. (Eur Ph). 2014.
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