Use of Lippia rotundifolia and Cymbopogon flexuosus essential oils , individually or in combination , in broiler diets

This study investigated the effects of Cymbopogon flexuosus and Lippia rotundifolia microencapsulated essential oils on broiler performance and carcass yield. One hundred and fifty mixed-sex Cobb broiler chicks were used, from one day up to 42 days of age, in a completely randomized design, with five treatments and three replicates of ten birds each. The treatments were: negative control (basal diet), positive control (diet with enramycin and salinomycin), and three diets with microencapsulated essential oils from lemongrass, L. rotundifolia, and combination with 50% of both. The performance and carcass yield were not affected by the treatments. The intestine absolute weight was lower in the combination treatment compared with the negative control treatment and the lemongrass essential oil. The intestine relative weight was higher in the treatments with lemongrass and L. rotundifolia essential oils in relation to the combination. The liver relative weight was lower with the lemongrass essential oil and the combination compared with the treatment with the L. rotundifolia essential oil. The trial could not find results enough to recommend the use of the lemongrass and L. rotundifolia essential oils as an additive in broiler diets.


Introduction
Antimicrobials have been used as additives in poultry for many years, contributing to improved growth performance and feeding efficiency of birds (Agostini et al., 2012).However, concern about the increase in cases of bacterial resistance and residues in products have resulted in the prohibition of performance enhancers by the European Union (Santos et al., 2005).Thus, Brazilian exporters must confirm to the standards, stimulating studies on substitutes for antibiotics, such as plant essential oils and other additives such as organic acids, probiotics, and prebiotics.
Essential oils and their main compounds can be used as additives and improve broiler performance and intestinal health (Cho et al., 2014).In general, they act by promoting selection for the beneficial microorganisms in the intestine (Traesel et al., 2011).
Cymbopogon flexuosus essential oil is extracted from the leaves of the plant also known as lemongrass.The mixture between neral isomers (citral b or cis-citral) and geranial (citral a or trans-citral) form citral, which is the major component of lemongrass and gives the essential oil its characteristic aroma, besides various long-known medicinal properties utilized in popular culture, including antimicrobial activity (Adukwu et al., 2012;Desai and Parikh, 2012).Lemongrass essential oil, due to the effect of the citral, can be cytotoxic and genotoxic to human lymphocytes, but is safe if used at low concentrations, under 400 µg/mL (Sinha et al., 2014).
Lippia rotundifolia is an endemic plant to the Brazilian Cerrado, from the area known as the "Serra do Espinhaço".Although little studied, it is a promising species that belongs to the family Verbenaceae.According to Gomide et al. (2013), the major component of its essential oil is β-myrcene.It also has proven antimicrobial activity (Souza et al., 2015).There are no reports in the literature of the use of these two species as additives in animal feed.Also, when used together, the plants can show synergistic effects on the performance of broilers.
Microencapsulation can be made to increase the stability of the essential oils (to circumvent volatility problems) and protect them during processing and feed passage through the stomach (Barreto et al., 2008;Traesel et al., 2011).The objective of this study was to evaluate the effect of the microencapsulated essential oils of lemongrass and L. rotundifolia as additives in the diet on performance, carcass yield, and organ weights of broilers.

Material and Methods
The research was conducted in Montes Claros, Minas Gerais, Brazil, between January and February, 2015.The procedures were performed in accordance with ethical standards and approved by the Ethics Committee on Animal Usage of the Universidade Federal de Minas Gerais.
The 150 one-day-old Cobb 500 ® chicks were used, mixed, housed in 30 cages (60 × 35 × 100 cm) with feeders and waterers.The experiment was conducted in a completely randomized design, with five treatments and three replicates of 10 animals.The treatments were: negative control, without antimicrobials and anticoccidials; positive control -feed supplemented with 10 ppm enramycin and 42 ppm salinomycin; lemongrass -feed containing 120 mg of lemongrass essential oil for each kg -1 of animal body weight; L. rotundifolia -feed containing 120 mg of L. rotundifolia essential oil for each kg -1 of animal body weight; combination -feed with the mixture of the two essential oils.The dose used was defined from the antimicrobial activity presented by the essential oils of L. rotundifolia (Souza et al., 2015) and lemongrass (Azevedo et al., 2016) in preliminary in vitro tests.
The nutrition plan was divided into three phases: starter (1-21 days), grower (22-33 days), and finisher (34-42 days).The diets were formulated to meet nutritional levels recommended by Rostagno et al. (2011) and offered ad libitum throughout the experimental period in a mashed form (Table 1).
The lemongrass essential oil was acquired from the Ferquima Industry and Trade LTD company (Vargem Grande Paulista, SP, Brazil) and the L. rotundifolia oil was purchased from producers of the Fundação Universidade do Vale do Jequitinhonha Cooperativa (Serro, district of São Gonçalo do Rio das Pedras, Minas Gerais, Brazil), both extracted by the steam distillation method and packaged in 500-mL bottles.
Subsequently, the oils were converted into microcapsules by the Croma Microencapsulados company (São Paulo, SP, Brazil) via the coacervation method with edible polymers.
After microencapsulation and inclusion to diet, new analysis was done to verify the permanence of the essential oil compounds.For the analysis of the volatiles, the feed was transferred to headspace-type glass vials (20 mL) and placed in the autosampler system (HS combi-PAL).The vial contents were homogenized (500 rpm), incubated (75 °C for 5 min) and subsequently injected (1000 µL) with a pre-heated syringe (75 °C).For identification of volatiles, analysis was performed using Agilent Technologies system (7890A), coupled with mass spectrometry (MS 5975C), fitted with a DB-5ms fused silica capillary column (30 m × 0.25 mm × 0.25 μm) and helium (flow of 1 mL•min -1 ) as carrier gas.The programming was 35 °C for 2 min, at 2 °C/min up to 80 ºC, then 4 °C min -1 up to 150 °C, for a total chromatographic run of 42 min.The column was heated at 300 °C during 1 min for cleaning.The system operated in scan mode (monitoring) with electron impact at 70 eV, in a range of 45 to 550 m/z.The identification of volatiles was performed by MSD Chemstation software for comparison of the mass spectrum with the library (National Institute of Standards and Technology, NIST 2002).
The birds and feed leftovers were weighed on the first day of the experiment and at 7, 21, 33, and 42 days of age.The variables analyzed were average body weight (BW), weight gain, feed intake, and feed conversion ratio (the relationship between intake and gain, calculated by BW gain/feed intake) of each phase.In the total period of the experiment, we calculated the production viability (VC), by the difference between the total number of housed birds and the number of dead birds, divided by the total birds housed and multiplied by 100, and productive efficiency index (PEI), through the daily average weight gain multiplied by the production viability and divided by the feed conversion multiplied by 10.
After 43 days, two birds of each experimental treatment were selected (one male and one female, weighing up to 10% above or below the average weight), after fasting for 8 h, then slaughtered by bleeding from the jugular vein, scalded, plucked, eviscerated, and then the commercial cuts were separated.Yields assessed were: carcass; breast; thighs + drumsticks; wings; back; feet; relative and absolute weight of the organs (intestine, caecum, liver, pancreas, gizzard, bursa, spleen, heart, and abdominal fat), plus the length of the intestines.Carcass yield was obtained by the ratio of hot carcass weight (gutted) and the fasting weight of the bird.The remaining yields were obtained by the ratio of the weight of the parts and the hot carcass weight.Data of performance, carcass yield, and organ weights were subjected to analysis of variance and means were compared by Tukey test at 0.05 probability.
Analysis by gas chromatography using the headspace system was carried out to evaluate the persistence of the components in the feed (Table 3).The compounds detected in the essential oils prior to microencapsulation remained after the process, as well as after incorporation in the feed.Even storage for ten days did not impair the composition of the oils.In the feed with combination of the two essential oils, component characteristic of both species used were detected.
Animal health is directly linked to the balance of intestinal microflora.Naturally, beneficial and pathogenic microorganisms colonize the gastrointestinal tract of  poultry.The beneficial microorganisms can collaborate in the synthesis of vitamins, reduce gas production, improve digestion and nutrient absorption, and inhibit the growth of pathogenic bacteria.However, the imbalance of microflora leads to an increase of pathogenic microorganisms that can cause much harm to the animal (diseases, mucosal lesions, digestion process deterioration) (Furlan et al., 2004).Essential oils can ensure the balance of the microflora and, consequently, the animal health (Cho et al., 2014).Body weight, weight gain, feed intake, feed conversion, and productive efficiency indices (Table 4) were not statistically different among treatments (P>0.05).Good hygiene and management practiced during animal rearing may have contributed to their low quantity of pathogenic microorganisms in the intestine, not worsening even in the negative control.Mortality (0.0%) and production viability (100%), which were similar among treatments, confirm the good experimental conditions.According to Freitas et al. (2001), the significant effects of the use of performance enhancers are best perceived when animals are subjected to hygiene-challenge conditions, with high population density, high contamination risk, poor hygiene, and exposure to diseases.The same applies to the use of essential oils.As a result, the lack of effect on performance   is common among works.Silva et al. (2011) also obtained similar results using essential oil of mastic-red, compared with the negative control (without feed additives) and the positive control (ration with added zinc bacitracin and salinomycin).Shiva et al., 2012 did not detect differences between the performance of animals receiving essential oil of oregano and those receiving antibiotics (bacitracin methylene disalicylate and colistin sulfate) or no additive.Both authors highlight the lack of hygiene challenge as a cause of the resemblance.However, Cho et al. (2014) improved feed efficiency in broilers challenged with Clostridium perfringens fed diets with thyme and star anise, compared with positive (avilamycin) and negative controls.
Treatments with essential oils did not differ statistically from the positive (with antimicrobials and anticoccidials) and negative (without additives) controls (P>0.05)for carcass and cut yields (Table 5).Rizzo et al. (2010) analyzed broiler chickens fed diets plus commercial products containing various essential oils and also found no differences in carcass and cut yields at 44 days in animals receiving essential oils and fed diets containing avilamycin or no additive.On the other hand, Khattak et al. (2014) observed higher carcass and breast yields in animals treated with a commercial combination of essential oils of basil, cumin, bay leaf, lemon, oregano, sage, thyme, and tea, compared with the diet without additives.
The absolute and relative weights of the intestines and liver were affected by the treatments (Table 6).The values for the absolute intestine weight were lower in the treatment with the combination of essential oils, compared with treatment without additives and treatment with the lemongrass essential oil (P<0.05).The relative weight of intestine was higher in treatments with lemongrass essential oil and L. rotundifolia essential oil in relation to their combination (P<0.05).The reduction in intestinal weight is expected, since the use of such additives reduces the thickness of the intestinal wall.Silva et al. (2011) also observed a reduction in weight of the intestines of birds supplemented with antimicrobials and essential oil of mastic-red.However, animals that received the control diet and the diet with lemongrass essential oil presented the highest absolute and relative intestinal weights.
The relative liver weight was lower with the essential oil of lemongrass and the mix of essential oils compared with treatment with essential oil of L. rotundifolia (P<0.05).This decrease may be due to some kind of toxicity, although the animals had normal livers without macroscopic lesions.
Data are consistent with those found by Barreto et al. (2008), who also observed lower liver weight relative to the control in chickens receiving red pepper essential oil.
The relative spleen weight was highest (P = 0.044) in the animals fed the essential oils (lemongrass, L. rotundifolia, and combination of the two oils), showing that there was a likely effect on the immune system.

Conclusions
The trial could not find results enough to recommend the use of the lemongrass and Lippia rotundifolia essential oils as additives in broiler diets.

Table 1 -
Composition of experimental diets

Table 2 -
Relative abundance (%) of the compounds detected in the essential oils of Cymbopogon flexuosus and Lippia rotundifolia by gas chromatography coupled to mass spectrometry

Table 3 -
Main compounds detected in the essential oils of Cymbopogon flexuosus and Lippia rotundifolia after microencapsulation and incorporation in feed

Table 4 -
Performance of broilers fed diets containing essential oils of Cymbopogon flexuosus and Lippia rotundifolia 1 Feed negative control without additives. 2 positive control with antimicrobials and anticoccidials. 3rol feed + essential oil of Cymbopogon flexuosus.4Controlfeed+essential oil of Lippia rotundifolia.5Controlfeed + association of Cymbopogon flexuosus and Lippia rotundifolia essential oils. C -coefficient of variation; NS -not significant by the Tukey test at 5% probability.

Table 6 -
Absolute and relative weight of organs of broiler chickens fed diets containing essential oils of Cymbopogon flexuosus and Lippia rotundifolia at 43 days of age Means followed by different letters in the same row differ by the Tukey test (P<0.05). 1 Feed negative control without additives. 2ositive control with antimicrobials and anticoccidials.3Controlfeed+essentialoil of Cymbopogon flexuosus.4Controlfeed+essential oil of Lippia rotundifolia.5Controlfeed + association of Cymbopogon flexuosus and Lippia rotundifolia essential oils.CV -coefficient of variation.