INTRODUCTION
Essential oils (EO) derived from spices and herbs, as single components or as mixed preparations, can play significant roles in supporting both performance and health status of poultry (Huyghebaert et al., 2011; Lee et al., 2011; Khattak et al., 2014). Beneficial effects of active plant substances in poultry nutrition include the stimulation of appetite and feed intake, the improvement of endogenous digestive enzyme secretion, the activation of immune response and antibacterial, antiviral, antioxidant and antihelminthic actions (Lee et al., 2004; Jamroz et al., 2005; Zeng et al., 2015b). The aforementioned biological effects of EO could then lead to efficient nutrient utilization in poultry. Indeed, the supplementation of EO into low energy or nutrient density diet significantly increased daily weight gain and enhanced the digestibilities of nutrients compared with the low energy or nutrient density based control diet and exhibited comparable performance compared with a standard energy or nutrient density diet in pigs (Yan et al., 2010; Zeng et al., 2015a). In line with the latter studies, it was reported that dietary EO exhibited identical growth performance of broiler chickens fed the low-nutrient density (LND) diets compared with those fed the high-nutrient density (HND) diet (Scheuermann et al., 2009). Unfortunately, the absence of the LND control diet in the latter experiment made it difficult to prove the observed effect. Buchanan et al. (2008) reported that improvement of feed conversion ratio was only observed when EO was added into a HND vs LND diet. In contrast, no significant effect of EO on growth performance was observed in broiler chickens fed a threonine-deficient diet (Muhl and Liebert, 2007). In contrast to pig trials (Yan et al., 2010; Zeng et al., 2015a), the effect of EO on growth performance in broiler chickens fed a diet containing LND vs HND diet is inconclusive. Thus, the present experiment was intended to validate whether there is interaction between nutrient density and EO on growth performance in broiler chickens. In addition to production traits, various parameters such as blood metabolites, blood immune responses, organ weights, and cecal microflora were evaluated to see the presence or absence of the interaction between EO and nutrient density.
MATERIALS AND METHODS
Experimental design
The experimental facility was thoroughly cleaned and disinfected before the initiation of the experiment. A total of eight hundred-forty 1-d-old male broiler chicks (Ross 308) were obtained from local hatchery. Upon arrival, they were individually weighed and randomly placed into one of twenty-eight pens (1.8 m×1.8 m) with fresh rice husks as a bedding material and stocking density was set at 0.108 m2 per bird. The present experiment consisted of four dietary treatments which were given for 35 days (starter 1 to 21 days, finisher 22 to 35 days). Each treatment consisted of seven pens and each pen had 30 chickens (n = 210 chickens/treatment). There were four dietary experimental diets with two nutrient density diets (HND vs LND) supplemented with or without EO at 150 mg/kg of diet. The commercially available EO preparation (Biostrong 510, Delacon, Steyregg, Austria) consisted of a mixture of EO with thyme and star anise, Quillaja extracts, and bulking and anti-caking agents. Corn-soybean meal based HND and LND based diets were formulated (Table 1). The LND vs HND diet contained less energy and nutrients by one percent point of crude protein (CP), 50 kcal of nitrogen-corrected true metabolizable energy, 0.05 percent point of available phosphate, and 0.05 percent point of lysine. These nutrient reduction can be considered moderate as compared with the previous studies (Brickett et al., 2007; Zhao et al., 2009; Li et al., 2010; Mirshekar et al., 2013), in which the LND vs HND diet was formulated to reduce CP levels by 2 percent points, and energy levels by more than 150 kcal/kg of diet. Diet and water were provided ad libitum. Continuous lighting program was used and the temperature of facility was maintained at 32°C during the first week posthatch and gradually decreased to reach 25°C at 3 weeks and kept thereafter. At days 14 and 28, all broiler chicks used in this study were vaccinated against Newcastle disease (ND) and infectious bronchitis (IB) by eye drop using the attenuated live mixed vaccine (Nobilis Ma5+Clone 30, MSD AH Korea Ltd., Seoul, Korea). All experimental protocols were approved by the Animal Care Committee of KonKuk University.
Sampling
Feed intake and body weight per pen were measured on a weekly basis and used to calculate feed conversion ratio. At 35 days, eight chickens per pen were randomly selected for blood sampling. Blood was collected from cardiac puncture immediately after cervical dislocation. Sera were obtained by gentle centrifugation (600 g for 15 min) and stored at −20°C prior to use. Immediately after blood sampling, organs such as liver, spleen, bursa of Fabricius and abdominal fat were excised, weighed and expressed as relative weight to live body weight. In addition, right breast and thigh meats were sampled and weighed. Finally, cecal contents were removed aseptically, placed into sterile tubes and kept on ice until used for gut microbiota analysis on the same day of the sampling.
Measurement of total cholesterol, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in serum samples
Blood parameters were measured as described elsewhere (Lee et al., 2010). In brief, total cholesterol concentration and activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) in sera sampled at 35 days were measured according to the colorimetric method using cholesterol diagnostic kit (Cholesterol E kit, Asan Phamaceutical Co., Seoul, Korea) and GOT-GPT assay kits (Asan Phamaceutical Co., Korea).
Measurement of infectious bronchitis- and Newcastle disease-reactive antibody response
Viral antibodies against IB and ND in sera sampled at 35 days were determined (Lee et al., 2012) using commercial enzyme-linked immunosorbent assay kits (IDEXX Laboratory, Westbrook, ME, USA) according to the manufacturer’s instructions. The results were expressed as antibody titer that was calculated using the formula provided by IDEXX.
Enumeration of intestinal microflora
Individual cecal contents were subjected to serial 10-fold (w/v) dilution with ice-cold phosphate-buffered saline as suggested by Noh et al. (2014). Total microbes were counted after grown on total plate agar (Difco, Detroit, MI, USA), lactic acid bacteria on Man Rosa-Sharpe agar (Difco, USA), and total coli forms on MacConkay agar (Difco, USA) at 37°C for 24 h. Results obtained were presented as base-10 logarithm colony forming unit (cfu) per gram of cecal digesta.
Statistical analysis
Pen was considered an experimental unit. Two-way analysis of variance was performed to define the effect of diet (LND vs HND), EO (0, 150 mg/kg of diet) and the diet by EO interaction on the variables using the general linear model procedure of SAS program (SAS, 2000). A statistical significance was preset at p<0.05 unless otherwise stated.
RESULTS
There was no interaction between nutrient density and EO on the measured parameters except for bursa of Fabricius and abdominal fat in broiler chickens. Body weights were affected (p<0.05) by nutrient density at 21 days and by EO supplementation at 35 days. HND vs LND diet significant increased (p<0.05) daily body weight gain at 1 to 21 days (Table 2). Dietary EO significantly increased (p<0.05) daily body weight gain during the periods of 22 to 35 and 1 to 35 days. Daily feed intake was not affected (p>0.05) by dietary EO, but significantly reduced (p<0.05) by the LND vs HND diet, especially during the periods of 1 to 21 and 1 to 35 days. Feed conversion ratio was significantly improved (p<0.05) by EO addition during the periods of 22 to 35 days and 1 to 35 days (Table 2). Either nutrient density or EO did not influence the relative weights of liver, spleen, breast and leg meat yields (Table 3), cecal microflora (Table 4) and blood parameters such as total cholesterol, GOT, GPT, and ND- and IB-specific antibodies (Table 5).
DISCUSSION
The present study was designed to investigate whether dietary EO added into different nutrient density diets could improve growth performance of broiler chickens. Initially, a positive effect was expected in the light of previous reports showing that dietary EO increased growth performance and nutrient utilization in broiler chickens (Hernandez et al., 2004; Mountzouris et al., 2011; Bravo et al., 2014; Cho et al., 2014). In this study, we confirmed the previous reports (Buchanan et al., 2008; Leeson, 2012) on the well-established negative effect of nutrient density on growth performance (e.g., decrease in live body weight, feed intake, and daily weight gain) in broiler chickens. It was observed that EO supplementation, regardless of nutrient density, significantly increased (p<0.05) daily body weight gain during the periods of 22 to 35 and 1 to 35 days, and also improved feed conversion ratio during the periods of 22 to 35 and 1 to 35 days. The consistent increase in daily weight gain and decrease in feed conversion ratio by dietary EO would be likely the consequence of increased nutrient utilization as reported elsewhere (Hernandez et al., 2004; Lee et al., 2004; Mountzouris et al., 2011; Bravo et al., 2014; Cho et al., 2014). However, we did not measure nutrients digestibility or digestive enzyme activities in this study.
It is of note that EO-mediated increase in growth performance was equally effective in chickens fed diet containing different nutrient densities. In previous studies with EO supplemented into different nutrient density, conflicted results have been reported. For example, Buchanan et al. (2008) observed an increase in feed efficiency in broiler chickens fed the EO-added, HND diet, but this effect was not seen when the LND diet was used. Bozkurt et al. (2012) reported that body weights were increased in broiler chickens fed the EO-fortified, wheat-based diet, but were significantly decreased when EO was added into the corn-based diet. Finally, dietary EO did not improve overall growth performance in broiler chickens fed a threonine-deficient diet (Muhl and Liebert, 2007). In this study, the LND vs HND diet contained less energy and CP, but formulated to keep the ratios of lysine to limiting amino acids constant. Whether that the effect of dietary EO on growth performance is more effective in low energy/nutrient density diets with balanced amino acids needs to be addressed. Nonetheless, our study provides evidence that dietary EO could improve growth performance of broiler chicken and the EO-mediated effect was independent to nutrient density. The latter finding is considered of practical relevance in feed formulation in poultry production. According to Leeson (2012), the LND vs HND diet will be feasible in poultry production as the feed prices are currently on the increase.
None of parameters measured in this study were affected by either nutrient density or dietary EO. However, it was found that there were significant interactions (p<0.05) between density and EO on the relative weight of bursa of Fabricius and abdominal fat. Dietary EO when added into a LND diet increased relative weight of bursa of Fabricius, which reached to the value shown in birds fed a HND diet alone. Whether this indicates the consequence of altered immune status by EO needs to be verified as no difference in humoral responses against ND and IB were observed in this study. To our surprise, addition of EO into the HND, but not LND diet, tended to increase the relative abdominal fat, thus leading to significant treatment interaction. At this stage, clear explanation on the confounding result is not readily available.
In conclusion, the present study showed that dietary EO, independent to nutrient density, effectively increased body weight, daily weight gains and feed conversion ratio in broiler chickens. In addition, there were significant treatment interactions on relative bursal of Fabricius and abdominal fats. Finally, none of parameters measured, i.e., relative organ weights, percentage of meat yields, cecal microflora, blood characteristics were affected by either nutrient density or dietary EO.
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