Use of Tea Tree Essential Oil ( Melaleuca alternifolia ) in Laying Hen’s Nutrition on Performance and Egg Fatty Acid Proﬁle as a Promising Sustainable Organic Agricultural Tool

: The level of production in a variety of organic production systems is often lower than in other traditional production systems. In poultry production, there is also a direct negative e ﬀ ect of the small scale regarding sustainable organic poultry production. Regardless of di ﬀ erences between organic and conventional production systems, this experiment aimed to investigate the usage of tea tree Melaleuca alternifolia (Maiden and Betche) Cheel essential oils as a natural alternative to antibiotics in hen nutrition on productive parameters, table egg quality and eggs fatty acid proﬁle as a promising sustainable organic agricultural tool. A total of 360 Lohmann Brown hens, aged 54 weeks, divided into three di ﬀ erent treatment diets, were supplemented with 0 (T1), 40 (T2) and 80 mg / kg (T3) of M. alternifolia essential oil, respectively. Experimental treatments were replicated four times within 30 birds each. The experiment lasted for a total of 56 days (55 to 62 weeks of hens age). A 56-day experimental had two timetable periods of 28 days each: period 1 (55 to 58 weeks of hen age) and period 2 (59 to 62 weeks of hen age). For compound feed supplemented with M. alternifolia essential oil, daily egg production and the e ﬃ ciency of nutrient utilization (FCR) was improved signiﬁcantly ( p < 0.05) until the end of week 58, with a signiﬁcant ( p < 0.05) increase in the thickness of eggshell, as well as egg production ( p < 0.05). However, egg mass, feed consumption, FCR and albumen height, Haugh unit, and eggshell strength did not show any signiﬁcant ( p > 0.05) di ﬀ erences inﬂuenced by essential oil feed supplementation. Lower concentrations of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA), and higher concentrations of polyunsaturated fatty acid (PUFA), were recorded with M. alternifolia essential oil supplementation, but without signiﬁcant ( p > 0.05) di ﬀ erences. At the end of the experiment, the obtained results showed that the addition of M. alternifolia essential oil to hen nutrition had a positive e ﬀ ect on production parameters and eggs fatty acid proﬁle, with increased eggshell thickness ( p < 0.05). p > 0.05) inﬂuence on the fatty acid proﬁle of the yolk. Lower concentrations of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA), while higher concentrations of polyunsaturated fatty acid (PUFA), were recorded with


Introduction
Nowadays, modern organic agriculture has been described as production in a variety of organic production systems, which is often lower than in other traditional production systems. In poultry production, there is also a direct negative effect of the small scale regarding sustainable organic poultry production [1]. Organic agriculture helps to make farming sustainable, safe, and environmentally friendly, and essential oils and botanicals as a natural remedy serve that purpose.
For more than a hundred years, M. alternifolia has been used as a natural remedy all over the world, and primarily in Australia. Nowadays, bioactive ingredients in M. alternifolia are used in the manufacture of homeopathic drugs, and thus in pharmaceuticals and agriculture, with a growing interest in Europe, Japan and the United States [1]. Myrtle family Myrtaceae, represents the most important and known species for the industrialization of M. alternifolia oil because of its beneficial properties [2]. For the distillation of essential oils, flowers, herbs, leaves, roots, and another plant parts could be successfully used. M. alternifolia essential oil is a mixture of terpenes, aldehydes, esters, alcohols, and other chemical molecules, and therefore has been used in poultry nutrition for its antimicrobial, antibacterial, antioxidant and digestive stimulant properties. In the last twenty years, essential oils have been ignored as a possible natural remedy or natural antibiotic alternative for poultry [3]. However, they are recently gaining spotlight in the scientific community [4,5]. M. alternifolia oil, because of its antibiotic properties, is widely used as an antiseptic tool, often is used to minimize inflammation and can be very useful in fungal infection treatments [2]. M. alternifolia oil, due to its toxicity, should not be used in animal nutrition in high concentrations. Deaths regarding the higher dose of consumed M. alternifolia oil were not recorded in the known literature. The aforementioned statement was confirmed in the research of Hammer et al. [2]. However, in the right concentrations, M. alternifolia oil addition in broiler chickens diet increases body weight gain by 7% and improve nutrient utilization by 6% when compared with results of chickens that were not fed rations with the supplementation of M. alternifolia [6]. Olgun [7] research has shown that eggshell thickness was positively affected by essential oil addition to hens' diet, as well as other quality parameters of eggs, while Nikolova and Kocevski [8] in their research have shown its influence on the technological aspects of eggshell quality. In the research of Nadia et al. [9], usage of essential oil increased egg production and improved feed utilization, when the results of experimental treatments were compared with control treatment. Investigations with hens have shown that the dietary addition of thyme, sage, and rosemary essential oil [10] and essential oil mixture [6] increase hens' production, and increases immunity and eggshell quality, in line with the results obtained by Spasevski et al. [11], with the usage of powder form of marigold, paprika, and carrot in hens' diet. Due to the link between dietary lipids and developing coronary heart disease, the lipid composition of table eggs is the primary consumer concern, which is the same regarding dairy products [12]. It has been proven that adequate and well-balanced compound feeds for hens can improve the fatty acid profile of the eggs. Although the essential oils have a positive effect on the body metabolism of lipids [13], there have been no studies reported, or very few, on the effect of M. alternifolia dietary essential oil addition on egg fatty acids profile in hens.
Therefore, this research aimed to evaluate the effects of M. alternifolia essential oil in hen nutrition on productive performance results, table egg quality, and egg fatty acid profile.

Materials and Methods
Ethical Approval: Biological experiment with laying hens was approved by the University EC board and performed following the EU legislation and principle of the Three Rs within Directive 2010/63/EU.
Animals and Experimental Design: The experiment with laying hens was conducted under the principles of the European Union Strategy for the Protection and Welfare of Animals. A total of 360 Lohmann Brown hens aged 54 weeks were divided into three different treatment diets supplemented with 0, 40 and 80 mg/kg of M. alternifolia essential oil (Planet Fresh d.o.o., Montenegro), respectively. M. alternifolia was provided, including Terpinen-4-ol 40.0%, γ-Terpinene 23.0% and α-Terpinene 10.4% as the active components. Each treatment was replicated four times with 30 hens in each. Hens were in an environmentally controlled experimental facility with a constant temperature of 22 • C. Environmental conditions in the facility were in line with hybrid specifications. Hens were provided with a diet of 110 g of feed/hen/day, while water of an average temperature of 20 • C was provided ad libitum. Compound feed in mash form was used for hen nutrition during the experimental period. The basal feed was balanced according to the hybrid recommendation with and without the on top addition of M. alternifolia essential oils in the mixtures. The nutritive value of the used diets is given in Table 1. Sample Preparation and Eggs Quality Assessment: Eggs were gathered every day and the egg production was recorded for both period 1 (55 to 58 weeks of hens age), and period 2 (59 to 62 weeks of hens age), and for the complete periods of research. During the experiment, egg mass was registered daily. Consumption of feed was registered daily, and the feed conversion ratio was calculated for different production periods. Every 28 days in a row, 12 eggs per treatment were randomly gathered: three eggs per each replicate to measure albumen height, calculate Haugh units, and measure eggshell thickness and eggshell strength. Albumen height measurement was performed with a digital caliper, while Haugh units were calculated (1): HU = 100 Log (h − 1.7 w 0.37 + 7.6) HU = Haugh unit; h = albumen height (mm) and w = egg weight (g) Eggshell thickness was measured at the three points of measurements, top end, bottom end and center, with a micrometer screw gauge. An average of three thickness values from each egg has been used to define the thickness of the eggshell. A test machine, Instron, was used to measure the break strength of uncracked eggs. An egg was laid down on a continually raised load until it broke down. The point of egg break under load is considered as the measured strength of the egg. Table Egg Yolk Fatty Acids Analyses: Egg yolk fatty acids profile was analyzed by the gas chromatography technique. First, lipids were extracted from egg yolk. The extraction of lipids from the yolk was performed according to the Folch extraction method. Extracted lipids with 14% (w/w) boron trifluoride-methanol solution were used for the preparation of fatty acid methyl esters. Upon obtaining the samples, analyzes by GC were conducted on an Agilent 7890A chromatograph (Agilent Technologies, USA) with an FID, auto-injection module for liquid, equipped with fused silica capillary column (Supelco SP-2560 Capillary GC Column 100 m × 0.25 mm, d = 0.20 µm). As a gas carrier, helium of 99.9997 vol % purity, with a flow rate of 1.5 mL/min and with 1.092 bar pressure was used. The fatty acid profile of egg yolk was determined based on retention times and standard comparison. The fatty acid profile was shown as the % of the total fatty acids.
Statistical Analyses: The data obtained in the experiment were analyzed by one-way analysis of variance within statistical software Statistica 13. When the analysis of variance showed statistical significance, Duncan's multiple range test was used. The significant difference was registered at p < 0.05.

Results and Discussion
The effect of M. alternifolia essential oil on hen productive performance results is given in Table 2. During the 56 days of the experimental trial, only egg production showed significant (p < 0.05) results, while egg weight, feed intake and feed conversion ratio did not record any significant (p > 0.05) influence of diet supplementation with M. alternifolia essential oils. Feed conversion ratio and hen-day egg production were significantly improved (p < 0.05) in weeks 59 to 62 of the experimental period. Generally, it is assumed that essential oils of herbs and spices may have a positive influence, and might improve the feed consumption, due to their aromatic properties [14,15]. Bozkurt et al. [16] compared four herbs (thyme, oregano, rosemary, turmeric) at an inclusion level of 5 or 10 g/kg diet in a study with 28-week old laying hens for 12 weeks. They found no differences in feed intake among the treated groups and did not observe any differences when comparing the treatments to the untreated control group, which is in agreement with our research. Similar results were obtained when garlic was included in diets fed to laying hens in the concentrations of 20, 40, 60, 80 or 100 g/kg diet for hens of nearly the same age (27-28 weeks), but for only 5-6 weeks [17]. However, Abdo et al. [18] found that the average feed intake was significantly reduced for hens which were fed either 10 to 50 g/kg of green tea leaves or 5 to 25 mg/kg of green tea essential oil when compared to an unsupplemented control treatment. In our research, feed conversion ratio and hen-day egg production were increased (p < 0.05) in weeks 59 to 62, however, during a total of 56 days of feeding, trial egg production record significant (p < 0.05) results, while dietary addition of essential oils did not show significant (p > 0.05) effects regarding egg weight, feed consumption and feed conversion ratio, in agreement with other research [9,11,16,19]. The essential oils used as "production stimulators" for hens were primarily introduced in their daily diet to increase the utilization of the limit-fed diet and, in turn, improve the production of eggs. Some studies showed positive effects on production results, such as egg production rate, as was the case in our research, and the egg mass, whereas some other studies demonstrated no effect of essential oil addition [20].
The effect of M. alternifolia essential oil on egg quality is shown in Table 3. Supplementation of M. alternifolia essential oil significantly increased the eggshell thickness (p < 0.05) at the end of the experimental period of 56 days, in line with the research of Bozkurt et al. [21], who concluded that dietary addition of essential oil mixture increases the weight of eggshell, eggshell thickness, and strength of shell breaking, while dietary supplementations of M. alternifolia essential oil did not affect eggshell thickness (p > 0.05) at the beginning of the hen laying period in our study. No significant differences in albumen height, Haugh unit, and eggshell strength between the different dietary treatments T1 and T2 (p > 0.05), compared to control treatment T1, were recorded. Bölükbaşi et al. [22] showed that the supplementation of bergamot essential oil at three inclusion levels (0.25, 0.50, 0.75 mL/kg) had no impact on the proportion of albumen and eggshell thickness of the eggs of hens at the age of 67 weeks. However, at any addition level, bergamot essential oil decreased eggshell percentage by 15% or more when compared with an untreated control group. The dietary addition of black cumin essential oil did not show a significant (p > 0.05) influence on shell weight when added in concentrations of 1; 2 or 3 mL/kg, to hens' diet, respectively. Similarly, essential oil of oregano in the amount of 50 or 100 mg/kg, did not show either positive or negative effects on yolk color score, Haugh unit or shell thickness when added to the feed at the age of 32 weeks [19], but when used in broiler chickens diet, oregano oil and powder form mixtures of medicinal plants, and have shown significant effects [4,23]. The Haugh unit value as the main measure of internal quality of eggs was not compromised by the dietary supplementation of bergamot essential oil [22], oregano essential oil [19], a mixture of essential oils [21], green tea leaves or essential oil [18], thyme, oregano, rosemary or turmeric powder [9], or a garlic-thyme combination [24], in agreement with our study with a dietary addition of M. alternifolia essential oil to hen nutrition in concentrations of 40 and 80 mg/kg, respectively.
The influence of M. alternifolia essential oil on the fatty acid profile of egg yolk of control and experimental treatments is given in Table 4. The addition of M. alternifolia essential oil in treatments T2 and T3 compared with control treatment, T1, did not show a significant (p > 0.05) influence on the fatty acid profile of the yolk. Lower concentrations of saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA), while higher concentrations of polyunsaturated fatty acid (PUFA), were recorded with M. alternifolia essential oil supplementation, but without significant (p > 0.05) differences. In our study, different dietary treatments did not show significant (p > 0.05) influence on the egg yolk fatty acid profile. The research of Galobart et al. [10] showed that the dietary addition of rosemary essential oil in hen nutrition had no effects on the fatty acid composition of egg yolk. Besides that, the research of Bölükbaşi et al. [22] showed that the ratio of docosahexaenoic fatty acid (DHA) and the ratio of n-3 fatty acids of egg yolk were elevated by the dietary addition of bergamot essential oils. Other studies have shown changes in the lipid metabolism of poultry fed with the addition of essential oils [22,25]. To our knowledge, there have been no reports on the effect of M. alternifolia essential oil on the fatty acid composition of egg yolk, which makes this research a very valuable asset to the scientific community. The adequate ratio between n-6 and n-3 polyunsaturated fatty acids is a very important standard in healthy human nutrition. Being aware of n-3 polyunsaturated fatty acids' benefits and health-promoting effects, the nutritionists recommend a diet rich in n-3 fatty acids, as well as a lower ratio between n-6 and n-3 fatty acids, from the currently common 15-20:1 to 1-4:1. According to Simopoulos [26] and Kralik et al. [27], the ratio of n-6 and n-3 polyunsaturated fatty acids in egg yolk lipids less than 4:1 is considered to be beneficial to human health. This desirable ratio of n-6 and n-3 polyunsaturated fatty acids was highlighted by other authors as well [28,29].

Conclusions
Obtained results have shown that the addition of M. alternifolia essential oil in the hen diet has a significant positive effect on egg production, but did not show a significant effect on the yolk fatty acid profile, except increased eggshell thickness. Overall, M. alternifolia essential oil in a lower concentration of 40 mg/kg in may be beneficial and recommended for hen nutrition, but further investigation about M. alternifolia essential oil's influence on laying hens' nutrition and its effects and mode of action is more than necessary.

Conflicts of Interest:
The authors declare no conflict of interest.