Evaluation of Essential Oil of Fennel and Garlic Separately or Combined with Bacillus licheniformis on the Growth, Feeding Behaviour, Hemato-biochemical Indices of Oreochromis niloticus (L.) Fry

Improving the bio-economic efficiency of aquaculture is dependent on advances in biology, nutrition and environmental management of the production cycle. Antimicrobials and other veterinary drugs are administered regularly as additives in fish food, are used as therapeutics or growth promoters [1]. Nevertheless, the use of veterinary drugs is becoming more restricted since they present numerous side-effects for the environment and health safety [2]. Use of antibacterial agents in aquaculture should be avoided as it leads to proliferation of resistant and creates a negative image for aquaculture industry. Use of essential oil extracted from herbs medicine, could be a valuable alternative instead of antibiotics because there are fewer chances for the development of resistant strains [3].


Introduction
Improving the bio-economic efficiency of aquaculture is dependent on advances in biology, nutrition and environmental management of the production cycle. Antimicrobials and other veterinary drugs are administered regularly as additives in fish food, are used as therapeutics or growth promoters [1]. Nevertheless, the use of veterinary drugs is becoming more restricted since they present numerous side-effects for the environment and health safety [2]. Use of antibacterial agents in aquaculture should be avoided as it leads to proliferation of resistant and creates a negative image for aquaculture industry. Use of essential oil extracted from herbs medicine, could be a valuable alternative instead of antibiotics because there are fewer chances for the development of resistant strains [3].
There are many aromatic plants such as oregano, rosemary, sage, peppermint, thymus, fennel and garlic in worldwide, especially in the Mediterranean area [4]. The extracted essential oil from aromatic plants are usually used for antioxidant [5], digestive stimulant [6].
Fennel, Foeniculum vulgare is a biennial medicinal plant belonging to the family Apiaceae (Umbelliferae). Essential oils of fennel have hepatoprotective effects [7], as well as anti-inflammatory, and antioxidant activities [8]. Many phytochemical studies have been conducted to study the chemical composition of the essential oil of fennel from different origins and have shown that the major components are phenylpropanoid derivatives and monoterpenoids [9].
Allium is the largest and most important genus of the Alliaceae family and comprises 450 species, which were widely distributed in the northern hemisphere. Among them, garlic (Allium sativum L.), is a well-known species being used in traditional medicine and food in many countries [10]. Garlic and its constituents have antimicrobial activity. It was showed that the antibacterial activity of garlic resulted from thio sulfinates, especially allicin, which is responsible for the antimicrobial activity, as well as its flavor and aroma [11]. Five types of garlic preparations are currently available on the market: garlic essential oils, garlic oil macerate, garlic powder, aged garlic extract and fresh garlic [12]. Among them, the antimicrobial activity of garlic oil is 200 times greater than garlic powder and 900 times the strength of fresh garlic [12].
Previous studies revealed that herbs medicine and essential extracted oil from herbs can be successfully used in fish nutrition such as Carum carvi seeds [13], Foeniculum vulgare seeds [14], Cuminum cyminum powder [15], ground garlic, Allium sativum [16,17], oil of oregano, Origanum heracleoticum [18,19], oil of thyme and fennel [20]. Furthermore, some aromatic plants and their extracts have been reported to stimulate the growth of certain bacteria where used a prebiotic, but these studies are scarce [21]. As aforementioned, many authors have attempted to examine various essential oil components from herbal plants, but information regarding fry culture, especially the effects of essential oil of fennel and garlic supplements and their combined with probiotics on fish performance and hematobiochemical blood parameters, is little. Therefore, the present study was designed to investigate the effect of fennel oil, garlic oil and Bacillus licheniformis alone and combined as dietary feed additives on growth

Fish, facility and feeding trial
The feeding trial was performed at an indoor installation of the Aquaculture Laboratory, faculty of agriculture Benha University. Nile tilapia, O. niloticus fries were obtained from fish hatchery of central laboratory for aquaculture research, Abbassa, Abou-Hammad Sharkia, Egypt. Fry were acclimated for two weeks to the experimental conditions in a fiberglass tanks. During this period fish fed daily on the commercial diet to be adapted to pelleted feeds. At the beginning of the experiment, 18 (100-L) glass aquaria were stocked with 20 fish with an initial average weight (1.88 ± 0.12 g). Each aquarium was supplied with compressed air via air-stones from air pumps. Overhead fluorescent illuminating was set at 14:10 (light:dark). All fish were fed, initially, at a rate of 6% of total body weight daily for the first 6-week and then decreased to 4% of total body weight daily for the remaining 12-week [22]. The fish were fed four times a day at 10:00, 12:00, 14:00, and 16:00, 6 days a week, for 84 days. All aquaria were siphoned twice a day to remove faecal materials then replaced by aerated fresh water. Total fish weight in each aquarium was determined every 2 weeks to check their growth and to adjust the feeding rate. Feeding was stopped 24 h before sampling. Water samples for chemical analysis were monitored weekly during the experimental period. Dissolved oxygen and temperature were measured on site with an YSI model 58 oxygen meter (Yellow Spring Instrument Co., Yellow Springs, OH, USA). Total ammonia was measured using DREL/2 HACH kits (HACH Co., Loveland, Co. USA). pH was measured using a pH meter (Digital Mini-pH Meter, model 55, Fisher Scientific, USA). During the period of the feeding trial, the water-quality parameters were averaged (± SD): Water temperature was 26.25 ± 0.9°C: dissolved oxygen, 5.7 ± 0.5 mg L -1 : pH 8.33 ± 0.4 and total ammonia, 0.07 ± 0.01 mg L -1 . All tested water quality criteria were suitable and within the acceptable limits for rearing the Nile tilapia O. niloticus [23].

Extraction of essential oils and analysis
Fresh fennel seeds were dried at 35°C and crushed to fine powders using a grinder. Essential oil was obtained by hydrodistillation of the powdered dry seeds using a Clevenger-type apparatus. The essential fennel oil (EFO) was separated, dried over anhydrous sodium sulfate. However, extraction of the essential garlic oils (EGO) from fresh cloves of garlic was performed with a hydrodistiller after mashed. The hydrodistillation lasted 3 hours. Essential oil with an unpleasant strong odor was obtained and dried by adding anhydrous sodium sulfate, filter sterilized through a 0.22-mmfilter. Essential fennel and garlic oil were stored in a dark glass bottle at 4°C until use. The analysis of EFO and EGO were analyzed using gas chromatography-mass spectrometry (GC-MS) to identify their chemical constituents according to Aazza and Douiri et al. [24,25], respectively.

Preparation inoculum of (B. licheniformis)
Strain of Bacillus licheniformis used was obtained from Microbiological Resources Center (MIRCEN), faculty of Agriculture, Ain Shams University, Egypt. Inoclum was prepared by adding 5 g lyophilized cells of B. licheniformis to 100 ml of specific media which containing 5 g L -1 peptone, 3 g L -1 beef extract at pH 7.0 then was incubation at 37 ο C for 24 hours. After this period, cells were centrifugation for 15 min (2000 g) then washed with phosphate buffered saline (PBS) with pH 7.3. The washed cells was added dropwise into the ingredients of diet before to pellet [26] until to reach the level 7 × 10 7 (CFU g -1 ) of 7×10 7 (CFU g −1 ) of B. licheniformis. The control diet received the same volume PBS to maintain an equal volume of PBS in all experimental diets.

Experimental diets
The basal practical diet was formulated to contain approximately (35% crude protein and 19.96MJ Kg -1 gross energy) ( Table 1). The basal practical diet was divided into six parts. The first part prepared without feed additives as control diet (D1). Other five parts prepared to contain B. licheniformis at level 7×10 7 colony forming unit (CFU g −1 diet) diet  under the light microscope using a Neubauer haemocytometer after blood dilution with phosphate-buffered saline (pH 7.2). Serum AST and ALT were determined colorimetrically using spectrophotometer using specific kits according to Reitman and Frankel [29]. Total serum protein and albumin and globulin were determined according to Doumas et al. [30].

Statistical analysis
Data were statistically analyzed by ANOVA using SAS ANOVA procedure (Statistical Analysis System 2004). The data were submitted to one ways classification variance analysis. Duncan's multiple range test was used to compare differences between treatment means when significant F values were observed [31], at (P < 0.05) level. All percentage data were arc-sin transformed prior to analysis [32], however data are presented untransformed to facilitate comparisons.

Growth and feed utilization
Survival at the end of the experiment was high (about 95%) and the effect of dietary treatments was significant ( Figure 1). No significant differences in survival of fish (P>0.05) were observed among fish groups fed diet (D2, D3 and D4). The survival of fish fed diet D6 (EGO + B. licheniformis) was significantly higher that of other treatments. The differences in body weight were first observed after the 4 weeks of the feeding and increased as the feeding period continued ( Figure 2).

Feeding behaviour
The observation of fish behaviour was conducted for 6 days from June 20 th until July 21 st to study the effects of experimental diets on fish behavior according to Hussein et al. [27] to study the effects of experimental diets on fish behaviour. The types of fish behavior included were mouth wrestling and chasing. Fish were fed manually their daily amount of the experimental diets four times a day at 10:00, 12:00, 14:00 and 16:00. Ten minutes after the third meal (14:00) all aquarium were observed individually and each aquarium was given 3 min to quantify the behavioural interactions (i.e., as mouth wrestling and chasing separately) between fish.

Sample collection and analysis
At the end of the trial a random sample of five individual fish were sampled from each aquarium, then oven-dried 105°C for 24 h, ground, and stored at −20°C for subsequent analysis. Proximate analysis was conducted on diets and fish samples. Dry matter, crude lipids, crude protein and ash content were determined by the standard [28]. Dry matter was determined after drying the samples in an oven (105°C) for 3 h. Crude protein was determined by micro-Kjeldhal method, N × 6.25 (using Kjeltech auto analyzer, Model 1030, Tecator, Höganäs, Sweden) [28] (method number 984.13), crude lipid by soxhlet extraction with diethyl ether (40-60°C) [28] (method number 920.39) and ash by incineration at 550°C for 12 h [28] (method number 942.05). Nitrogenfree extract was computed by taking the sum of values for crude protein, crude lipid and ash then subtracting this sum from 100. At the end of the feeding trial all fish were counted and weighed to calculate final body weight (FBW), weight gain (WG), specific growth rate (SGR%), feed conversion ratio (FCR) and protein efficiency ratio (PER) using the following equations: WG (g/ fish) = FBW -IBW; SGR% = [ln FBW -ln IBW]/t×100, where IBW is initial body weight (g); ln = natural logarithmic; t = time in days. FCR = FI /WG, where FI is feed intake (g); PER=WG/protein intake (g).

Blood samples, hematological and biochemical blood parameters
At the end of experimental period, fish were fasted for 24 h prior to blood sampling. Fish were anaesthetized by tricaine methanesulfonate (MS222) at 250 mg L -1 in water. Blood was drawn near caudal peduncle from five fish from each treatment by a sterile syringe and transferred into a heparinized tube for hematological study. Blood was drawn from another five fish from each treatment kept outside without anticoagulant at room temperature (26 ο C) for few minutes then centrifuged at 5,000 g for 15 min for separation of serum (collected serum was stored at -20°C) for biochemical study. Hematocrit (Hct) was determined as described by Reitman and Frankel [29]. Hb was determined by the hemoglobin kits which is a standardized procedure of the cyanomet hemoglobin method. RBCs were counted

Chemical composition
Proximate analysis of whole body of O. niloticus fed the experimental diets was presented in Table 5. The chemical composition of whole body appeared that no significant differences were found in dry matter and ash content of fish fed control and treated diets.

Discussion
The present study indicated that the highest growth and feed utilization were obtained at 1ml of EFO and EGO combined with B. licheniformis 7 × 10 7 (CFU g −1 ). This study of the application of EFO and EGO in diet showed the first report about the growth indices of Nile tilapia. Whereas, fennel seed as a powder was used as the growth Moreover, a significant reduction in the final body weight of fish control was observed compared with fish fed other diets. Results of growth and feed utilization are presented in (Table 2) and indicated that the highest weight gain (WG) and specific growth rate (SGR) were recorded by fish fed D5 (EFO + B. licheniformis) and D6 (EGO + B. licheniformis). Furthermore, no significant differences were found in WG and SGR among D2 (B. licheniformis), D3 (EFO) and D4 (EGO). The highest FI was observed in fish fed D4 (EGO), D5 (EFO + B. licheniformis) and D6 (EGO + B. licheniformis). While, the best FCR and PER were observed in D3 (EFO), D4 (EGO), D5 (EFO + B. licheniformis) and D6 (EGO + B. licheniformis) in comparison to the other treatments groups.

Feeding behavior
The behavioural observation were recorded at week 7 ( Figure 3) and the results showed significant differences among all treatments in terms of mouth wrestling and chasing behaviour. It showed that mouth wrestling and chasing behaviour were higher in fish feed D6 (EGO + B. licheniformis) resulting in more aggressive for pellets feed than all treatments diets. The opposite trend were observed in D1 (control diet).

Hematological analysis and biochemical blood
The changes in hematological parameters of Nile tilapia fed treated diets are presented in Tables 3 and 4 [14,33]. Besides, most of the authors have studied the effect of garlic as a powder form on growth and nutrient utilization [17,26,34,35]. The growth rates for the previous studies were significantly higher in fish fed diets supplemented with garlic than control groups. Garlic contains allicin, which promotes biogenic performance due to its positive effect on the intestinal flora, thereby improving digestion and enhancing nutrients utilization which influences the growth of fish [36]. Steam distillation of mashed garlic produces garlic oil containing methyl and allyl-sulphides of allicin and having the practical advantage of being more stable than allicin itself [37]. Due to the fact that the extracted essential oil from herbal plants has much higher contents of active substance than dried herbal plants resulting in the essential oil is usually used in low percentages in the diet compared to dried herbal plants.

. The highest hematocrit (Hct), hemoglobin (Hb) and red blood cells (RBCs) values were
Little information has been reported the effect of application of different essential oil in fish growth. These studies indicated that diets supplemented with essential oil of weet orange peel (Citrus sinensis) and Aloysia triphylla ( [19,38] with different dose could enhance weight gain and specific growth rate. Supplementation of the essential oil of fennel or garlic in diet may improve digestibility, availability of nutrients and lead to a higher protein synthesis, which in turn could explain the better growth in this study. Meanwhile, stimulation of growth by B. licheniformis in the present study may attribute to improve digestibility by enhancing the synthesis of vitamins, enzymatic activity [39]. These results are in agreement with Bacillus subtilis [40]; carp [41], Tilapia O. nilotica. On the other hand, the increase in growth for the combined application of EFO and/or EGO with B. licheniformis may be due to the EFO and EGO promoted the growth of exogenous probionts bacteria B. licheniformis in gastrointestinal of fish more than other endogenous populations bacteria, which improved the growth performance of Nile tilapia. The control of the microflora in intestinal fish by addition of oregano essential oil (Origanum heracleoticum L) can positively effect on growth performance [18]. The main way of the essential oil work seems to be through the regulation of intestinal microflora [42]. From aforementioned, the essential oil of fennel and garlic are good potential alternatives to the growth promoters.
The feeding behaviour of fish is complex [43], however, the acceptance or rejection of diet is physiologically dependent on inputs from chemoreception [44]. Fish have several chemosensory systems including gustation (taste), olfaction (smell), chemical sensory and chemoreceptor cells. The role of herbal plants for feeding behaviour represented in the flavor, thereby influences of eating patterns, the secretion of digestive fluids and total feed intake [45]. It seems that fish fed EGO combined with B. licheniformis could be stimulated the gustatory system is the most important in acquisition and ingestion of food. Allicin has an intense garlic flavor with a strong effect on olfaction in the most aquatic animals such as Pelodiscus sinensis, Ctenopharyngodon idellus, Cyprinus carpio, Carassius auratus, and Oreochromis niloticus [45]. Harada [46] indicated that garlic had a strong food calling effecting on loach (Oriental weatherfish) and Japanese amberjack, Seriola quinqueradiata Temminck et Schlegel.
Hematological variables are good predictors for explaining the health status of fish [47]. The highest Hct, Hb and RBCs count were obtained at D5 (EFO + B. licheniformis) and D6 (EGO + Bacillus licheniformis). Erythrocytes content in fish blood gives a guide to the health status of fish and can be helpful to determine any abnormalities Results are presented as means ± SD of triplicate observations. Means in the same row with different superscripts were significantly different (p < 0.05).  Results are presented as means ± SD of triplicate observations. Means in the same row with different superscripts were significantly different (p < 0.05).  Results are presented as means ± SD of triplicate observations. Means in the same row with different superscripts were significantly different (p < 0.05).  arising from the use of immunostimulants. Accordingly, the elevate number of RBCs multiplies the concentration of hemoglobin ultimately resulting in a high capacity for oxygen carrying which improved the health of fish and consequently enhancing growth [48]. Furthermore, Fazllolahzadeh et al. [37] assumed that the increase of blood indices such as, Hct, Hb, and RBCs may be attributable to a defense reaction against garlic which occurs by stimulation of erythropoiesis. The number of RBCs was significantly (P<0.05) higher in Asian sea bass, Lates calcarifer [49], Labeo rohita [50] and Nile tilapia, O. niloticus fed diet supplemented with garlic as a powder form. The present study is inconsistent with Soltan and El-Laithy [51]. They reported that fennel seeds and garlic powder did not effect on Hct and Hb values for Nile tilapia, O. niloticus. Yılmaz and Ergün [52] reported that garlic oil did not produce any undesired effects on hematological characteristics for Sea Bass Dicentrarchus labrax. With regard to the different essential oil addition Acar et al. [19] showed that O. mossambicus fed a basal diet containing 1% thyme, rosemary or fenugreek for 45-day increased RBCs count and Hct value.
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are liver enzymes and they have the function of transferring the amino group from alpha-amino acids to alpha-keto acids. A large amount of ALT and AST is released in the blood mostly during liver cell damage [53]. Thus, detection of the serum level of ALT and AST allows monitoring liver cell damage. The diets containing (EFO + B. licheniformis) and (EGO + B. licheniformis) could decrease the activity of ALT and AST compared to other treatment groups. Similar results were obtained by El-Dakar [33] who showed a significant lower (P<0.05) in levels of ALT and AST in Nile tilapia, O. niloticus fed fennel seed meal supplemented with diet. Also, Soltan and El-Laithy [51] found that ALT and AST levels were significantly decreased when Nile tilapia fed diets supplemented with 1% garlic powder or 1% fennel seed with compared to other diets. Application of garlic (Allium sativum) may cause stabilization cell membrane and protect the liver against deleterious agents and free radical-mediated toxic. This is reflected in the reduction of liver enzymes [37]. Also, Metwally [54] indicated that garlic-addition helps the liver to maintain its normal function by accelerating the generative capacity of its cells. The different active components found in essential oil of fennel and garlic may play an important role in enhancing the activity of liver enzymes but the exact mechanism for this need more advanced study. Globulin fractions are certainly important for maintaining a healthy immune system and immune function in the blood [55]. The increase in total protein and globulin contents is thought to reflect strong innate immunity [17]. In this study, diets supplemented with (EFO + B. licheniformis) and (EGO + B. licheniformis) showed a significant elevate in total protein and globulin in Nile tilapia. It can be inferred that the EFO and EGO which combined with B. licheniformis may enhance the non-specific immune response and more tolerant of stressful conditions of Nile tilapia. These results are similar with [20] who reported that the highest content of total protein was determined in fish fed diet supplemented with mixed oil of (thyme and fennel) with level 100 ml kg -1 diet, but serum albumin was not significantly different from the control group. The addition of garlic products, fresh oil and powdered forms increased the total protein in Nile tilapia O. niloticus, even at low level 0.25% of garlic oil [54]. Also, Shalaby et al. [16] reported that the highest total protein was determined in Nile tilapia O. niloticus when garlic powder was supplemented to the diet at 10 g kg -1 diet. Serum total protein and albumin contents decreased in sea bass exposed to garlic oil (0.005 ml L -1 water) via bath immersion for 96 h, but serum globulin was decreased at (0.02 ml of garlic oil L -1 ) [52].
The present study indicated that no significant differences in chemical composition of body of fish fed different treated diets. In this regard, Abd El Hakim [14] found no significant differences in the chemical body composition of Nile tilapia fed diet supplemented with different level of fennel seed. Contrarily, Maniat et al. [56] showed that garlic supplemented in diets induce higher protein and lower fat content in body of benni fish (Mesopotamichthys sharpeyi). Ahmad and Abdel-Tawwab [13] reported that no significant differences (P>0.05) in moisture and crude protein in Nile tilapia, Oreochromis niloticus body meanwhile total lipids increased by increasing level of caraway seed (belonging to family Apiaceae as fennel). Shalaby et al. [16] reported the highest crude protein and the lowest crude lipid content were obtained by Nile tilapia fed diet supplemented with 30 g kg -1 garlic. Furthermore, Talpur and Ikhwanuddin [49] showed that sea bass fed diet supplemented with garlic was decreased in lipid content in body. Also, Metwally [54] showed that crude protein and ash was increased significantly in O. niloticus while crude lipid decreased significantly with diets containing different sources of Allium sativum.
In conclusion, addition of essential oil of fennel combined with B. licheniformis and/or essential oil of garlic combined with B. licheniformis resulting in increased growth performance and enhancement of hematological and biochemical blood parameters in Nile tilapia O. niloticus.