Herbal medicinal products are widely used around the world (Jordan et al., 2010). A growing interest has emerged in using herbs in animal feeds by both researchers and feed companies (Logambal and Michael, 2000). Since ancient times, garlic (Allium sativum)has been used worldwide as a seasoning, spices, and herbal remedies or folk medicine (Rivlin, 2001).Garlic has been used in ancient Egypt in treating many ailments such as heart diseases, headache, bites worms and tumors (Aly et al., 2008), as well as ancient Chinese medicine indications that garlic were used to treat, improve male potency and enhance sexual prowess (Kahn, 1996). It is known to possess a vast variety of biological functions such as antithrombotic (Block et al., 1986), anticancer (Mousa, 2001), antioxidant (Wuet al., 2001), improving immune-system (Kanget al., 2001), lower serum lipid and glucose levels (Lawson et al., 2001) and blood pressure (Ali et al., 2000). Also, Agarwal (1996) revealed that garlic has been widely used as foodstuff since antiquity. Dietary garlic has a potential effects as a growth promoter or immune, and physiological enhancer agent for fish, Clarias gariepinus (Thanikachalam et al., 2010); Oncorhynchus mykiss (Nya and Austin, 2011) and Cyprinus carpio (Naeiji et al., 2013).Partially, dietary garlic not only improved the growth performance (Soltan and El-Laithy, 2008; Mahfouz et al., 2009),but also enhancement both of the immune-system (Ahmed et al., 2008;Metwally, 2009), and physiological responses (Diab et al., 2002; Shalaby et al., 2006) of Oreochromis niloticus.
Bioactive components of garlic, including several sulfur-containing compounds such as Allicin (responsible for the distinctive odor), diallyl-disulfied, diallyl-trisulfied and others, which are responsible for most of the pharmacological properties of garlic (Amagase et al., 2001). Allicin (dially thiosulfinate) is the most abundant compound representing about 70% of all thiosulfinates present, or formed in crushed garlic. Also, garlic is a rich source of calcium (Ca), phosphorus (P), zinc (Zn), iron (Fe) ; has a high content of carbohydrates and as a consequence a high nutritive value; contains silica (Si), sulfate (S) salts, B1, B complex, A and C vitamins (Han et al., 1995).Therefore, the objectives of the present work were to evaluate the effects of graded levels (0, 1, 2 and 3% / kg diet) of dried garlic (A. sativum) lobes(DGL) on growth performance, feed and nutrients utilization, fish carcass composition, and hematological and biochemical parameters of male monosex Nile tilapia O. niloticus for 60 days in an indoor feeding experimental diets.
MATERIALS AND METHODS
Fish and experimental management:
This study was conducted in Fish Research Laboratory, Animal Production Department, Faculty of Agriculture, MansouraUniversity, Al Dakahlia Governorate, Egypt. Fish were stocked in rearing tank for two weeks as adaptation period during this time they were offered the basal diet. A total of 84 all male monosex O. niloticus fingerlings at average initial body weight of 17.14 ± 0.35 g were randomly distributed. The fish were stocked at rates of 7 fish / glass aquarium (90 x 50 x 40 cm), where three aquaria were referred to every dietary treatment. Each aquarium was supplied with 108 L dechlorinated tap water and an air stone connected with electric compressor. The replacement of the aquaria water was done partially every day to re-new the water and to remove the wastes. Light was controlled by a timer to provide a 14h light: 10h dark as a daily photoperiod.
The ingredients and chemical composition of a basal diet (BD) are shown in Table 1.The lobes of garlic were cleaned and shade-dried in a drying oven at 50°C for 72 hours. Dried garlic lobes (DGL) were milled into fine particle size (< 250 µm), and kept in a dry, air-tight transparent plastic container. All ingredients and additive were milled and mixed, then pressed by manufacturing machine (pellets size 1mm). DGL were added in experimental diet at levels 0, 1, 2 and 3% / kg diet. During the experimental period (60 days), the fish were fed a rate of 3% of the live body weight daily, six days a week and twice daily, at 8.0 am and 2.0 pm. The fish were weighed every two weeks by a digital scale (accurate to ± 0.01 g) to adjust their feed quantity every aquarium according to the actual body weight changes.
Table 1. Ingredients and proximate chemical analysis (% on dry matter basis) of the experimental diet
At the end of the experiment, the remained fish were sampled from each aquarium and kept frozen (- 20°C) for chemical analysis. The chemical analysis of the basal diet and whole fish body were carried out according to AOAC (2004). Water quality parameters included temperature (via a thermometer), pH (using Jenway Ltd., Model 350-pH-meter) and dissolved oxygen (using Jenway Ltd., Model 970- dissolved oxygen meter) were measured weekly according to Abdelhamid (1996). All tested water quality was suitable for rearing Niletilapia (O. niloticus) fingerlings as cited by Abd El-Hakim et al. (2002). Since, water temperature ranged between 24 and 28oC, pH values 6.55 – 7.60 and dissolved oxygen 5.50 – 8.80 mg/l. Body weight of individual fish was measured biweekly to calculate the growth performance parameters such as final weight (FW, g); total weight gain (TWG, g); average daily gain (ADG, mg/fish/day); relative growth rate (RGR); specific growth rate (SGR, % / day) and survival rate (SR, %), and feed utilization parameters such as feed intake (FI, g / fish); feed conversion ratio (FCR); feed efficiency (FE, %); protein efficiency ratio (PER); protein productive value (PPV, %) and energy utilization (EU, %) according to Abdelhamid (2000).
Blood samples were collected from caudal vein in the caudal peduncle at the end of the experiment. Adequate amounts of whole blood in small plastic vials containing heparin were used for the determination of hemoglobin (Hb) by using commercial kits (Diamond Diagnostic, Egypt). Also, total red blood cells count (RBCs) and total white blood cells count (WBCs) were counted on an Ao Bright -Line Hämocytometer model (Neubauer imroved, Precicolor HBG, Germany), as well as the packed cell volume (PCV %) was measured according to Stoskopf (1993). Other blood samples were collected and transferred for centrifugation at 3500 rpm for 15 min to obtain blood plasma for determination of the total protein according to Gornall et al. (1949), albumin according to Weichsebum (1946) and globulin by difference according to Doumas and Biggs (1972).
All data were statistically analyzed using general liner models (GLM) procedure according to SAS (2001) for users guide (SAS version 9.2) to detect the overall effects of treatments. All ratios and percentages were arcsine-transformed prior to statistical analyses. The differences between mean were compared for the significance (P ≤ 0.05) using Duncan's multiple rang test (Duncan, 1955).
Data in Table 2 showed that fish fed DGL were significantly (P ≤ 0.05) improved all growth performance parameters compared to the control (0 %). Fish fed 1% DGL was the highest significant (P ≤ 0.05) in all growth performance parameters among other treatments. However, increasing levels of DGL up to 1% caused significantly (P ≤ 0.05) decreased of all growth performance parameters. No significant (P ≥ 0.05) differences in SR % among all levels.
Table 2. Growth performance parameters of male monosex O. niloticus fed different levels of dried garlic lobes.
Feed utilization parameters (FI, FCR, FE, PER, PPV and EU) of O. niloticus illustrated in Table 3. Results revealed that the addition of 1% DGL led to improve significantly (P ≤ 0.05) of FCR, FE, PER, PPV and EU compared to other treatments. While, fish fed 2% DGL gave the highest FI (P ≤ 0.05) among other levels. On the other hand, results indicated that the increasing levels of DGL up to 1% had impaired effects (P ≤ 0.05) on all feed utilization parameters except FI.
Table 3. Feed utilization parameters of male monosex O. niloticus fed different levels of dried garlic lobes.
Chemical analysis of the experimental fish:
The chemical analysis of the whole fish body at the start and the end of the experiment was presented in Table 4. Dry matter (DM), crude protein (CP) and Ash at the end were highest than at the beginning of the experiment, while the ether extract (EE) and energy content (EC) decreased at the end than at the beginning of the experiment. Also, results showed that fish fed 1% DGL recorded the lowest significant (P ≤ 0.05) values of EE and EC, and the highest significant value of CP among other levels. On the other hand, the control group was the highest significant (P ≤ 0.05) in DM compared with all levels. However, no significant (P ≥ 0.05) differences in ash contents among all levels of DGL.
Table 4. Chemical composition of the whole fish body of male monosex O. niloticus fed different levels of dried garlic lobes.
Hematological and blood biochemical parameters:
Data of hematological and blood biochemical parameters were illustrated in Figure 1. Results indicated that the addition of 1% DGL gave the highest significant (P ≤ 0.05) values of Hb concentration, RBCs count, PCV% and WBCs count compared to other levels. While, the control group recorded the highest significant (P ≤ 0.05) values in blood indices (MCV, MCH and MCHC) among other groups. On the other hand, fish fed 1% DGL gave the highest significant (P ≤ 0.05) values in total protein, albumin, and globulin among all levels. However, no significant (P ≥ 0.05) differences of albumin / globulin ratio among all DGL levels.
Fig. 1. Hematological and blood biochemical parameters of male monosex O. niloticus fed different levels of dried garlic lobes. Mean having different small letters are significantly different (P ≤ 0.05). Hb, hemoglobin; RBCs, red blood cells; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBCs, white blood cells.
Attempts to use the natural materials such as medicinal plants could be widely accepted as feed additives to enhance efficiency of feed utilization and animal productive performance (Mohamed et al., 2003). In the present study, results indicated that addition of dietary DGLled to significantly (P ≤ 0.05) improve of all growth performance and feed utilization parameters of O. niloticus compared to the control group. The present findings are in agreement with those obtained by Khattab et al. (2004) and Shalaby et al. (2006). In this respect, Soltan and El-Laithy (2008) reported that the FW, SGR, FI, and FCR of O. niloticus increased significantly with increasing levels of A. sativum. In addition, Aly et al. (2008) and Aly and Mohamed (2010) found the same results of O. niloticus after feeding with garlic (10 and 20 g/kg diet fed) for 8 months compared to the control group of fish fed free diet of garlic. Likewise, Abdel-Hakim et al. (2010) found that the highest growth performance parameters were recorded with the group fed on the basal diet supplemented with fresh garlic at 3g/kg level, while the diet supplemented with dried garlic (5g/kg) recorded significantly the best feed efficiency parameters compared with the control group. In contrast, negative effects have been reported by Ndong and Fall (2007) who found that hybrid tilapia fed a garlic-supplemented diet at 0.5 and 1% exhibited no improvement in growth compared to those fed a control diet after 2–4 weeks. The differences of results obtained from previous studies and the current study may be due to the difference in the experiments conditions, size and age of fish, addition of garlic…etc.
The positive effects in fish growth performance and feed utilization attributed to the bioactive components of garlic including sulphur containing compounds, such as allin, diallylsulphides, and allicin (Amagase and Milner, 1993).In addition, allicin has an intense garlic flavor with a strong stimulatory effect on olfaction in most aquatic animals, including Pelodiscus sinensis, Ctenopharyngodon idellus, Cyprinus carpio, Carassius auratus, and Oreochromis niloticus (Lee and Gao, 2012). On the other hand, sulfur compounds in garlic are considered as active antimicrobial agents and improve immunity and therefore stimulate growth (EL-Afify, 1997). Also Sivam (2001) suggested that the improvements due to garlic may due to its antimicrobial, antioxidant, and antihypertensive properties, which reflected to improved growth performance and feed utilization.
In the present study, fish fed 1% of DGL significantly (P ≤ 0.05) increased the protein content in their whole body; whereas the total lipid and ash contents significantly decreased in the same group compared to the other groups. These results are in agreement with those obtained by Khattab et al. (2004); Shalaby et al. (2006); Metwally (2009);Mehrim (2009). Likewise, Luo et al. (2008) found that garlic could improve the flesh quality of grass carp, Ctenopharyngodon idellus. In addition, Aly et al. (2008) reported that the post-harvest flesh quality and shelf-life of O. niloticus fed a garlic-supplemented diet were improved. On the other hand, there were no significant changes in fish body composition caused by different garlic levels (Diab et al., 2002). Also, Abdel-Hakim et al. (2010) indicated that supplementation of O. niloticus diets with fresh or dried garlic had no significant effects on moisture; dry matter and ash contents of fish whole bodies, while it released significant effects on the whole body protein and fat compared to the control group. The promising findings in the present study on chemical composition of O. niloticus may be due to the dietary supplementation with 1% DGL, which was enhanced plasma total protein (Fig. 1d), as well as may be increased the growth performance (Table 2), and improved PER and PPV (Table 3).
Knowledge of the haematological characteristics is an important tool that can be used as an effective and sensitive index to monitor physiological and pathological changes in fishes. Normal ranges for various blood parameters in fish have been established by different investigators in fish physiology and pathology (Xiaoyun et al., 2009). Results in Figure1 showed that fish fed 1% DGL significantly (P ≤ 0.05) increased Hb, RBCs, PCV, WBCs, total protein, albumin, globulin and albumin / globulin ratio compared to the control group. These results are in agreement with those obtained by Martins et al. (2002) and Shalaby et al. (2006) who verified that addition of A. sativum to fish diets increased erythrocytes number, hemoglobin content, hematocrit value, leucocytes, and thrombocytes. Also, Ndong and Fall (2007) found that hybrid tilapia fed a garlic-supplemented diet at 0.5 enhanced in total leucocyte count, respiratory burst, phagyctic activity, phagocytic index and lysozyme activity. Furthermore, Nwabueze (2012) found that RBC, PCV and Hb were significantly higher in Clarias gariepinus fed 0.5% garlic supplemented diets than other concentrations.
These hopeful current results in hematological and blood biochemical parameters led to increase the immune responses and healthy status of fish, which reflected to improve the growth performance, survival rate and feed efficiency. Where, A. sativum has some constituents that may play a role in the immune system stimulation and in the function of organs related to blood cell formation such as thymus, spleen, and bone marrow (Jeorg and Lee, 1998).Also, A. sativum has been antioxidant properties, which could have a protective nature against gastrointestinal neoplasias, against blood clots (anti-platelet action) due in part to the compounds alliin and ajoene, which have fibrinolytic activity. Ajoene inhibits thromboxane synthesis through the inhibition of the cyclo-oxygenase and lipoxygenase enzymes(Schulz et al., 2004).In addition, S-allyl cysteine, present in the crushed garlic, was found to inhibit tumor metabolism and enhance the immune response (Sumiyoshi, 1997). The allyl sulfides enhance the glutathione s-transferase enzyme system, which through their dependent biochemical pathway enhance the liver's detoxification of carcinogenic substances.The allium pecies show immune enhancing activities that include promotion of lymphocyte synthesis, cytokine release, phagocytosis and natural killer cell activity (Kyo et al., 1998).
From the foregoing results, it could be concluded that dietary supplementation of dried garlic (A. sativum L.) lobes is useful at levels 1% / kg diet for enhancing growth performance, feed utilization, chemical composition of the whole fish body, hematological and blood biochemical parameters of Nile tilapia O. niloticus.
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