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Effects of dietary onion (Allium cepa) powder on growth, innate immune response and hemato–biochemical parameters of beluga (Huso huso Linnaeus, 1754) juvenile
Fish & Shellfish Immunology 45 (2015) 828e834
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Effects of dietary onion (Allium cepa) powder on growth, innate immune response and hematoebiochemical parameters of beluga (Huso huso Linnaeus, 1754) juvenile Raza Akrami a, *, Ahmad Gharaei b, Majid Razeghi Mansour c, Ali Galeshi a a
Department of Fisheries, Azadshahr Branch, Islamic Azad University, Azadshahr, Iran Department of Fisheries, Faculty of Natural Resources and Hamoun International Wetland Research Institute, University of Zabol, Zabol, Sisatan and Baluchestan, Iran c Young Researchers and Elite Club, Azadshahr Branch, Islamic Azad University, Azadshahr, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online 8 June 2015
The present study was aimed at determining the effects of dietary onion powder on growth, innate immune response and hematoebiochemical parameters of beluga juvenile (Huso huso). Basal diets containing onion powder 0 (control), 0.5 and 1% of feed were fed to beluga juvenile. At the end of the experiment, the highest weight gain (WG%) and specific growth rate (SGR) was observed in group fed with 1% onion (P < 0.05). There were no significant difference (P > 0.05) about feed conversion ratio (FCR) in treatment groups that fed diets containing various levels of onion powder. After 8 weeks, serum lysozyme activity, superoxide dismutase activity (SOD), respiratory burst activity and serum total immunoglobulin (Ig) showed a significant increase in treatment group with 1% onion powder compared to other groups (P < 0.05). The group fed 1% onion showed a significantly increases in the number of erythrocytes (RBC), leucocyte (WBC), haematocrit (Hct) levels compared to the control group (P < 0.05). Haemoglobin, monocyte, lymphocyte and neutrophil had no significant change (P > 0.05) in treatment groups and control. The analysis of AST and LDH levels showed a significant decrease in 1% onion compared to the control and 0.5% onion diet (P < 0.05), while ALT and ALP levels were not influenced (P > 0.05). The blood glucose, total protein, triglyceride, cholesterol, albumin and globulin levels were lower in treated groups compared with the control (P < 0.05). The results of this study demonstrated that dietary onion powder could be an improvement in growth, hematological parameters and immune function of beluga juvenile. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Onion powder Blood parameters Immune response Huso huso
1. Introduction The use of dietary additives in fish farms is one of the methods commonly used to improve weight gain, feed efficiency, and/or disease resistance in cultured fish. Therefore, several kinds of additives for aquafeed that are used to improve the performance of fish have been studied [1]. Immunostimulant plants or their byproduct contain several phenolic, polyphenolic, alkaloid, quinine, terpenoid, lectine and polypeptide compounds [2]. Many of which shown to be very effective alternative to antibiotics, chemicals or synthetic compounds and vaccines. The most important advantage of using immunostimulant plants in aquaculture is that they
* Corresponding author. E-mail address: [email protected] (R. Akrami). http://dx.doi.org/10.1016/j.fsi.2015.06.005 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
contain natural organic materials that do not cause any threat to fish health or to the environment or to human health [2]. Therefore, instead of antibiotics and biocides, increasing attention is being paid to the use of immunostimulants for disease-control strategies in aquaculture [2]. The development of new additives for aquafeed, however, still attracts the attention of many researchers and fish farmers. In this study, attention focused on onion. Onion (Allium cepa L.) has a high content of free and glycosidically bonded quercetin and oxidized quercetin derivatives [3,4]. Onion has been known to have antibacterial, antioxidant, and/or anticancer effects [5,6], and it reduces endogenous lipogenesis and increases catabolism of lipids [7]. Onions contain a wide variety of microconstituents such as trace elements, vitamins, flavonoids and sulfur compounds [8], which may have protective effects against cancer. Additionally, a previous study revealed that onion powder was one of the most effective dietary additives tested that improve
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lysozyme activity of the Olive flounder (Paralichthys olivaceus) juvenile [1]. The beluga (Huso huso) is the largest species of sturgeon and one of the most important species in the Caspian Sea [9]. This species is suitable for aquaculture because of its fast growth, easy reproduction and high tolerance to adverse environmental conditions [10]. Therefore, further identification of commercially available dietary additives to improve performance and disease resistance of these fish is still highly desired. The purpose of the present study was to examine the effects of onion powder in the diet of beluga on growth, immune response and hematoebiochemical parameters.
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from each tank and were anesthetized with clove solution, and about 4 mL of blood was drawn from the caudal vein, using a syringe. Then, blood samples were introduced to both heparinized and non-heparinized tubes in order to perform haematological and immunological studies, respectively. Blood sera were obtained by centrifuging blood samples at 3000 rpm (15,609 g) for 10 min using a Heraeus Labofuge 400, and the sera were removed with a disposable transfer pipette [16]. And stored at _20 C until analysis for biochemical and immunological studies. 2.5. Hematological assays
2. Materials and methods 2.1. Experimental conditions Healthy beluga juveniles were obtained from Shahid Marjani Fisheries Center (Gorgan province, Iran) and stocked in the experimental fiberglass tanks (2 m 2 m 0.5 m) for 2 weeks before the beginning of the treatment regime, in order to condition the fish to the laboratory system and handling procedures and then, 108 fish with mean weight of 48.95 ± 7.66 g were randomly allocated in 9 tanks, 12 fish in each tank with three replicates per diet. The control group received no onion powder. The tanks were connected to continuous circulating system. Continuous aeration was provided to each tank through air stone connected to a central air compressor. During the experimental period, water temperature, dissolved oxygen and pH recorded 24.41 ± 1.9 C, 6.54 ± 0.98 mg l1 and 7.5 ± 0.5, respectively. 2.2. Preparation of onion powder and the experimental diets A commercially available onion powder (Hamishak Industrial Co., Ltd. Abbas abad, Tehran, Iran) was used as a dietary additive. For each experiment, the required concentration 0 (control), 0.5% and 1% onion powder was mixed in the basal diet. To prepare the diets, a commercial pellet diet (containing 39% protein, 18% lipid, 7.2% ash and 22.71 MJ kg1 GE) was crushed, mixed with the appropriate onion powder concentration and water, and made again in to the pellets, which were allowed to dry for 18 h at 45 C by air circulation and stored at 4 C until use. Control diet was prepared adding only water [11,12]. Juveniles were fed the experimental diet for 8 weeks in rate of 3e5% of the body weight per day, spread across two feeding times (07:00 and 19:00). 2.3. Growth performance In order to analyze the growth indices of the juveniles, all of fish from each tank were biometried and weighted once every 15 days during the experiment, at least 12 h after the last feeding [12e14]. At the end of the feeding trial, weight gain (WG%), specific growth rate (SGR; % ady1) and feed conversion ratio (FCR) were calculated according to the following formulas [15]: WGð%Þ ¼ 100 ðWf Wi Þ=Wi FCR ¼ dry feed intake (g)/wet WG (g)
SGR % day1 ¼ ðLn Wf Ln WiÞ 100=t where Wf and Wi are final and initial body weights, respectively and t is time in days. 2.4. Blood sample collection At the end of the experiment, three fish were sampled randomly
The erythrocyte (RBC) and leukocyte (WBC) counts were determined using a Neubaeur hemocytometer according to Martins et al. [17]. Hemoglobin levels (Hb) were obtained by the cyanomethemoglobin spectrophotometry method. Hematocrit (Hct) was measured using the standard microhematocrit method [18]. Differential leukocyte counts (monocyte, lymphocyte and neutrophil) were determined using Giemsa staining method and detected blood smears under light microscope [19]. 2.6. Biochemical assays Biochemical analysis was performed in the sequence: glucose (glucose oxidase), total protein (Biuret method), triglycerides using lipase (lipase/GPO-PAP), cholesterol (cholesterol oxidase), albumin (Bromocresol Green method) [20]. Globulin was calculated by subtracting albumin value from total plasma protein [21]. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) enzymes were determined colorimetrically and alkaline phosphatase (ALP) was determined using an enzymatic method [20]. 2.7. Immunological assays 2.7.1. Lysozyme activity Lysozyme level was determined by turbidometric assay according to the method of Ellis [22] with little modifications. Aliquots (1.75 mgl1) of Micrococcus lysodeikticus suspension (Sigma) (0.375 mg ml1, 0.05 M PBS, pH 6.2) were mixed with 250 ml1 of each sample, and the optical density was measured after 15 and 180 s by spectrophotometer (Biophotometer Eppendorf) at 670 nm. PBS was used as a blank, and results were expressed according to amounts of lysozyme (mg) per mg of sample calibrated using a standard curve that determined with hens' egg white lysozyme (Sigma) in sterile sodium phosphate buffer. 2.7.2. Serum superoxide dismutase (SOD) activity Serum Superoxide dismutase (SOD) activity was measured spectrophotochemically by the ferricytochrome C method using xanthine/xanthine oxidase as the source of superoxide radicals [23]. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 mM xanthine, 0.013 mM cytochrome C and 0.024 IU ml1 xanthine oxidase. The reaction was triggered after the addition of the xanthine oxidase. One activity unit was defined as the amount of enzyme necessary to produce 50% inhibition of the ferricytochrome C reduction rate that measured at 550 nm [24]. Enzyme activity was expressed as units per ml serum (U ml1). 2.7.3. Respiratory burst activity The generation of intracellular superoxide radicals by sole phagocytes was determined by the reduction of nitro-blue tetrazolium (NBT) according to the technique described by Secombes
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[25] and Boesen et al. [26]. Phagocyte monolayers were washed with L-15 medium and HBSS (Hank's Balanced Salt Solution) to remove any trace of antibiotics. Then, 100 ml NBT dissolved at 1 mg ml1 in HBSS and added to the wells and the phagocytes incubated at 22 C for 30 min. After incubation, the medium was removed and the culture was washed twice with isotonic PBS and fixed with 100 ml methanol (100%) per each well for 3 min, then washed twice with 70% methanol. The cells were air dried. Formazan was solubilized in 120 ml of KOH (2 M) plus 120 ml of dimethyl sulfoxide (DMSO) and the absorbance was measured spectrophotometrically (Hitachi) at 620 nm. 2.7.4. Serum total immunoglobulin (Ig) Serum total immunoglobulin (Ig) levels were determined according to the method described by Siwicki and Anderson [27]. Briefly, serum total protein content was measured base on micro protein determination method (C-690; Sigma), prior to and after precipitating down the immunoglobulin molecules, using a 12% solution of polyethylene glycol (Sigma). 2.8. Statistical analysis Values were expressed as Mean ± standard error (SE). Growth performance, haematological, biochemical and immunological parameters were tested using one-way ANOVA and Duncan's multiple range test was used for comparison of the mean values at the 5% level of significance using software SPSS (Version 16.0).
Table 2 Effects of dietary onion powder on Haematological parameters of beluga juveniles for 8 week using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control 1
6
RBC (10 ml ) WBC (103 ml1) Hb(g/dl) Hct (%) Monocyte (%) Lymphocyte (%) Neutrophil (%)
0.11 10.13 4.8 31.4 12.3 65.7 21.1
± ± ± ± ± ± ±
0.5% a
0.03 1.4a 0.98a 3.8a 1.4a 3.3a 1.53a
0.13 11.36 4.5 34.7 13.6 67.6 20.6
1% ± ± ± ± ± ± ±
ab
0.02 1.4 ab 0.82a 4.1 ab 1.9a 5.8a 2.1a
0.17 12.43 5.1 39.3 14.1 69.8 18.8
± ± ± ± ± ± ±
0.01b 1.2b 0.79a 1.2b 2.5a 3.51a 3.5a
Data are represented as mean ± SE. Values of means in columns were significantly different (P < 0.05).
Table 3 Effects of dietary onion powder on the blood serum biochemical parameters of beluga juveniles for 8 weeks using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control
Glucose (mg l1) Total protein (mg l1) Triglycerides (mg dl1) Cholesterol (mg dl1) Albumin (mg l1) Globulin (mg l1) AST (IU dl1) ALT (IU dl1) ALP (IU dl1) LDH (IU dl1)
63.5 1.2 497.2 56.4 0.52 0.56 69.8 3.1 449.3 381.2
± ± ± ± ± ± ± ± ± ±
0.5% 0.8a 0.7a 20.1a 3.3a 0.3a 0.1a 2.5a 0.5a 14.1a 15.5a
59.33 1. 6 464.5 49.2 0.83 0.78 70.3 3.2 464.8 387.8
1% ± ± ± ± ± ± ± ± ± ±
1.2b 0.9 ab 14.4a 4.1 ab 0.2b 0.1b 1.9a 0.4a 9.5a 9.1a
53.1 2.1 387.3 46.5 0.98 0.81 59.8 2.4 429.6 313.5
± ± ± ± ± ± ± ± ± ±
1.4c 0.2b 13.8b 1.4b 0.3b 0.2b 2.6b 0.24a 08.2a 9.3b
Data are represented as mean ± SE. Values of means in columns were significantly different (P < 0.05).
The comparison of the given data on the weight gain, specific growth rate (SGR) and food conversion ratio (FCR) of the treatment groups are shown in Table 1. According to the tests, the difference in the mean values of SGR and weight gain among the treatment groups compare with the control group is statistically significant (P < 0.05). Highest weight gain (394.23 ± 26.6) and specific growth rate (3.01 ± 0.9) were recorded on treatment group fed with 1% dietary onion powder. The feed conversion ratio (FCR) was improved in those fish fed with onion powder diet over the control (P > 0.05), although there wre no significant differences between treatment compare with the control group (Table 1). After 8 weeks of treatments, statistical analysis of data showed that there were no significant differences of Hb and differential leukocyte counts (P > 0.05), but the RBC, WBC count and Hct showed a significant increasing in 1% dietary onion powder treatment group compared with the control group (P < 0.05) (Table 2). The effects of dietary onion powder on juvenile beluga observed through some biochemical parameters are shown in Table 3. In all treated groups, only glucose level decreased significantly (P < 0.05) when compared with control group. Whereas total protein, triglycerides, cholestrol, AST and LDH levels showed significant differences (P < 0.05) in high-dose group (1% onion powder) when compared with control group at the end of experiment.
Table 1 Effects of dietary onion powder on growth parameters of beluga juveniles for 8 week using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control
0.5%
1%
Weight gain % SGR (%/day) FCR
308.97 ± 24.7a 2.65 ± 0.11a 0.95 ± 0.05a
334.4 ± 19.9 ab 2.77 ± 0.01 ab 0.82 ± 0.17a
394.23 ± 26.6b 3.01 ± 0.09b 0.85 ± 0.05a
Data expressed as mean ± SE. Values in the same column sharing the same superscript letter are not significantly different (P > 0.05).
By the way, albumin and globulin levels showed noticeable difference (P < 0.05) in all treatment groups when compared with control group (Table 3). The effects of dietary onion powder on some Immunological parameters are shown in Figs. 1e4. In all treatment groups, lysozyme activity, SOD activity, Respiratory burst and total immunoglobulin (Ig) levels showed significant increasing (P < 0.05) when compared with control group at the end of experiment. 4. Discussion The use of immunostimulants is highly recommended in aquaculture and has become one of routine procedures in fish farming. However, due to their high costs, a large number of these additives are not economical. There are numerous plants that have been used in traditional medicine [28]. Our results indicate that
70 Lysozym activity (unit ml -1)
3. Results
c
60
b
50 40
a
30 20
10 0 Control
0.5%
1%
Fig. 1. Effects of dietary onion powder on the serum lysozyme activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SD, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
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c
SODactivity (unit ml-1)
60
50
b
a
40
30 20 10
0 Control
0.5%
1%
Respiratory burst activity (OD620)
Fig. 2. Effects of dietary onion powder on the serum superoxide dismutase (SOD) activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
c
1400
b
1200
a
1000 800 600 400 200
0 Control
0.5%
1%
Fig. 3. Effects of dietary onion powder on the serum respiratory burst activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
60
c
Ig (mg ml-1)
50 40
b a
30 20 10
0 Control
0.5%
1%
Fig. 4. Effects of dietary onion powder on the total immunoglobulin (Ig) levels of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
feeding of fish with 1% onion powder significantly improved growth performance. Some compounds in medicinal plants including bioflavonoids can positively induce effects on growth performance and general health of fish [29]. It has been reported that onion stimulates the digestive process, accelerating digestion and reducing food transit time in the gastrointestinal tract [30].
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Onion prebiotic activity is also being investigated [31,32] due to their high soluble fiber content, specially inulin and fructooligosaccharides which stimulate in the colon the growth of specific microorganisms, as bifidobacteria and lactobacilli, with a general positive health effect [33e35]. Similar to the present study, the increase of growth performance and feed efficiency were recorded in juvenile beluga after feeding ginger (Zingiber officinale) [28] and nettle (Urtica dioica) [35]. Additionally, a previous study revealed that onion extract was one of the most effective dietary additives that improve weight gain of juvenile olive flounder (P. olivaceus) [36]. However, unlike this study, dietary inclusion of various 0, 0.5, 1, 2, 3, and 5% onion powder had no distinctive improvement on weight gain, specific growth rate and feed efficiency of the juvenile olive flounder. The effects of dietary additives on fish performance may vary depending on fish species, size, the dose of the additive, fish nutritional/ physiological status, and/or ambient culturing conditions. To our knowledge, there was no information on effects of dietary onion powder on blood parameters in the Chondrostean fish. The WBC (leucocytes) serves as one of the first lines of body defense and their numbers increase sharply when infections arise. Many data were published to show that the herbal plant could be act as immunostimulants and increase the total WBC [37]. The increase in WBC counts, and other blood cells, following feeding of onion diet, demonstrates the immunostimulatory effects and anti-infection properties of onion which is in line with the previous work of Gholipour kanani et al. [28] and Binaii et al. [35] who obtained increased WBC after feeding juvenile beluga with ginger and nettle diet respectively. It could be explained that the bioactive compounds polyphenols, flavonoids and quercetin found in onion prevented fish from infection by triggering immune system [38]. As a result of feeding onion diet, RBC (erythrocyte) count and Hct % increased in treated groups, exhibiting positive health effect on fish because of bioactive compounds in onion. In previous studies, it has been reported that immunostimulant herbal plants could increase immune functions by affecting the blood cells [39e43]. The obtained results agree with those obtained in beluga after receiving nettle [35] and ginger [26]. Although an increased level of lymphocyte, neutrophil and monocyte in juvenile beluga fed with diets enriched with ginger and nettle was reported by Gholipour kanani et al. [28] and Binaii et al. [35], no significant change was observed between control and onion supplementation diets (P > 0.05). The present study showed an enhancement of total protein in group fed with the highest dose of onion. Since there is a close relationship between the level of protein synthesized in liver tissue and plasma protein pools, total protein levels in plasma maybe elevated due to the increased levels of protein synthesis in liver tissue of fish treated with onion powder. Similarly, the highest serum protein level was recorded in juvenile beluga fed with 1% ginger [28] and 12% nettle [35]. Total protein in the plasma induce albumin and globulin. The increase in the levels of serum protein, albumin and globulin in fish is thought to be associated with a stronger innate immunity response [44]. In our study, globulin and albumin content was higher in onion group. Gholipour kanani et al. [28] and Binaii et al. [35] reported that the administration of some herbal medicine such as ginger and nettle increase globulin and albumin in juvenile beluga similar to the present study, the increase of globulin and albumin were recorded in onion. Al-Salahy [45] found that serum albumin of Clarias lazare treated with onion juices increased after 24 h but serum total protein decrease after 5 h. Unlike this study, Cho and Lee [1] observed dietary inclusion of various level of onion powder did not affect serum total protein in juvenile olive flounder. The onion and garlic effectiveness as hypoglycaemic agents has been scarcely investigated. Recently, it
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has been reported that long-term absorption of natural flavonoids as quercetin could be useful to prevent advanced glycation of collagens, which contributes to development of cardiovascular complications in diabetic patients [46]. In the current study, glucose in fish fed with 1% onion diet improved significantly. The bioactive constituents from onion and garlic, such as methiin and S-allyl cysteine sulphoxide (SACS), exert their anti-diabetic action by stimulating the insulin production and secretion by pancreas, interfering with dietary glucose absorption, and favoring the insulin saving [47]. Binaii et al. [35] observed that beluga supplemented with the nettle showed no significant difference in concentration of glucose serum after 8 weeks. It seems that the difference between our results and the findings of other studies may result from different fish size, water quality, rearing condition, herbal medicine, etc. Some investigations have demonstrated that onion also has compounds with capacity to reduce blood triglycerides levels and to inhibit cholesterol biosynthesis in vitro [48]. Allicin and its derivative compounds are the main active substances responsible for the hypolipidemic and hypocholesterolemic effects of onion and garlic, as much in humans as in experimentation animals [49,50]. On the other hand, quercetin is the most important flavonoid in onion that prevents the biosynthesis of cholesterol by inhibiting the activity of fatty acid synthesis [51]. Therefore decreased cholesterol and triglycerides in fish fed with diets having 1% onion levels at the end of the test may be attributed to the influence of quercetine on the synthesis of cholesterol. This is in agreement with Binaii et al. [35] who reported significant decrease in serum cholesterol and triglycerides levels in beluga fed with basal diet incorporated with nettle. Also Al-Salahy [45] recorded decrease in cholesterol level in Clarias lazera after 5 days of repeated dose of oral administraton onion juice, while it did not show any significant change in triglyceride. On the contrary, Cho and Lee [1] found that the level of cholesterol and triglyceride were not affected by inclusion of the onion powder in the diet of juvenile olive flounder. AST, ALT, LDH, ALP enzymes are used as indices of liver damage. Elevated levels may indicate degeneration, necrosis, and destruction of the liver due to cellular damage. In addition, these enzymes may be used as indices to assess whether experimental materials and diet treatments create toxicity [52]. ALT and ALP revealed a non-significant difference in all beluga fed diet containing onion compared to the control group. As differences in metabolic enzymes levels were not observed in the experimental groups, the consumption of 1% onion did not appear to induce liver toxicity in fish. LDH and AST activity significantly decreased after the feeding administration of 1% onion. Due to anti-radical and anti-oxidant properties of the onion, its administration might prevent lipid peroxidation of cell membranes and inhibit the release of foresaid enzymes into the plasma. Therefore, onion may be useful and prevention of diseases caused by oxidative stress derived from its rich content in flavonoids. Although, dietary garlic and ginger in diet of beluga led to a negligible effect on the serum ALT, a significant decrease in AST was obtained in garlic group compared to control group [28]. Studies of Cho and Lee [1] showed that no significant changes were observed in AST and ALT of cultured juvenile olive flounder fed with diets enriched with onion powder. Moreover, the ALT, AST, and ALP of juvenile beluga were not influenced by different level of nettle [35]. Decrease of both serum ALT and AST was reported in Clarias lasare in response to the repeated dose (1 dose every 24 h for 5 days). Lysozymes are extensively distributed throughout the animal kingdom and catalyse the hydrolysis of peptidoglycans of bacterial cell walls and acts as non-specific innate immunity molecules against the incursion of detrimental bacteria [53,54]. The serum lysozyme activity is considered as a defence barrier against
bacterial pathogens thus resulting in the reduction of disease [55]. In this study lysozyme activities showed significant increase in onion groups. These data are analogous with the increased serum lysozyme activity recorded in olive flounder which fed with the 0.5% onion powder [1]. Moreover, elevated lysozyme activity have been reported in tilapia (Oreochromis niloticus) after supplementing diets with Chinese herbs, Astragalus radix and Scutellaria radix [56] and rainbow trout treated with 10 g/kg ginger [2]. However, unlike this study, lysozyme activity was not influenced in juvenile beluga fed with ginger and garlic [28] and nettle [35]. Teleost IgM resembles mammalian IgM in structure, physiological characteristics, soluble forms and membrane-bound forms [57,58]. The soluble forms which are present in the blood and other fluids play a role as an immune effector molecule [59]. Previous studies have reported a considerable individual variation in serum Ig level among fish. Some changes may be related to size and/or [60,61], environmental conditions [62] or disease status [63]. Moreover, using immunostimulants showed an enhancement in Ig production, for example; Ig production in rainbow trout fed with cumin seed oil (Nigella sativa) and nettle extract (quercetin) as immunostimulants showed significant increase compared to control [64]. Similarly, our study showed increasing Ig level in fish fed with onion powder in all treatment groups. This result coincide with the investigations of Binaii et al. [35] who reported enhanced levels of serum Ig level in beluga after feeding with nettle for 8week. The productions of oxygen free radicals by neutrophils via the respiratory burst are important events in bactericidal pathways in fish [65] and superoxide anion is the first product to be released from the respiratory burst; therefore the measurement of O2 has been accepted as an accurate method of measuring this activity [22]. The results showed respiratory burst activity in the beluga fed by the 0.5% and 1% onion diet significantly rose compared to control and the highest dose showed the highest respiratory burst activity. This is in agreement with the work of Binaii et al. [35], who noticed greater respiratory burst activity in beluga administered with dietary nettle after feeding for 8 weeks. Teyssier et al. [66] reported that the modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder. It seems that the same happen could be occurred in respiratory burst activity of beluga. In conclusion, our study has shown that onion treatment might have an interesting, potential efficacy on immunity and it is able to modulate some immune parameters in beluga. Acknowledgment This study was supported by the fund of Islamic Azaud University of Azadshahr Branch (Grant Number: 142) and the authors wish to thank the staff of Shahid Marjani propagation and cultivation center of sturgeon fish for their kind help during the experiment. References [1] H.C. Cho, S.M. Lee, Onion powder in the diet of the Olive flounder (Paralichthys olivaceus): Effects on the growth, body composition and lysozyme activity, J. World Aquac. Soc. 43 (2012) 30e38. [2] A.D. Talpur, M. Ikhwanuddin, A. Ambok Bolong, Nutritional effects on ginger (Zingiber officinal Roscoe) on immune response of Asian sea bass (Lates calcarifer) and disease resistance against Vibrio harveyi, Aquacultur 400e401 (2013) 46e52. [3] H.J. Suh, J.M. Lee, J.S. Cho, Y.S. Kim, S.H. Chung, Radical scavenging compounds in onion skin, Food Res. Int. 32 (1999) 659e664. [4] G. Griffiths, L. Trueman, T. Crowther, B. Thomas, B. Smith, Onions-a global benefit to health, Phytotherapy Res. 16 (2002) 603e615. [5] F.A. Ramos, Y. Takaishi, M. Shirotori, Y. Kawaguchi, K. Tsuchiya, H. Shibata, T. Higuti, T. Tadokoro, M. Takeuchi, Antibacterial and antioxidant activities of
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Effects of dietary onion (Allium cepa) powder on growth, innate immune response and hematoebiochemical parameters of beluga (Huso huso Linnaeus, 1754) juvenile Raza Akrami a, *, Ahmad Gharaei b, Majid Razeghi Mansour c, Ali Galeshi a a
Department of Fisheries, Azadshahr Branch, Islamic Azad University, Azadshahr, Iran Department of Fisheries, Faculty of Natural Resources and Hamoun International Wetland Research Institute, University of Zabol, Zabol, Sisatan and Baluchestan, Iran c Young Researchers and Elite Club, Azadshahr Branch, Islamic Azad University, Azadshahr, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 April 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online 8 June 2015
The present study was aimed at determining the effects of dietary onion powder on growth, innate immune response and hematoebiochemical parameters of beluga juvenile (Huso huso). Basal diets containing onion powder 0 (control), 0.5 and 1% of feed were fed to beluga juvenile. At the end of the experiment, the highest weight gain (WG%) and specific growth rate (SGR) was observed in group fed with 1% onion (P < 0.05). There were no significant difference (P > 0.05) about feed conversion ratio (FCR) in treatment groups that fed diets containing various levels of onion powder. After 8 weeks, serum lysozyme activity, superoxide dismutase activity (SOD), respiratory burst activity and serum total immunoglobulin (Ig) showed a significant increase in treatment group with 1% onion powder compared to other groups (P < 0.05). The group fed 1% onion showed a significantly increases in the number of erythrocytes (RBC), leucocyte (WBC), haematocrit (Hct) levels compared to the control group (P < 0.05). Haemoglobin, monocyte, lymphocyte and neutrophil had no significant change (P > 0.05) in treatment groups and control. The analysis of AST and LDH levels showed a significant decrease in 1% onion compared to the control and 0.5% onion diet (P < 0.05), while ALT and ALP levels were not influenced (P > 0.05). The blood glucose, total protein, triglyceride, cholesterol, albumin and globulin levels were lower in treated groups compared with the control (P < 0.05). The results of this study demonstrated that dietary onion powder could be an improvement in growth, hematological parameters and immune function of beluga juvenile. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Onion powder Blood parameters Immune response Huso huso
1. Introduction The use of dietary additives in fish farms is one of the methods commonly used to improve weight gain, feed efficiency, and/or disease resistance in cultured fish. Therefore, several kinds of additives for aquafeed that are used to improve the performance of fish have been studied [1]. Immunostimulant plants or their byproduct contain several phenolic, polyphenolic, alkaloid, quinine, terpenoid, lectine and polypeptide compounds [2]. Many of which shown to be very effective alternative to antibiotics, chemicals or synthetic compounds and vaccines. The most important advantage of using immunostimulant plants in aquaculture is that they
* Corresponding author. E-mail address: [email protected] (R. Akrami). http://dx.doi.org/10.1016/j.fsi.2015.06.005 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
contain natural organic materials that do not cause any threat to fish health or to the environment or to human health [2]. Therefore, instead of antibiotics and biocides, increasing attention is being paid to the use of immunostimulants for disease-control strategies in aquaculture [2]. The development of new additives for aquafeed, however, still attracts the attention of many researchers and fish farmers. In this study, attention focused on onion. Onion (Allium cepa L.) has a high content of free and glycosidically bonded quercetin and oxidized quercetin derivatives [3,4]. Onion has been known to have antibacterial, antioxidant, and/or anticancer effects [5,6], and it reduces endogenous lipogenesis and increases catabolism of lipids [7]. Onions contain a wide variety of microconstituents such as trace elements, vitamins, flavonoids and sulfur compounds [8], which may have protective effects against cancer. Additionally, a previous study revealed that onion powder was one of the most effective dietary additives tested that improve
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lysozyme activity of the Olive flounder (Paralichthys olivaceus) juvenile [1]. The beluga (Huso huso) is the largest species of sturgeon and one of the most important species in the Caspian Sea [9]. This species is suitable for aquaculture because of its fast growth, easy reproduction and high tolerance to adverse environmental conditions [10]. Therefore, further identification of commercially available dietary additives to improve performance and disease resistance of these fish is still highly desired. The purpose of the present study was to examine the effects of onion powder in the diet of beluga on growth, immune response and hematoebiochemical parameters.
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from each tank and were anesthetized with clove solution, and about 4 mL of blood was drawn from the caudal vein, using a syringe. Then, blood samples were introduced to both heparinized and non-heparinized tubes in order to perform haematological and immunological studies, respectively. Blood sera were obtained by centrifuging blood samples at 3000 rpm (15,609 g) for 10 min using a Heraeus Labofuge 400, and the sera were removed with a disposable transfer pipette [16]. And stored at _20 C until analysis for biochemical and immunological studies. 2.5. Hematological assays
2. Materials and methods 2.1. Experimental conditions Healthy beluga juveniles were obtained from Shahid Marjani Fisheries Center (Gorgan province, Iran) and stocked in the experimental fiberglass tanks (2 m 2 m 0.5 m) for 2 weeks before the beginning of the treatment regime, in order to condition the fish to the laboratory system and handling procedures and then, 108 fish with mean weight of 48.95 ± 7.66 g were randomly allocated in 9 tanks, 12 fish in each tank with three replicates per diet. The control group received no onion powder. The tanks were connected to continuous circulating system. Continuous aeration was provided to each tank through air stone connected to a central air compressor. During the experimental period, water temperature, dissolved oxygen and pH recorded 24.41 ± 1.9 C, 6.54 ± 0.98 mg l1 and 7.5 ± 0.5, respectively. 2.2. Preparation of onion powder and the experimental diets A commercially available onion powder (Hamishak Industrial Co., Ltd. Abbas abad, Tehran, Iran) was used as a dietary additive. For each experiment, the required concentration 0 (control), 0.5% and 1% onion powder was mixed in the basal diet. To prepare the diets, a commercial pellet diet (containing 39% protein, 18% lipid, 7.2% ash and 22.71 MJ kg1 GE) was crushed, mixed with the appropriate onion powder concentration and water, and made again in to the pellets, which were allowed to dry for 18 h at 45 C by air circulation and stored at 4 C until use. Control diet was prepared adding only water [11,12]. Juveniles were fed the experimental diet for 8 weeks in rate of 3e5% of the body weight per day, spread across two feeding times (07:00 and 19:00). 2.3. Growth performance In order to analyze the growth indices of the juveniles, all of fish from each tank were biometried and weighted once every 15 days during the experiment, at least 12 h after the last feeding [12e14]. At the end of the feeding trial, weight gain (WG%), specific growth rate (SGR; % ady1) and feed conversion ratio (FCR) were calculated according to the following formulas [15]: WGð%Þ ¼ 100 ðWf Wi Þ=Wi FCR ¼ dry feed intake (g)/wet WG (g)
SGR % day1 ¼ ðLn Wf Ln WiÞ 100=t where Wf and Wi are final and initial body weights, respectively and t is time in days. 2.4. Blood sample collection At the end of the experiment, three fish were sampled randomly
The erythrocyte (RBC) and leukocyte (WBC) counts were determined using a Neubaeur hemocytometer according to Martins et al. [17]. Hemoglobin levels (Hb) were obtained by the cyanomethemoglobin spectrophotometry method. Hematocrit (Hct) was measured using the standard microhematocrit method [18]. Differential leukocyte counts (monocyte, lymphocyte and neutrophil) were determined using Giemsa staining method and detected blood smears under light microscope [19]. 2.6. Biochemical assays Biochemical analysis was performed in the sequence: glucose (glucose oxidase), total protein (Biuret method), triglycerides using lipase (lipase/GPO-PAP), cholesterol (cholesterol oxidase), albumin (Bromocresol Green method) [20]. Globulin was calculated by subtracting albumin value from total plasma protein [21]. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) enzymes were determined colorimetrically and alkaline phosphatase (ALP) was determined using an enzymatic method [20]. 2.7. Immunological assays 2.7.1. Lysozyme activity Lysozyme level was determined by turbidometric assay according to the method of Ellis [22] with little modifications. Aliquots (1.75 mgl1) of Micrococcus lysodeikticus suspension (Sigma) (0.375 mg ml1, 0.05 M PBS, pH 6.2) were mixed with 250 ml1 of each sample, and the optical density was measured after 15 and 180 s by spectrophotometer (Biophotometer Eppendorf) at 670 nm. PBS was used as a blank, and results were expressed according to amounts of lysozyme (mg) per mg of sample calibrated using a standard curve that determined with hens' egg white lysozyme (Sigma) in sterile sodium phosphate buffer. 2.7.2. Serum superoxide dismutase (SOD) activity Serum Superoxide dismutase (SOD) activity was measured spectrophotochemically by the ferricytochrome C method using xanthine/xanthine oxidase as the source of superoxide radicals [23]. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.1 mM xanthine, 0.013 mM cytochrome C and 0.024 IU ml1 xanthine oxidase. The reaction was triggered after the addition of the xanthine oxidase. One activity unit was defined as the amount of enzyme necessary to produce 50% inhibition of the ferricytochrome C reduction rate that measured at 550 nm [24]. Enzyme activity was expressed as units per ml serum (U ml1). 2.7.3. Respiratory burst activity The generation of intracellular superoxide radicals by sole phagocytes was determined by the reduction of nitro-blue tetrazolium (NBT) according to the technique described by Secombes
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[25] and Boesen et al. [26]. Phagocyte monolayers were washed with L-15 medium and HBSS (Hank's Balanced Salt Solution) to remove any trace of antibiotics. Then, 100 ml NBT dissolved at 1 mg ml1 in HBSS and added to the wells and the phagocytes incubated at 22 C for 30 min. After incubation, the medium was removed and the culture was washed twice with isotonic PBS and fixed with 100 ml methanol (100%) per each well for 3 min, then washed twice with 70% methanol. The cells were air dried. Formazan was solubilized in 120 ml of KOH (2 M) plus 120 ml of dimethyl sulfoxide (DMSO) and the absorbance was measured spectrophotometrically (Hitachi) at 620 nm. 2.7.4. Serum total immunoglobulin (Ig) Serum total immunoglobulin (Ig) levels were determined according to the method described by Siwicki and Anderson [27]. Briefly, serum total protein content was measured base on micro protein determination method (C-690; Sigma), prior to and after precipitating down the immunoglobulin molecules, using a 12% solution of polyethylene glycol (Sigma). 2.8. Statistical analysis Values were expressed as Mean ± standard error (SE). Growth performance, haematological, biochemical and immunological parameters were tested using one-way ANOVA and Duncan's multiple range test was used for comparison of the mean values at the 5% level of significance using software SPSS (Version 16.0).
Table 2 Effects of dietary onion powder on Haematological parameters of beluga juveniles for 8 week using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control 1
6
RBC (10 ml ) WBC (103 ml1) Hb(g/dl) Hct (%) Monocyte (%) Lymphocyte (%) Neutrophil (%)
0.11 10.13 4.8 31.4 12.3 65.7 21.1
± ± ± ± ± ± ±
0.5% a
0.03 1.4a 0.98a 3.8a 1.4a 3.3a 1.53a
0.13 11.36 4.5 34.7 13.6 67.6 20.6
1% ± ± ± ± ± ± ±
ab
0.02 1.4 ab 0.82a 4.1 ab 1.9a 5.8a 2.1a
0.17 12.43 5.1 39.3 14.1 69.8 18.8
± ± ± ± ± ± ±
0.01b 1.2b 0.79a 1.2b 2.5a 3.51a 3.5a
Data are represented as mean ± SE. Values of means in columns were significantly different (P < 0.05).
Table 3 Effects of dietary onion powder on the blood serum biochemical parameters of beluga juveniles for 8 weeks using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control
Glucose (mg l1) Total protein (mg l1) Triglycerides (mg dl1) Cholesterol (mg dl1) Albumin (mg l1) Globulin (mg l1) AST (IU dl1) ALT (IU dl1) ALP (IU dl1) LDH (IU dl1)
63.5 1.2 497.2 56.4 0.52 0.56 69.8 3.1 449.3 381.2
± ± ± ± ± ± ± ± ± ±
0.5% 0.8a 0.7a 20.1a 3.3a 0.3a 0.1a 2.5a 0.5a 14.1a 15.5a
59.33 1. 6 464.5 49.2 0.83 0.78 70.3 3.2 464.8 387.8
1% ± ± ± ± ± ± ± ± ± ±
1.2b 0.9 ab 14.4a 4.1 ab 0.2b 0.1b 1.9a 0.4a 9.5a 9.1a
53.1 2.1 387.3 46.5 0.98 0.81 59.8 2.4 429.6 313.5
± ± ± ± ± ± ± ± ± ±
1.4c 0.2b 13.8b 1.4b 0.3b 0.2b 2.6b 0.24a 08.2a 9.3b
Data are represented as mean ± SE. Values of means in columns were significantly different (P < 0.05).
The comparison of the given data on the weight gain, specific growth rate (SGR) and food conversion ratio (FCR) of the treatment groups are shown in Table 1. According to the tests, the difference in the mean values of SGR and weight gain among the treatment groups compare with the control group is statistically significant (P < 0.05). Highest weight gain (394.23 ± 26.6) and specific growth rate (3.01 ± 0.9) were recorded on treatment group fed with 1% dietary onion powder. The feed conversion ratio (FCR) was improved in those fish fed with onion powder diet over the control (P > 0.05), although there wre no significant differences between treatment compare with the control group (Table 1). After 8 weeks of treatments, statistical analysis of data showed that there were no significant differences of Hb and differential leukocyte counts (P > 0.05), but the RBC, WBC count and Hct showed a significant increasing in 1% dietary onion powder treatment group compared with the control group (P < 0.05) (Table 2). The effects of dietary onion powder on juvenile beluga observed through some biochemical parameters are shown in Table 3. In all treated groups, only glucose level decreased significantly (P < 0.05) when compared with control group. Whereas total protein, triglycerides, cholestrol, AST and LDH levels showed significant differences (P < 0.05) in high-dose group (1% onion powder) when compared with control group at the end of experiment.
Table 1 Effects of dietary onion powder on growth parameters of beluga juveniles for 8 week using a one-way ANOVA (n ¼ 3 tank per treatment group with 12 fish per tank). Parameter
Control
0.5%
1%
Weight gain % SGR (%/day) FCR
308.97 ± 24.7a 2.65 ± 0.11a 0.95 ± 0.05a
334.4 ± 19.9 ab 2.77 ± 0.01 ab 0.82 ± 0.17a
394.23 ± 26.6b 3.01 ± 0.09b 0.85 ± 0.05a
Data expressed as mean ± SE. Values in the same column sharing the same superscript letter are not significantly different (P > 0.05).
By the way, albumin and globulin levels showed noticeable difference (P < 0.05) in all treatment groups when compared with control group (Table 3). The effects of dietary onion powder on some Immunological parameters are shown in Figs. 1e4. In all treatment groups, lysozyme activity, SOD activity, Respiratory burst and total immunoglobulin (Ig) levels showed significant increasing (P < 0.05) when compared with control group at the end of experiment. 4. Discussion The use of immunostimulants is highly recommended in aquaculture and has become one of routine procedures in fish farming. However, due to their high costs, a large number of these additives are not economical. There are numerous plants that have been used in traditional medicine [28]. Our results indicate that
70 Lysozym activity (unit ml -1)
3. Results
c
60
b
50 40
a
30 20
10 0 Control
0.5%
1%
Fig. 1. Effects of dietary onion powder on the serum lysozyme activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SD, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
R. Akrami et al. / Fish & Shellfish Immunology 45 (2015) 828e834
c
SODactivity (unit ml-1)
60
50
b
a
40
30 20 10
0 Control
0.5%
1%
Respiratory burst activity (OD620)
Fig. 2. Effects of dietary onion powder on the serum superoxide dismutase (SOD) activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
c
1400
b
1200
a
1000 800 600 400 200
0 Control
0.5%
1%
Fig. 3. Effects of dietary onion powder on the serum respiratory burst activity of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
60
c
Ig (mg ml-1)
50 40
b a
30 20 10
0 Control
0.5%
1%
Fig. 4. Effects of dietary onion powder on the total immunoglobulin (Ig) levels of beluga juveniles for 8 weeks. Values are expressed as mean ± SE, n ¼ 6. Mean values bearing different superscripts at the different stage were statistically significant (P < 0.05).
feeding of fish with 1% onion powder significantly improved growth performance. Some compounds in medicinal plants including bioflavonoids can positively induce effects on growth performance and general health of fish [29]. It has been reported that onion stimulates the digestive process, accelerating digestion and reducing food transit time in the gastrointestinal tract [30].
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Onion prebiotic activity is also being investigated [31,32] due to their high soluble fiber content, specially inulin and fructooligosaccharides which stimulate in the colon the growth of specific microorganisms, as bifidobacteria and lactobacilli, with a general positive health effect [33e35]. Similar to the present study, the increase of growth performance and feed efficiency were recorded in juvenile beluga after feeding ginger (Zingiber officinale) [28] and nettle (Urtica dioica) [35]. Additionally, a previous study revealed that onion extract was one of the most effective dietary additives that improve weight gain of juvenile olive flounder (P. olivaceus) [36]. However, unlike this study, dietary inclusion of various 0, 0.5, 1, 2, 3, and 5% onion powder had no distinctive improvement on weight gain, specific growth rate and feed efficiency of the juvenile olive flounder. The effects of dietary additives on fish performance may vary depending on fish species, size, the dose of the additive, fish nutritional/ physiological status, and/or ambient culturing conditions. To our knowledge, there was no information on effects of dietary onion powder on blood parameters in the Chondrostean fish. The WBC (leucocytes) serves as one of the first lines of body defense and their numbers increase sharply when infections arise. Many data were published to show that the herbal plant could be act as immunostimulants and increase the total WBC [37]. The increase in WBC counts, and other blood cells, following feeding of onion diet, demonstrates the immunostimulatory effects and anti-infection properties of onion which is in line with the previous work of Gholipour kanani et al. [28] and Binaii et al. [35] who obtained increased WBC after feeding juvenile beluga with ginger and nettle diet respectively. It could be explained that the bioactive compounds polyphenols, flavonoids and quercetin found in onion prevented fish from infection by triggering immune system [38]. As a result of feeding onion diet, RBC (erythrocyte) count and Hct % increased in treated groups, exhibiting positive health effect on fish because of bioactive compounds in onion. In previous studies, it has been reported that immunostimulant herbal plants could increase immune functions by affecting the blood cells [39e43]. The obtained results agree with those obtained in beluga after receiving nettle [35] and ginger [26]. Although an increased level of lymphocyte, neutrophil and monocyte in juvenile beluga fed with diets enriched with ginger and nettle was reported by Gholipour kanani et al. [28] and Binaii et al. [35], no significant change was observed between control and onion supplementation diets (P > 0.05). The present study showed an enhancement of total protein in group fed with the highest dose of onion. Since there is a close relationship between the level of protein synthesized in liver tissue and plasma protein pools, total protein levels in plasma maybe elevated due to the increased levels of protein synthesis in liver tissue of fish treated with onion powder. Similarly, the highest serum protein level was recorded in juvenile beluga fed with 1% ginger [28] and 12% nettle [35]. Total protein in the plasma induce albumin and globulin. The increase in the levels of serum protein, albumin and globulin in fish is thought to be associated with a stronger innate immunity response [44]. In our study, globulin and albumin content was higher in onion group. Gholipour kanani et al. [28] and Binaii et al. [35] reported that the administration of some herbal medicine such as ginger and nettle increase globulin and albumin in juvenile beluga similar to the present study, the increase of globulin and albumin were recorded in onion. Al-Salahy [45] found that serum albumin of Clarias lazare treated with onion juices increased after 24 h but serum total protein decrease after 5 h. Unlike this study, Cho and Lee [1] observed dietary inclusion of various level of onion powder did not affect serum total protein in juvenile olive flounder. The onion and garlic effectiveness as hypoglycaemic agents has been scarcely investigated. Recently, it
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has been reported that long-term absorption of natural flavonoids as quercetin could be useful to prevent advanced glycation of collagens, which contributes to development of cardiovascular complications in diabetic patients [46]. In the current study, glucose in fish fed with 1% onion diet improved significantly. The bioactive constituents from onion and garlic, such as methiin and S-allyl cysteine sulphoxide (SACS), exert their anti-diabetic action by stimulating the insulin production and secretion by pancreas, interfering with dietary glucose absorption, and favoring the insulin saving [47]. Binaii et al. [35] observed that beluga supplemented with the nettle showed no significant difference in concentration of glucose serum after 8 weeks. It seems that the difference between our results and the findings of other studies may result from different fish size, water quality, rearing condition, herbal medicine, etc. Some investigations have demonstrated that onion also has compounds with capacity to reduce blood triglycerides levels and to inhibit cholesterol biosynthesis in vitro [48]. Allicin and its derivative compounds are the main active substances responsible for the hypolipidemic and hypocholesterolemic effects of onion and garlic, as much in humans as in experimentation animals [49,50]. On the other hand, quercetin is the most important flavonoid in onion that prevents the biosynthesis of cholesterol by inhibiting the activity of fatty acid synthesis [51]. Therefore decreased cholesterol and triglycerides in fish fed with diets having 1% onion levels at the end of the test may be attributed to the influence of quercetine on the synthesis of cholesterol. This is in agreement with Binaii et al. [35] who reported significant decrease in serum cholesterol and triglycerides levels in beluga fed with basal diet incorporated with nettle. Also Al-Salahy [45] recorded decrease in cholesterol level in Clarias lazera after 5 days of repeated dose of oral administraton onion juice, while it did not show any significant change in triglyceride. On the contrary, Cho and Lee [1] found that the level of cholesterol and triglyceride were not affected by inclusion of the onion powder in the diet of juvenile olive flounder. AST, ALT, LDH, ALP enzymes are used as indices of liver damage. Elevated levels may indicate degeneration, necrosis, and destruction of the liver due to cellular damage. In addition, these enzymes may be used as indices to assess whether experimental materials and diet treatments create toxicity [52]. ALT and ALP revealed a non-significant difference in all beluga fed diet containing onion compared to the control group. As differences in metabolic enzymes levels were not observed in the experimental groups, the consumption of 1% onion did not appear to induce liver toxicity in fish. LDH and AST activity significantly decreased after the feeding administration of 1% onion. Due to anti-radical and anti-oxidant properties of the onion, its administration might prevent lipid peroxidation of cell membranes and inhibit the release of foresaid enzymes into the plasma. Therefore, onion may be useful and prevention of diseases caused by oxidative stress derived from its rich content in flavonoids. Although, dietary garlic and ginger in diet of beluga led to a negligible effect on the serum ALT, a significant decrease in AST was obtained in garlic group compared to control group [28]. Studies of Cho and Lee [1] showed that no significant changes were observed in AST and ALT of cultured juvenile olive flounder fed with diets enriched with onion powder. Moreover, the ALT, AST, and ALP of juvenile beluga were not influenced by different level of nettle [35]. Decrease of both serum ALT and AST was reported in Clarias lasare in response to the repeated dose (1 dose every 24 h for 5 days). Lysozymes are extensively distributed throughout the animal kingdom and catalyse the hydrolysis of peptidoglycans of bacterial cell walls and acts as non-specific innate immunity molecules against the incursion of detrimental bacteria [53,54]. The serum lysozyme activity is considered as a defence barrier against
bacterial pathogens thus resulting in the reduction of disease [55]. In this study lysozyme activities showed significant increase in onion groups. These data are analogous with the increased serum lysozyme activity recorded in olive flounder which fed with the 0.5% onion powder [1]. Moreover, elevated lysozyme activity have been reported in tilapia (Oreochromis niloticus) after supplementing diets with Chinese herbs, Astragalus radix and Scutellaria radix [56] and rainbow trout treated with 10 g/kg ginger [2]. However, unlike this study, lysozyme activity was not influenced in juvenile beluga fed with ginger and garlic [28] and nettle [35]. Teleost IgM resembles mammalian IgM in structure, physiological characteristics, soluble forms and membrane-bound forms [57,58]. The soluble forms which are present in the blood and other fluids play a role as an immune effector molecule [59]. Previous studies have reported a considerable individual variation in serum Ig level among fish. Some changes may be related to size and/or [60,61], environmental conditions [62] or disease status [63]. Moreover, using immunostimulants showed an enhancement in Ig production, for example; Ig production in rainbow trout fed with cumin seed oil (Nigella sativa) and nettle extract (quercetin) as immunostimulants showed significant increase compared to control [64]. Similarly, our study showed increasing Ig level in fish fed with onion powder in all treatment groups. This result coincide with the investigations of Binaii et al. [35] who reported enhanced levels of serum Ig level in beluga after feeding with nettle for 8week. The productions of oxygen free radicals by neutrophils via the respiratory burst are important events in bactericidal pathways in fish [65] and superoxide anion is the first product to be released from the respiratory burst; therefore the measurement of O2 has been accepted as an accurate method of measuring this activity [22]. The results showed respiratory burst activity in the beluga fed by the 0.5% and 1% onion diet significantly rose compared to control and the highest dose showed the highest respiratory burst activity. This is in agreement with the work of Binaii et al. [35], who noticed greater respiratory burst activity in beluga administered with dietary nettle after feeding for 8 weeks. Teyssier et al. [66] reported that the modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder. It seems that the same happen could be occurred in respiratory burst activity of beluga. In conclusion, our study has shown that onion treatment might have an interesting, potential efficacy on immunity and it is able to modulate some immune parameters in beluga. Acknowledgment This study was supported by the fund of Islamic Azaud University of Azadshahr Branch (Grant Number: 142) and the authors wish to thank the staff of Shahid Marjani propagation and cultivation center of sturgeon fish for their kind help during the experiment. References [1] H.C. Cho, S.M. Lee, Onion powder in the diet of the Olive flounder (Paralichthys olivaceus): Effects on the growth, body composition and lysozyme activity, J. World Aquac. Soc. 43 (2012) 30e38. [2] A.D. Talpur, M. Ikhwanuddin, A. Ambok Bolong, Nutritional effects on ginger (Zingiber officinal Roscoe) on immune response of Asian sea bass (Lates calcarifer) and disease resistance against Vibrio harveyi, Aquacultur 400e401 (2013) 46e52. [3] H.J. Suh, J.M. Lee, J.S. Cho, Y.S. Kim, S.H. Chung, Radical scavenging compounds in onion skin, Food Res. Int. 32 (1999) 659e664. [4] G. Griffiths, L. Trueman, T. Crowther, B. Thomas, B. Smith, Onions-a global benefit to health, Phytotherapy Res. 16 (2002) 603e615. [5] F.A. Ramos, Y. Takaishi, M. Shirotori, Y. Kawaguchi, K. Tsuchiya, H. Shibata, T. Higuti, T. Tadokoro, M. Takeuchi, Antibacterial and antioxidant activities of
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