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Jan 1, 2014 - 0.6 g /kg diet of ginseng extract, the 4th group was fed a basal diet mixed with ... American Ginseng (Panax quinquefolius L.) are the two most ...

Vol 4 (1) Jan’14

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Immunomodulatory and Growth Performance Effects of Ginseng Extracts as a Natural Growth Promoter in Comparison with Oxytetracycline in the Diets of Nile Tilapia (Oreochromis niloticus) Sabry A.A. El-Sayeda,b* , Sarah Y.A. Ahmed c, d, Neveen R. Abdel-Hamidc Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, P.O. Box 44511, Zagazig, Egypt. b

Department of Clinical Nutrition, College of Applied Medical Sciences, University of Hail, Hail 2440 Saudi Arabia. c Bacteriology, Mycology and Immunology Dept., Faculty of Veterinary Medicine, Zagazig University, P.O. Box 44511, Zagazig, Egypt. d Department of Clinical Laboratory Sciences, College of Applied Medical Science, University of Hail, Hail 2440 Saudi Arabia. *Corresponding Author: [email protected] Rec. Date: Jan 01, 2014 00:19, Accepted Date: Jan 09, 2014 08:43

Abstract Ginseng is a traditional herbal medicine, used as an immunomodulator producing several cytokines which stimulate lymphoid cells to proliferate. Using of oxytetracycline in fish diets as a growth promoter make immunosuppression to both cellular and humoral immunity. This study was aimed to study the immunomodulatory role of Ginseng extracts in Nile tilapia (Oreochromis niloticus) and provide a natural alternative to the use of oxytetracycline as growth promoter. Two hundred Nile tilapia (Oreochromis niloticus) were randomly distributed into equal 4 groups, each one contained 50 fish (25 fish / replicate). The 1st group (control group) was fed with a basal diet (32.16% protein) without any additives, the 2nd group was fed with a basal diet mixed with 1g /kg diet oxytetracycline, the 3rd group was fed with a basal diet mixed with 0.6 g /kg diet of ginseng extract, the 4th group was fed a basal diet mixed with 0.6 gm/kg diet ginseng extract plus 1g /kg diet oxytetracycline. At the end of the feeding experiment, the growth performance and different immunological parameters of experimental fish were assessed. Fish was challenged with pathogenic Aeromonas hydrophila to evaluate the effect of the used additives on its ability to withstand the infection. The results revealed that the growth performance and different immunological parameters were significantly higher in all fish groups that received ginseng extracts than the other groups. So, addition of Ginseng extracts to the Nile tilapia diets can be used as immunostimulants and served as a natural alternative growth promoter for oxytetracycline.

Keywords: Immunomodulator, Oxytetracycline, Oreochromis Niloticus.

Ginseng

extracts,

Growth

promoter,

Introduction

added to fish feed, provide proteins, lipids, carbohydrates and energy; they were also used for the purpose of disease treatment and immune enhancement. Asian Ginseng (Panax Ginseng) and [email protected]/index.php/ijlr

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replace antibiotic growth promoters (Hashemi and Davoodi, 2011). When plant materials were

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Herbal plants serve as a new class of growth promoters that provide an alternative feeding strategy to

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Vol 4 (1) Jan’14

American Ginseng (Panax quinquefolius L.) are the two most recognized Ginsengs (Yuan et al., 2010).The main active agents in Panax Ginseng are ginsenosides,which are triterpene saponins (Kiefer and Pantuso, 2003). The active ingredients of Ginseng are ginsenosides, mono and polysaccharides (Tan and Vanitha, 2004). Ginsenosids are generally considered the main active component of ginseng roots that are used for medicinal purposes (Dennis and Micheal, 2008). Ginseng saponins called ginsenosides are the major active constituents in both ginsengs (Yuan et al., 2010). The steroidal saponins (ginsengsoides) enhance both B and T-cell mediated immune responses (Tan and Vanitha, 2004). Pretreatment with the ginseng extract (ginsana) led to an increase in the number of antibody forming cells, as well as of the titers of circulating antibodies (Singh et al., 1984). Also modulates the cell mediated immune response, it is also significantly elevate natural killer (NK) cell activity and the production of interferon when compared with control animals. Immunomodulatory effect of Ginseng is enhanced lymphocyte proliferation only in the mitogen stimulation assay (Wilasrusmee et al., 2002).There are significance increase in the immune response which induced by dietary supplementation of 2% panax Ginseng on Nile tilapia (Oreochromis niloticus) for 84 days (Nakagawa et al., 2007), as well as increase in the average body weight, complement activity, bactericidal activity against Escherichia coli, lysozyme activity and adherent phagocyte activity. Antibiotics have been used to prevent diseases and to improve feed efficacy for long time, but there are many problems associated with the use of antibiotics in aquaculture (Subasinghe , 1997). Interaction of antibiotics with the immune system may result in immunosuppression (Salauze et al., 1994). Oxytetracycline interfere with normal immunological processes in fish and has a suppressive effect on specific and non-specific immune system parameters such as leucocyte counts, nitroblue tetrazolium activity, total plasma protein and immunoglobulin levels, and phagocytic activity (Yonar et al.,2011). Material and Methods Expremintal fish Two hundred Nile tilapia (Oreochromis niloticus) with an average body weight of 41.57±0.3 gm were kept in two concrete pond (3 X 1 X 1 m) for two weeks to be acclimatized before the start

Each 100 g of Muv-Oxytetracycline contains Oxytetracycline Hydrochloride 20 gm (Equivalent to18.53 Oxytetracycline base). [email protected]/index.php/ijlr

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Muv-Oxytetracycline (muvco)

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of the experiment.

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Vol 4 (1) Jan’14

Ginseng extraxt Ginseng capsules (Pharco pharmaceuticals), each capsule contains 100 mg Ginseng root extract that contains ginsenosides (saponin glucosides). Experimental fish design A total number of 200 Nile tilapia were randomly distributed into equal 4 groups, each one contain 50 fish (25 fish / replicate), and all were fed a basal diet that contain (32.16%) protein .The 1st group was fed a basal diet without any additives and served as control group, 1g /kg diet oxytetracycline to the 2nd group, 0.6 g /kg diet Ginseng extract to the 3rd group, 0.6 g /kg diet Ginseng extract plus 1g /kg diet oxytetracycline to the 4th group, The experiment was extended for 3 months in the summer season(June-September). Feeding formulation A balanced dietary ration formulation was prepared as mentioned by NRC (1993) (Table 1). Table 1. Diet Composition. Ingredients Yellow corn Wheat flour

Composition (%) 31 10

Soybean meal, (44%) 22 Fish meal, (60%) 16 Poultry by-products 14 Vegetable oils 5.5 Vitamins and mineral mixture* 1.5 Total 100 DE, Kcal / Kg diet 3123 CP% 32.16 *Vitamins and mineral mixture (Amcozink): Each kg contains 20000000 I.U vit. A , 2000000 I.U vit. D3 ,10g vit.E,500 g zink bacitracin and calcium carbonate up to 1000g. Feeding of fish The amount of feed (on dry matter basis) delivered per day was adjusted at the beginning and

dispersed by hand to avoid the loss of feed and its accumulation in the bottom of the pond, and so the amount of the given diets was also the amount of intake. [email protected]/index.php/ijlr

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respective diets three times daily (at 10.00 h a.m., 12.00 h p.m and 2.00 h p.m), the feed was

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after one month of the experiment as (3% of body wt.) (Eliott, 1975). The fish was fed their

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Vol 4 (1) Jan’14

Measurement of survival and growth performance parameters Average body weight Fish of each group was weighed after one and two month of the feeding experiment. It was calculated according to (Siddiqui et al., 1988) as follows: Average body weight = the total weight of fish / the number of fish in each group. Weight gain % It was calculated according to (Jauncey and Ross,1982) as follows: Weight gain % = Final average body weight – Initial average body weight/ Initial average body weight x100. Specific growth rate (SGR) It was calculated according to (Sveier et al.,2000) as: Specific growth rate (SGR) = 100 (ln W2 – ln W1) / T Where W1 and W2 are the initial and final fish weight, respectively, and T was the number of days in the feeding period. Evaluation of feed utilization Feed intake Diet was provided regularly daily as a percent of body weight and feed intake was calculated as the total weight diet offered in a given period divided by the number of survival fish. Feed conversion ratio (F.C.R.) was calculated according to (Sveier et al., 2000) as follows: FCR = (dry feed intake by gm) / (live weight gain by gm) Condition factor (CF) Calculated according to (Gjedrem and Gunnes,1978) as follows: CF = (body weight by gm) / (total length cm)3 x 100. Survival rate Survival % = (No. of fish counted) / (No. of stocked fish) x100. Blood and serum samples Samples were collected from 6 fish which were randomly selected from each replica, samples

Evaluation of Non specific immune parameters Total leukocytic count according to the method described by Stoskopf (1993). [email protected]/index.php/ijlr

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collected after one and two months of the feeding experiment.

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collected from caudal vein after fish anathestheia by using benzocaine 50 mg/L. Samples were

Vol 4 (1) Jan’14

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Nitroblue tetrazolium activity (NBT) (Studnicka et al., 1985). Lysozyme activity(Iwama and Nakanishi, 1996). Serum bactericidal activity (SBA) It examines the ability of the fish's serum that fed some plant extracts and oxytetracycline with its diet to kill the pathogenic organisms (A.hydrophila) (Maqsood et al., 2010). Determination of total globulin The total serum protein concentration was estimated by the method of (Lim et al., 2009) Evaluatoin of Specific immune parameters Determination of IgM The quantitative determination of IgM was made by using nephelometry technique in which the immune complex formed by IgM that present in the serum sample with specific antiserum to it. These complexes scattered a beam of light passed through the sample. The amount or intensity of scatter was determined according to (Murray et al. 2009). Lymphocyte transformation test Lymphocyte transformation index was determined according to (Barta,1984) as following: Separation of lymphocytes By using of lymphocyte separation medium ficoll hypaque sterile , the separated lymphocytes were suspended in 1 ml of RPMI 1640 medium containing 10% foetal bovine serum (RPMI-10). Viability of lymphocytes It was determined by staining of lymphocytes by using 0.4% trypan blue according to (Hanks and Wallace,1985). Standardization of the lymphocytes concentration The required final concentration could be adjusted to reach 2x106 lymphocytic cell/ml by adding RPMI-1640 medium with 10% foetal bovine serum (RPMI-10) (Chi et al,1984). Preparation

and

standardization

of

mitogenic

solution

(non

specific

mitogen

phytohaemagglutinin (PHA) Glucose consumption assay

Evaluation of the lymphocyte transformation test: The extent of lymphocyte transformation rate was calculated as follows: [email protected]/index.php/ijlr

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glucose in the supernatant of tissue culture medium (Decock et al. 1980).

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Evaluation of the lymphocyte blastogenesis was estimated by determination of the residual

Vol 4 (1) Jan’14

International Journal of Livestock Research ISSN 2277-1964 ONLINE

Glucose concentration in the tested sample (C): OD of the medium C=

OD of the standard

x 100

C1 – C3 Stimulation index by PHA (SI) = While

C1 – C2

C1 = concentration of glucose in medium C2 = concentration of glucose in control non stimulated cells C3 = concentration of glucose in stimulated cells with PHA Challenge test At the end of the feeding experiment 20 fish from each group(10 fish/replicate) were transferred 8

-1

to glass aquaria , then were inoculated with 0.5 ml culture suspension (1x10 bacteria ml ) of pathogenic A.hydrophila via intraperitoneal route. The challenged fish were observed for 7 days in order to record the mortalities (Ruangroupan et al. 1986). Statistical analysis Data were statistically analyzed using the Analysis Of Variance (ANOVA) and Duncan multiple range test to determine differences between treatments and standard errors of treatment means. Differences were considered significant when (P

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