Growth Composition And Indices In Great Sturgeon Biology Essay

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Growth performance, carcass composition and immunophysiological indices in juvenile great sturgeon (Huso huso) fed prebiotic Immunoster>(IS) were investigated. After a 4-week acclimatization period, 270 beluga juveniles weighing 95.68 >10.05 g were randomly distributed into 9 fiberglass tanks (2 >2 >0.53 m) in three replicates and kept at a density of 30 fish per tank for a period of 8 weeks at water temperature 20.55 >5.11 >C and dissolved oxygen 6.73 >0.35 mg/l. IS was added at two levels of 1% and 3% to the basal diet in place of cellulose, except the control. At the end of trial, blood sampling and carcass analysis were conducted. Final weight, final length, BWI, SGR, FCR, PER and CF were affected significantly by IS at level of 3% compared with IS 1% group and the control group (P<0.05). HSI was insignificantly higher in fish fed IS 3% (P>0.05). No mortality was observed. There was significant difference (P<0.05) in crude protein of carcass composition between the experimental groups. MCV and MCH showed significantly higher levels in both experimental treatments compared with the control (P<0.05). IgM level and lysozyme activity in fish fed IS 1% were insignificantly (P>0.05) higher than the control group. Based on obtained results, it can be declared that IS can enhance growth performance and improve some immunophysiological indices of great sturgeon.

Keywords: Huso huso, Prebiotic, Immunoster, Growth Performance, Immunophysiological indices

* Corresponding author's email: taati919@yahoo.com

Introduction

Among sturgeons of the southern Caspian sea, beluga, Huso huso is a good candidate for aquaculture because of its market price, fast growth, reproduction in captivity and accelerating decline as a result of overfishing. The sharp decline of natural beluga populations that has been observed in the past decades, has promoted several countries to initiate juvenile production programmes for re-stocking and for caviar and meat production purposes (Chebanov & Billard, 2001).

A prebiotic is a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and /or activity of one or a limited number of bacteria in the colon that can improve host health (Gibson & Roberfroid, 1995). There is an increased attention on the use of prebiotics as a key strategy to maintain and expand the aquaculture industries in recent years (Genc et al., 2007a).

Mannan oligosaccharides (MOS) are complex carbohydrates derived from yeast cell walls. These compounds contain mannose as the primary carbohydrate element. MOS has a variety of beneficial effects on livestock ranging from growth promotion in cattle to immunostimulation in swine and avian species (Moran, 2004).

Immunostimulants are biological extracts and synthetic chemical which stimulate the immune response by promoting phagocytic cell function, increasing their bacterial activity and /or non-specific cytotoxic cells and antibody production (Sakai, 1999).

>-glucans are polymers of glucose found in the cell walls of plants, fungi and bacteria, which have been shown to have immunostimulatory activities in fish (Anderson, 1996).

Immunoster>(IS) is a prebiotic and an immunostimulant derived from the cell wall of a single source of brewers yeast, Saccharomyces cerevisiae. This substance contains MOS and >-glucans.

The effects of MOS on the growth performance, hematological parameters and immune responses have been studied in several aquatic species including gulf sturgeon, Acipenser oxyrinchus (Pryor et al., 2003), rainbow trout, Oncorhynchus mykiss (Yilmaz et al., 2007), tiger shrimp, Penaeus semisulcatus (Genc et al., 2007a), hybrid tilapia, Oreochromis niloticus >O. aureus (Genc et al., 2007b), European sea bass, Dicentrachus labrax (Torrecillas et al., 2007), channel catfish, Ictalurus punctatus (Welker et al., 2007), rainbow trout, O. mykiss (Staykov et al., 2007), cobia, Rachycentron canadum (Salze et al., 2008), red drum, Sciaenops ocellatus (Burr et al., 2008), Nile tilapia, Oreochromis niloticus (Sado et al., 2008), Atlantic salmon, Salmo salar (Grisdale-Helland et al., 2008), rohu, Labeo rohita (Andrews et al., 2009), western king prawn, Penaeus latisulcatus (Van Hai & Fotedar, 2009) and European sea bass, D. labrax (Torrecillas et al., 2010).

Although the above cited scientists have studied the effects of MOS in different species, data about the effect of MOS on sturgeons is very rare. Hence, the objective of the present study is to evaluate the influence of IS on the growth performance, carcass composition and immunophysiological indices of Huso huso.

Materials and methods

Experimental design

The study was conducted in Shahid Dr. Beheshti Sturgeon Fish Propagation and Rearing Center, Rasht, Iran and Dr. Dadman International Sturgeon Research Institute, Rasht, Iran. Prior to the feeding trials, fish were fed the basal diet to apparent satiation four times per day for a 4-week acclimatization period. Then, 270 beluga juveniles with mean body weight of 95.68 >10.05 g were randomly allocated into 9 fiberglass tanks (2 >2 >0.53 m) and kept at a density of 30 fish per tank, with three replicates per treatment (completely randomized design). The tanks were equipped with aeration through air stone connected to a central air compressor. All groups were fed their respective diets four times daily (08.00, 14.00, 20.00 and 02.00) at the same fixed rate (initially 4% of body weight per day and gradually reduced to 2%). Water of the tanks (using water from the Sefidroud river) replaced every 12 h to prevent accumulation of faeces and uneaten food. All tanks were kept under natural photoperiod, 11 h L-13 h D. The feeding trial was conducted for 8 weeks. During the trial, mean water temperature, mean oxygen concentration and mean pH value were 20.55 >5.11 >C, 6.73 >0.35 mg/l and 7.92 >0.09, respectively.

Experimental diets

The ingredients of the experimental diets (based on the formulation of Dr. Dadman International Sturgeon Research Institute, Rasht, Iran) are presented in Table 1. IS was supplied by Awill company, Victoria, Australia. IS was added at two levels of 1% and 3% to the basal diet in place of cellulose, except the control. All dry ingredients were thoroughly mixed for 30 min in a food mixer. Then, liquid ingredients were added in the diets and ingredients were mixed again for 20 min. This was placed into a commercial meat grinder for through mixing and extruded through a 4 mm diameter strand and dried in a drier at 30 >C for 24 h. The pellets were packed in sterile bags and sealed and stored at -15 >C until used.

Table 1 Ingredients of the experimental diets

Ingredients (%) Control IS 1% IS 3%

Kilka fish meal 42 42 42

Meat meal 9 9 9

Soybean meal 19.5 19.5 19.5

Wheat flour 11 11 11

Sunflower oil 9 9 9

Molasses 1.5 1.5 1.5

Lecithin 0.2 0.2 0.2

L-Methionine 0.5 0.5 0.5

L-carnitine 0.1 0.1 0.1

Salt 1.5 1.5 1.5

Vitamin C 0.1 0.1 0.1

Vitamin E 0.1 0.1 0.1

Cellulose 3 2 0

Vitamin premix* 1.5 1.5 1.5

Mineral premix** 1 1 1

Immunoster 0 1 3

* Vitamin premix (g/100 g vitamin premix except vitamins A and D3): A, 160000 IU; D3, 40000 IU; E, 4; K3, 0.2; B1, 0.6; B2, 0.8; B3, 1.2; B5, 4; B6, 0.4; B9, 0.2; B12, 0.8; H2, 0.02; C, 6; Inositol, 2; BHT (butylated hydroxyl toluene), 2.

** Mineral premix (g/100 g mineral premix): Fe, 2.6; Zn, 1.25; Se, 0.2; Co, 0.048; Cu, 0.42; Mn, 1.58; I, 0.1; Cholin chloride, 1.2.

Proximate composition of diets

Analysis of the experimental diets is showed in Table 2. Proximate analysis of the diets was conducted according to the standard methods of the Association of Official Analytical Chemists (1995) in the laboratory of veterinary organization, Rasht, Iran. Moisture content was estimated by drying the samples to constant weight at 105 >C in an oven (Memmert, Germany), crude protein content (N>6.25) was measured using a Kjeldahl system (Buchi, Switzerland), crude lipid content was determined by a Soxhlet system (Buchi, Switzerland), ash content was measured by weight after incinerating at 550 >C for 6h in a hotspot furnace (Gallenkamp, England). In order to determine the energy content, bomb calorimeter (Parr, USA) was utilized.

Table 2 Analyzed proximate composition of experimental diets

Ingredients Control IS 1% IS 3%

(% dry weight)

Dry matter 93.9 94.1 93.8

Crude protein 42 41.3 42.2

Crude lipid 15 15.2 14.8

Fibre 2.4 2.2 2.3

Ash 10.1 10.2 10.1

NFE* 30.5 31.1 30.6

Gross energy (MJ/kg) 14.65 14.69 14.62

* NFE, nitrogen free extract = 100- (Protein + Lipid + Fibre + Ash).

Growth performance

All biometric data were taken only after feeding had been ceased for 24h. Following these bi-weekly inventories feed rates were adjusted to reflect the new biomass gain in each tank. The growth performance of juveniles such as body weight increase (BWI), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), condition factor (CF), hepatosomatic index (HSI) and survival rate were calculated based on the standard formulae: BWI = (final body weight- initial body weight) >100/ initial body weight, SGR = (ln final weight- ln initial weight) >100/days, FCR = dry feed fed/ body weight gain, PER = weight gain / protein intake, CF = (body weight / body length3) >100, HSI = (liver weight / body weight) >100 and survival rate = (final number of fish / initial number of fish) >100. (Hung et al., 1993, 1997; Luo et al., 2010).

Sample collection and analysis

At the end of trial, six fish per treatment (two fish per replicate) were randomly selected and carcass analysis was carried out. Proximate analysis of carcass was performed according to AOAC (1995). Livers were excised and weighed in order to calculate HSI.

At the end of trial, in order to study immunophysiological indices, nine fish per treatment (three fish per replicate) were randomly captured and blood samples were collected using a 2>mL syringe from the caudal vein. The extracted blood was divided in two sets of Eppendorf tubes. One set contained heparin for hematology studies and the other one (non-heparinized) was centrifuged at 3000 rpm for 10 min in order to measure biochemical and immune indices. All sera were stored at -80 >C until analyzed. Before the blood samplings, all fish were starved for 24 h.

Hematocrit (Hct) values were determined using method described by Snieszko (1960). The amount of hemoglobin (Hb) was measured according to cyanmethemoglobin procedure suggested by Houston (1990). The number of red blood cell (RBC) and white blood cell (WBC) were enumerated in an improved Neubauer hemocytometer according to Klontz (1994). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated as described by Klontz (1994). In order to the differential leucocyte count, blood smears were prepared, air-dried, fixed in methanol, and stained using Giemsa (Merck, Germany). Leucocytes in blood smears were categorized into lymphocytes, neutrophils, eosinophils and monocytes (Klontz, 1994).

Total serum protein was evaluated using the Biuret reaction (Doumas et al., 1981). Albumin was measured using the bromocresol green binding method (Doumas et al., 1971). In order to assess osmolarity, a digital freezing osmometer (Roebling, Germany) was utilized. Ca2+ and Mg2+ values were determined using colorimetric method using an autoanalyzer (Technicon RA-1000, USA) according to (Kazemi et al., 2006). Na+ and K+ concentrations were measured by means of flame photometer (Jenway, England). IgM content was estimated according to the method described by Siwicki & Anderson (1993). Also, lysozyme levels were determined based on the method of Ellis (1990).

Statistical analysis

Levene's test was used to determine the homogeneity of variance. The mean values of all parameters were subjected to one-way analysis of variance ANOVA, and comparisons among treatment means were made by Tukey's test as a post-hoc test using SPSS software (version 17). Statistical significance was accepted at the P<0.05 level. All data in the text are presented as mean >SD.

Results

Growth indices are summarized in Table 3. Fish fed the IS 1% and 3% had better growth performance during the 8-week feeding trial. Final weight, final length, BWI, SGR, FCR, PER and CF were significantly affected by IS at level of 3% compared to IS 1% group and the control group (P<0.05). HSI was insignificantly higher in fish fed IS 3% (P>0.05). Survival rate was 100% in all experimental treatments.

Table 3 Growth performance of Huso huso in the 8-week feeding trial

Growth indices Control IS 1% IS 3%

Initial weight (g) 95.08 >10.30a 96.32 >9.82a 95.65 >10.11a

Final weight (g) 290.28 >58.23a 306.51 >58.18a 354.17 >57.28b

Initial length (cm) 30.84 >1.09a 30.96 >0.89a 30.85 >1.11a

Final length (cm) 42.26 >2.57a 42.67 >2.20a 44.52 >2.34b

BWI (%) 207.15 >13.85a 219.52 >19.78a 271.08 >10.68b

SGR (% day-1) 1.99 >0.87a 2.06 >0.11a 2.34 >0.51b

FCR 1.70 >0.10b 1.58 >0.11b 1.30 >0.49a

PER 1.39 >0.08a 1.52 >0.10a 1.84 >0.06b

CF 0.38 >0.005a 0.39 >0.005ab 0.41 >0.010b

HSI (%) 3.59 >0.37a 3.38 >0.35a 3.66 >0.38a

Survival (%) 100 100 100

Means in the same row with different superscripts are significantly different (P<0.05).

Whole carcass composition is presented in Table 4. There was significant difference in crude protein of carcass composition among the experimental groups (P<0.05). Crude protein of fish that fed IS 1% and 3% was significantly (P<0.05) higher than the fish fed the control diet. Lipid, moisture and ash content of whole body were not significant (P>0.05).

Table 4 Carcass composition of Huso huso in the 8-week feeding trial

Ingredients (%) Control IS 1% IS 3%

Crude protein 14.69 >0.61a 14.91 >0.68ab 15.52 >0.20b

Crude lipid 9.20 >1.04a 9.74 >1.96a 9.44 >1.35a

Ash 1.06 >0.04a 1.01 >0.04a 1.00 >0.04a

Moisture 73.84 >1.46a 73.48 >1.80a 73.06 >1.47a

Means in the same row with different superscripts are significantly different (P<0.05).

Samples were analyzed in duplicate.

Table 5 shows the levels of hematological indices of beluga juveniles at the end of trial. MCV and MCH were significantly higher in fish fed IS 1% and 3% compared with the control (P<0.05). There was an insignificant increase in Hct, WBC and neutrophil in fish fed the IS 1% and 3% (P>0.05). Significant differences (P<0.05) were observed in lymphocyte and eosinophil among the treatments.

Table 5 Hematological indices of Huso huso in the 8-week feeding trial

Hematological indices Control IS 1% IS 3%

Hct (%) 23.00 >1.73a 24.55 >2.00a 24.55 >3.20a

Hb (g dL-1) 5.35 >0.61a 5.27 >0.76a 5.57 >0.62a

RBC (>106 mm-3) 0.79 >0.08a 0.72 >0.10a 0.69 >0.14a

WBC (>103 mm-3) 64.05 >15.58a 66.66 >15.08a 70.66 >16.63a

MCV (Fl) 292.27 >22.05a 342.22 >51.62ab 364.68 >56.83b

MCH (pg) 67.83 >5.77a 73.13 >11.67ab 84.01 >19.48b

MCHC (%) 23.20 >1.37a 21.38 >1.89a 22.82 >2.58a

Lymphocyte (%) 47.44 >8.29b 46.00 >4.84ab 37.33 >11.22a

Neutrophil (%) 22.67 >6.81a 28.77 >6.26a 25.55 >8.07a

Eosinophil (%) 26.00 >5.36ab 22.22 >6.18a 35.66 >14.39b

Monocyte (%) 3.89 >1.90b 2.77 >2.22ab 1.44 >1.13a

Means in the same row with different superscripts are significantly different (P<0.05).

Differential counts were conducted in duplicate.

Biochemical indices are given in Table 6. Concentration of total protein in fish fed IS 1% and 3% was insignificantly higher than the control group (P>0.05).

Table 6 Biochemical indices of Huso huso in the 8-week feeding trial

Biochemical indices Control IS 1% IS 3%

Total protein (g dL-1) 1.50 >0.10a 1.56 >0.16a 1.51 >0.24a

Albumin (g dL-1) 0.60 >0.03a 0.62 >0.03a 0.59 >0.10a

Osmolarity (mOsmo L-1) 314.88 >10.55a 314.66 >9.24a 318.55 >7.71a

Na+ (meq L-1) 131.00 >2.06a 131.66 >2.29a 130.00 >2.95a

K+ (meq L-1) 1.96 >0.35a 2.04 >0.24a 2.12 >0.28a

Ca2+ (mg dL-1) 5.09 >0.60a 5.20 >0.53a 4.96 >0.95a

Mg2+ (meq L-1) 1.22 >0.21a 1.15 >0.17a 1.14 >0.20a

Means in the same row with the same superscript indicate no significant difference (P>0.05)

Table 7 shows immune indices of Huso huso. IgM concentration and lysozyme level in diet containing IS 1% were insignificantly higher than the control (P>0.05)

Table 7 Immune indices of Huso huso in the 8-week feeding trial

Immune indices Control IS 1% IS 3%

IgM (mg dL-1) 10.13 >4.65a 11.41 >3.46a 9.71 >4.84a

Lysozyme (>g mL-1) 0.38 >0.78a 1.81 >3.65a 0.45 >1.37a

Means in the same row with the same superscript indicate no significant difference (P>0.05)

Discussion

Results indicate that IS significantly improved growth indices such as final weight, final length, BWI, SGR, FCR, PER and CF. Survival rate was 100% in all experimental groups. These results are consistent with following similar studies: Staykov et al. (2007) declared that a supplement of 0.2% MOS in rainbow trout, Oncorhynchus mykiss extruded diets significantly increased growth and reduced the FCR and overall mortality in the treated groups. Torrecillas et al. (2007) reported that European sea bass, Dicentrachus labrax, fed MOS at 2>and 4>showed a significant growth improvement. They also observed a positive correlation between the dietary MOS inclusion levels and feed intake. In rainbow trout, O. mykiss and hybrid tilapia, Oreochromis niloticus >O. aureus the body protein concentration has been reported to increase as the level of 1 g/kg MOS was increased in the diet from 1.5 to 4.5 g/kg (Yilmaz et al., 2007; Genc et al., 2007b). Enrichment of live feeds such as rotifers and Artemia with 0.2% MOS was caused an overall greater ability to withstand hyposaline stress in larval cobia, Rachycentron canadum (Salze et al., 2008). In another study, Andrews et al. (2009) evaluated a diet supplemented with 1%, 2% and 4% MOS improved the weight gain, SGR and FCR in rohu, Labeo rohita fingerlings but the growth performance tended to decrease as dietary supplementation of immunostimulants increased to 2% or higher. In another report, dietary inclusion of MOS at 4 and 6 g/kg markedly improved feed utilization in juvenile sea bass, D. labrax (Torrecillas et al., 2010).

In contrast, Pryor et al. (2003) observed no differences in CF, SGR and FCR between control and 3 g/kg MOS supplemented groups in gulf sturgeon, Acipenser oxyrinchus. Grisdale-Helland et al. (2008) demonstrated that supplementing the diet with 10 g/kg MOS resulted in a decrease in the protein concentration in the body of Salmo salar. They also mentioned there were no significant effects of supplementing the diet with MOS on digestibility, feed intake or growth of S. salar.

The use of MOS as prebiotic to improve growth performance in fish still needs further research for better explanation of contradictory results. It may be because of the different basal diet, inclusion levels, animal characteristics (species and age), period of trial and circumstances of culture. According to Newman (2007), the complexity of carbohydrate structure in yeast's cell wall, yeast's different strains, fermentation and processing methods can all alter their function.

IS is considered as an immunostimulant for its potential (having >-1,3 glucans). The use of natural immunostimulants is a promising area in aquaculture because they are biodegradable, biocompatible and safe both for the environment and human health. Consequently, several substances, including vitamins, chitin, glucans and different animals and plants components, as well as yeast cells have been tested as immunostimulants in fish (Sakai, 1999; Ortuno et al., 2002).

The analysis of blood indices is a valuable guide in assessing the condition of aquatic organisms. For example, in response to stress, pollutants and nutrition as well as ecological and physiological conditions (Bahmani et al., 2001). Leucocytes are one of the main parts of the cellular immunity system and fluctuation of them is increasingly used as indicators of stress response in fish (Stoskopf, 1993). In the current study, the WBC count was higher in the IS 3% but no significant difference was seen. The increase WBC count in yeast-supplemented groups is due to >-1,3 glucan, which can recognize a specific receptor on WBCs (Andrews et al., 2009). >-glucans are polymers of glucose found in the cell walls of plants, fungi and bacteria, which have been shown to have immunostimulatory activities in fish (Anderson, 1996). In fish, glucan receptors have also been reported to exist on macrophages (Engstad & Robertsen, 1993; Ainsworth, 1994). When the receptor is engaged by glucans, the cells become more active and they secrete signal molecules (cytokines) that stimulate the formation of new WBCs (Raa, 1996). Andrews et al. (2009) observed that the leucocyte count was higher in L. rohita which treated with MOS at 1%, 2% and 4%. WBC counts in channel catfish, Ictalurus punctatus fed Bio-MOS>were higher compared to the control but no significant difference was recorded (Welker et al., 2007).

The increase in serum protein and albumin levels is considered to be associated with a stronger innate response in fish (Wiegertjes et al., 1996). Total serum protein values in fish fed IS 1% and 3% were higher than the control but no notable difference was found (P> 0.05). Albumin value was high in IS 1%.

Immunization of sturgeons against pathogenic micro-organisms has not been developed as it has for other cultured species such as cyprinids and salmonids (Khoshbavar-Rostami et al., 2007). Immunoglobulin M (IgM) is a major component of humoral immune system. Four immunostimulants such as vitamin A, chitin, yeast cells and levamisole administered in the diet to the sea bream, Sparus aurata specimens increased total seric IgM levels (Cuesta et al., 2004). In current study, IS 1% increased IgM levels in beluga juveniles although it was not significant.

Administration of IS 1% resulted an increase in serum lysozyme which can contribute to the enhancement in the non-specific immunity. Staykov et al. (2007) reported that O. mykiss treated dietary supplementation of MOS at 0.2% showed a significant difference in lysozyme level. However, lysozyme activity was lower in S. salar fed diets containing MOS compared with those fed the control diet (Grisdale-Helland et al., 2008).

In the present study, the effects of IS did not show significant differences in some hematological and biochemical parameters of H. huso because there were no physical, chemical or bacterial stresses. Also, the trial period was short to show more stimulation of immune responses. Similar to the current results, Sado et al. (2008) observed no significant effects of dietary MOS supplementation at 0.2%, 0.4%, 0.6% 0.8% and 1% on hematological parameters (RBC, Hb, Hct, WBC, MCV, MCHC, MCH and total protein) in juvenile Nile tilapia, Oreochromis niloticus. Furthermore, any difference in the hematology variables such as RBC, Hb, Hct and WBC in I. punctatus fed MOS-supplemented diet at 2 g/kg was not found (Welker et al., 2007). It had been documented that different factors are effective on the hematological and biochemical parameters of fishes, from which the species, environmental conditions, age, maturation and nutrition are very important (Ross & Ross, 1999).

In conclusion, the results of this trial indicate that IS can enhance growth performance and affect some immunophysiological variables of H. huso. Further research is needed to clarify the action mechanisms of MOS, as well as the appropriate inclusion dose and feeding period in great sturgeon.

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