Effects of prebiotic Immunogen on growth performance, intestinal bacteria colonization, and survival rate in Rutilus kutum fry

Effects of prebiotic Immunogen on growth performance, intestinal bacteria colonization, and survival rate in Rutilus kutum fry

A. Keramat Amirkolaie

Dept. of fisheries, Faculty of animal sciences and fisheries, Sari Agricultural Sciences and Natural Resources University of Sari, Sari, Iran

Email: amirkola@yahoo.com

(Received: Aug. 11.2014.   Accepted: Jan. 04.2015)

INTRODUCTION

The shortage of natural resources such as freshwater and land has led to the intensification of aqu+aculture system (Piedrahita 2003). However, increasing fish density may also increase the incidences of diseases in fish farms due to deteriorated water quality (Losordo et al. 1999).  Intensification can also increase stress level in farmed fish, which, in turn, may threat their immune system and exacerbate the incidences of bacterial infections (Liltved and Cripps 1999). Antibiotics have long been introduced as a solution to treat those infections (Burr et al. 2005), but long-term administration of antibiotics to treat bacterial diseases of farmed fish has widely been criticized because of bacterial resistance to antibiotics, elimination of gut microbial flora, high cost of these drugs, and potential side effects (Boyd and Massaaut 1999; Esiobu et al. 2002). The public’s concern about the negative impacts of antibiotics have sharply reduced their uses for aquaculture industry in the United States and Europe (Burr et al. 2005). Therefore, alternative techniques have been introduced to replace chemical drugs, among which the contribution of prebiotics may be significant. Prebiotics are potentially food supplements that decrease infectious adverse effects and increase feed efficiency by stimulating the growth of beneficial bacteria (Gibson and Roberfroid 1995). Prebiotics generally include nutrients such as non-digestible carbohydrate, resistant starch, nutrient fiber, sugars, some peptides, and proteins as well as some certain lipids that enter the intestine (Fooks and Gibson 2002). Several studies have demonstrated that prebiotics can improve growth parameters, disease resistance, villi surface area and microvilli length, and also modulate intestinal micro-biota in various aquatic animals (Genc et al. 2007; Li et al. 2007; Staykov et al. 2007; Zhou et al.  2007; Torrecillas et al. 2007; Burr et al. 2008; Salze et al. 2008). Moreover, dietary supplementation of polysaccharide prebiotic seems to reform bacterial community in fish intestine, which was found to improve health and feed efficacy in the host (Dimitroglou et al. 2009). Yet, there is still little information on beneficial bacteria and the by-products produced by dietary non-digestible carbohydrate. Immunogen as a commercial prebiotic contains various stimulating components such as mannan-oligosaccharide and β-glucans, which have been used as feed additives in various animals. Supplementation of Immunogen improved performance and disease resistance of Huso huso (Mohajer Esterabadi et al. 2010), common carp, Cyprinus carpio, (Ebrahimi et al. 2012), and reproductive performance of platy, Xiphophorus maculatus (Hajibeglou and Sudagar 2011).  R. kutum is one of the most commercially valuable fish species in the southern coast of the Caspian Sea (Abedi et al. 2012; Khara et al. 2012), which migrates from the sea to freshwater inlets (Heidari et al. 2009). Due to a sharp decline observed in the natural population, establishment of R. kutum restocking centers has been recognized as a solution to recover the natural stocks (Heyrati et al. 2006). At restocking centers, dietary prebiotics supplementation may improve fish growth, immune response, and survival rate. While some literature is available on the effects of prebiotic on fish performance and micro-flora characteristics, there is little information related to Immunogen on R. kutum. Hence, the main objective of the current study was to identify the effects of Immunogen on growth-related parameters and gut micro-flora in R. kutum fry.

MATERIALS AND METHODS

Experimental system and animal

This study was carried out using the experimental facility of Shahid Rajaee aquaculture complex located in the northern part of Iran. Fry R. kutum were bred using the reproduction facility of the complex and adapted to the experimental conditions a few days before the start of the experiment. Afterward, the fry with an average weight of 300 ± 1.7 mg were divided randomly into twelve 0.5 m3 tanks through individual counting with an initial stocking density of 500 fish per tank. The experiment lasted for 8 weeks. The prebiotic Immunogen used in this study is composed of mannan-oligosaccharide (18%) and β-glucans (1-3, 1-6)(30%) (Provided by Soroush Radian Co., Tehran, Iran). A basal diet was formulated using locally grown feed ingredients (Table 1). The inclusion of Immunogen into the basal diet at the rates of 0, 1, 1.5, and 2 g kg-1 was led to four experimental diets. 

Experimental procedure

Fish were weighed on the 1st and the last days of the experiment. The experimental diets were given to the fish at a rate of 10% of biomass (Takeuchi et al. 2002) three times per day (8.00, 13.00, and 18.00 hrs). The diets were randomly assigned to one of the 12 tanks, with three replications per diet. Randomization was done by numbering the diets and selecting three tanks randomly for each diet. Water quality parameters were monitored daily to ensure they were in appropriate range for the fish. The water temperature and pH ranged between 20-23 °C and 7.3-7.9, respectively, during the experiment. Oxygen concentration was measured in a randomly selected tank by a digital oxygen detector, which always remained above 6.1 mg/ l.On Day 56, all fish were weighed. Afterward, 30 fish were randomly selected from each tank and sacrificed using overdosed (400 mg l^-1) clove essence solution for measurement of body composition.

Table 1. The percentage of ingredients used in the basal diet on % dry matter weight basis.

Premix consisted of equal proportions of vitamins and minerals

Vitamin premix consisted of (g/kg premix):1200000 IU Vitamin A, 400000 IU Vitamin D3, 3000 IU Vitamin E, 1200 mg Vitamin K3, 5400 mg Vitamin C, 200 mg Vitamin B1, 3360 mg Vitamin B2, 7200 mg Vitamin B3, 9000 mg Vitamin B5, 2400 mg Vitamin B6, 600 mg Vitamin B9, 4 mg Vitamin B12, 500mg Antioxidant, up to 1kg carrier.

Mineral premix consisted of (g/kg premix): 2600 mg Mn, 600 mg Cu, 6000mg Fe, 4600mg Zn, 50mg Se, 100mg Iu, 50 mg Co, 100000 mg choline chloride, up to 1 kg carrier (composed of wheat bran).

Chemical analysis and bacteria count

Feed and fish body were analysed for dry matter through drying samples for 24 h at 103°C until constant weight (ISO 6496 1983). Ash content was determined by incineration in a muffle furnace for 4 h at 550°C (ISO 5984 1978). Crude protein (N×6.25) was measured applying the Kjeldahl method after acid digestion according to ISO 5983 (1979). Lipid was extracted by petroleum ether extraction in a Soxhlet apparatus (ISO 6492 1999).  At the  end  of  the  experiment, 10 fries were collected from each treatment and their intestine samples

were tested for bacterial counts. Prior to dissection and homogenization, the fry were rinsed with sterilized distilled water, cleaned with ethanol (70.0%) and then washed again with sterilized distilled water to eliminate all exterior bacteria. The intestine samples were dissected out in sterile conditions. For microbiological analysis, three samples from the middle part of the intestine were taken.

All samples were diluted using sterilized normal saline solution (0.85% NaCl w/v) and then placed into nutrient agar plates for bacterial counts (Ebrahimi et al. 2012).

Fish performance

Weight gain was determined by the difference between initial and final body weights (Amirkolaie et al. 2005). Feed conversion ratio (FCR) was calculated per tank from feed intake data and weight gains: FCR = feed consumed (g) / wet body weight gain (g) (Amirkolaie et al. 2005). Specific growth rate (SGR) was calculated as follows and expressed as a percentage:

SGR = 100 (Ln W_final- Ln W_initial) × Days ^-1 (Amirkolaie et al. 2005). The calculations were based on the dry weight of the diets.

Statistical analysis

Data are presented as means of each treatment with standard deviations. All data were verified for normality (Kolmogorov-Smirnov test) after ArcSine transformation. One-way ANOVA was used to determine the effects of Immunogen on fish performance and bacteria community. Tukey's test was used to compare differences between the means. For all statistical analyses, each tank was considered as the experimental unit.

RESULTS

Data on the growth performance of R. kutum are presented in Table 2. R. kutum fry significantly gained higher weight with the administration of 1 g/kg of Immunogen (p<0.05). Along with the growth, FCR and SGR were also significantly improved in R. kutum fed the prebiotic diets in comparison to control diet (p<0.05).

Nevertheless, growth-related parameters were similar to control diet in fish fed prebiotic levels of 1.5 and 2 g kg-¹.

Similar to growth parameters, fish displayed higher survival rates with 1 g kg^-1 prebiotic in comparison to the other treatments (p<0.05). The body composition results demonstrated that the inclusion of dietary prebiotic did not affect the carcass composition of R. kutum (Table 3; p>0.05). Bacterial counts were also affected by the prebiotic inclusion (Table 4). Total count of bacteria together with Gram positive bacteria count were increased by the addition of Immunogen (1 g kg^-1);  but, bacterial counts in fish fed prebiotic levels of 1.5 and 2 g/kg were similar to those in control diet (p<0.05).

Table 2. Growth performance in R. kutum fry feeding on different levels of Immunogen (IMU) over 56 days of experimental period. All values are means ± standard deviation of triplicate tanks/treatment (N=3).

Different superscript letters in the same row show significant differences (P<0.05).

Table 3. Body composition in R. kutum feeding on different levels of Immunogen (IMU) over 56 days of experimental period. All values are means ± standard deviation of triplicate tanks/treatment (N=3).

Table 4. Total counts of bacteria in the intestine of R. kutum feeding on different levels of Immunogen over 56 days of experimental period. All values are means ± standard deviation of triplicate tanks/treatment (N=3).

Different super script letters in the same row show significant differences (P<0.05).