The common carp, Cyprinus carpio, is one of the most important commercial fish in Cyprinidae^[1]. Due to the fact that about 50 to 60 percent of the culture costs are dedicated to nutrition; major challenges in commercial aquaculture of this species are improvements in formulated diets for growth optimization and health promotion. One way to deal with such a challenge is to use food supplements such as probiotics, prebiotic, and synbiotics, which, in addition to growth promotion, have beneficial effects on the host immune system^[2].

  Prebiotics are non-digestible food stuff that stimulates growth through activation of one or a limited number of intestinal bacteria leading to beneficial effects to the host and its health improvement^[3]. The prebiotic Mito (MHF-Y) known in Japan as an animal feed mixed with sugar extract, contains 1 to 4% dextran granular powder. Dextran is a component of glucose produced from sugar fermentation. This sugar is obtained from binding of 1 and 6-α-glycosides, while starch and cellulose are achieved by binding 1 and 4-α or β-1 and 4- glycosides. Since the binding between 1 and 6 is the most resistant to acidic degradation, the digestion of dextran would be difficult by the gastric secretions in animals. This polymer is broken to smaller dextran molecules called Isomalto Oligosaccharides (IMO) in the course of stomach to the intestine by specific enzymes in the intestinal mucosa. The residue of IMO is used as a food source for enteric bacteria; it also accelerates the growth of beneficial lactic acid bacteria such as Lactobasillus and Bifidus, which inhibit the growth of bacteria E .coli and Salmonela through the production of lactic acid resulting in decreased intestinal pH^[2].

  Among the studies conducted to date on the use of prebiotic in the diet of fish, nearly all researchers have focused on a few specific compounds (including inulin and mannan oligosaccharides or MOS)^[4-6]. Therefore, we have tried in this researchto use a new commercial prebiotic in carp diet in order to introduce the compound to aquaculture industry. The aim of this study was, therefore, to evaluate the effects of different levels of prebiotic Mito on the growth, survival, carcass composition, and hematological parameters in the common carp fry as one of the most important farmed fish in Iran.

Materials and Methods

  The fry of Cyprinus carpio (n = 600; 6.5 ± 0.5 g) were obtained from a hatchery in Nasr Fish Propagation Center located in the north of Iran and transferred to the experiment place. After the initial adaptation to the new temperature and test diet for one week, fish samples (n = 25) were measured for the length and weight and randomly placed in each tank previously arranged in 4 rows of 3 each with random numbering. The food used in this experiment was the pellets for carp fry (FFT) (Table 1). The experimental diets were prepared by manual addition of 1, 2 and 3 g prebiotic MHF-Y (Mito Carp., Japan) to kg diet. So each amount of prebiotic added to the diet separately and after the even distribution of prebiotic in the diet, the pellets became by meat grinder. The pellets were dried in the shade after 24 hr and were stored at 4°C in the refrigerator.

Table 1: Ingredients and composition of compounds in experimental diets.

  During the experimental period, the carp fry were fed based on a percentage of body weight (4%) three times (8, 13 and 18 hr) daily. All physicochemical conditions (such as temperature, oxygen content, pH, etc.) of water in the tanks were monitored daily during the experiment and maintained within optimum levels (Table 2). At the completion of the experiment (60 days), the fry were not fed for 24 hours. Afterward, all fish were caught and weighed. Then 36 fry (three per replicate) were randomly sampled, their heads and fins and skin were removed and minced three times. Each homogeneous mixture sample was packed individually and kept frozen at -20°C for 10 days. Finally, these samples were analyzed for carcasses chemical analysis. Crude protein (based on Kjeldahl), fat (by soxhlet), and ash (by burning samples in an electric oven at 550°C for 4 h) were measured through the standard method of AOAC^[7].

Table 2: Physiochemical parameters in water under which the fish were cultured.

  In order to measure hematological parameters, at the end of experiment four fish per tank were randomly collected and anesthetized with clove extract (1.0 g per liter)^[6]. Blood samples were taken from the caudal vein by 2 ml plastic syringe. The blood samples were transferred to heparinated tubes and stored at 4°C. Red and white blood cells (RBC & WBC) were counted using a Neubauer hemocytometer^[8], haematocrit was assessed through microhematocrit method^[9], and hemoglobin level was evaluated by a kit and spectrophotometers(540 nm)^[10]. To calculate the growth and nutritional parameters, the following formula were applied^[11]:

Weight gain (g) = Final weight (g) – Initial weight (g)
Percentage of body weight increase = 100 × [(Final weight- initial weight) /initial weight]
Specific growth rate (SGR) as % per day = 100 × [(Ln final weight - Ln initial weight)/ experimental period]
Condition factor (CF) % = 100 × [weight of fish (g)/ (length in cm)³]
Net fish production = the number of remaining fish at the end of experiment × (final weight- initial weight)
Daily food eaten (% per day) = [100 × (total food eaten per fish)/ (average initial weight × average final weight) / time)
Feed conversion ratio (FCR) = fish weight (g) / ingested food (g)
Protein efficiency ratio (PER) = weight gain (g) / protein consumed (g)
Survival rate (%) = 100 × (Final number of fish /Initial number of fish)

  In order to analyze the data, first the normality test was performed by Shapiro-Wilk test. The data on changes in growth, nutritional and chemical factors of fish carcasses were analyzed through one-way analysis of variance (ANOVA). Differences between the treatments were compared by Duncan’s multiple range tests. Raw data were first processed in Excel and the presence or absence of a significant difference was verified at 5% confidence using SPSS version 19.

Results

Effect of prebiotic on growth and survival
  The highest final weight (14.25 ± 0.12 g) was recorded in treatment with 2 g/kg of dietary prebiotic Mito and the lowest value (11.97 ± 0.16 g) was measured in the control with significant differences (P < 0.05). The greatest increase in body weight (7.73 ± 0.11 g) was also detected in fish fed 2 g/kg of prebiotic, and the lowest level (5.49 ± 0.19 g) was observed in the control group (Table 3). The percentage increase in body weight was fully similar to the body weight gain. In addition, the fry of carp received 2 g/kg of prebiotic Mito showed the highest values of SGR (1.13 ± 0.00), CF (1.52 ± 0.02), and net fish production (116 ± 1.72) in comparison with those estimated in the control (Table 3).

  In the whole experimental period, there was no mortality in the treatments and fry survival rate in prebiotic treatments showed no significant differences compared with the control group (P > 0.05) (Table 3).

Table 3: Growth parameters and survival rate measured at treatments in carp fry after 60 days (value ± standard deviation).

Values with similar superscript letters are not statistically different (P > 0.05).

Effect of prebiotic on feed utilization
  Significant differences (P < 0. 05) were observed among some treatments in nutritional parameters measured (ingested food, FCR and PER) (Table 4). The fry in 2 g/kg of Mito displayed the utmost amount (4.06 ± 0.04) of food eaten whereas the treatment received 1.0 g/kg of prebiotic was not different from the control (P > 0.05). FCR level was lowest (3.01 ± 0.04) in fish fed 2.0 g/kg of prebiotic Mito and the highest level was found in control. The PER of fish fed 2 g of dietary prebiotic was significantly different from the other treatments (P < 0.05).

Table 4: Nutritional parameters measured at treatments in carp fry after 60 days (value ± standard deviation).

Values with similar superscript letters are not statistically different (P > 0.05).

Effect of prebiotic on body composition
  From Table 5, there was no significant differences between body protein in fish fed with different levels of prebiotic and the control (P > 0.05). The highest and lowest amounts of body protein, respectively, were detected in fry fed 2 g of prebiotic and in control. There were no significant differences in body fat (P > 0.05) with the greatest and lowest levels in 2 and 3 g treatments, respectively.

Table 5: Analysis of carcass composition at treatments in carp fry after 60 days (value ± standard deviation).

Values with similar superscript letters are not statistically different (P > 0.05).

Effect of prebiotic on hematological parameters
  From Table 6, the number of RBC in fish showed significant differences between the control and the other groups (P < 0.05) with the greatest values in fish fed 2 g/kg of Mito; however, the treatments with 2 and 3 g/kg of prebiotic Mito were almost similar in RBC counts (P > 0.05). The WBC counts were no statistically dissimilar between the treated groups and the control (P > 0.05) with the highest and lowest numbers in group with 2 g/kg of prebiotic and the control. Hemoglobin and haematocrit values in 2 and 3 g treatments significantly differed with those detected in control (P < 0.05).

Table 6: Blood parameters measured in carp fry fed Mito prebiotic (value ± standard deviation).

Values with similar superscript letters are not statistically different (P > 0.05).

Discussion

  Based on the results of this study, the addition of prebiotic MHF-Y at 1, 2, and 3 g/kg in diet of carp fry led to significant changes in final weight gain, percentage of body weight increase, length elongation, SGR, daily food consumption, FCR, PER, CF, ingested protein, and final biomass. Improvements in the growth and nutritional performances are probably because prebiotics stimulate the immune system through binding to lectin-like receptors on leukocytes rendering increased proliferation of macrophages^[12]. It was also reported that immunity stimulators attach to specific receptors on the surfaces of immune phagocytes and lymphocytes and destroy pathogens by producing enzymes. In addition, they can produce some chemical transmitters such as interferon, interleukins, and complement proteins that stimulate the immune system and increase the activity of T and B lymphocytes^[13]. In research evaluated Mannan Oligo Saccharide (MOS) prebiotic effect on growth, survival, carcass composition and some hematological parameters in common carp fry, no significant differences were observed between the treatments, but most of the growth and nutritional performances were detected in the diet treated with 1 g/ kg of prebiotic^[6]. Also, research showed that addition of 1.5 percent inulin to a commercial diet of carp fry could have positive impacts on the growth and survival, which corresponds our results^[14]. The effects of Fermacto prebiotic on growth and nutritional factors in common carp were studied. The results showed a significant increase in body weight as well as the best SGR, FCR, and PER in fish fed with 3 g of dietary prebiotic compared to the control^[15]. Our finding agrees with those results. one research examined MOS levels (zero, 2 and 4 mg) in European sea bass, Dicentrachus labrax, and stated that both the 2 and 4 g of MOS significantly increased growth rate^[16]. In an experiment on the farmed juvenile tilapia, Oreochromis niloticus, the researchers reported that by increasing MOS, daily food intake decreased and that a level of 4.0 percent MOSled to greater weight gain^[17]. In this regard, a study surveyed MOS effects on the same tilapia species and reported that the fish fed with diets containing 4 and 6 g of MOS showed significant increases in weight, length and average daily growth than the control group, but no significant differences were found in FCR and survival among treatments^[18]. Similarly, no significant differences were observed between treatments in terms of survival in the present study. Since there were no significant differences between different treatments in the numbers of WBCs, it can partly explain the absence of a significant difference in the survival rate. It should be noted that the effects of growth and immunity stimulators on the survival rate of fish can usually cause significant changes during a period of longer than six months^[19].

  A research reported that addition of 0.5% inulin to a commercial diet of Caspian lake kutum (Kutum kutum) could have beneficial effects on the growth and survival, however, it did not report significant differences between treatments in carcass nutrient composition; the highest levels of carcass protein, fat and ash was found in control^[20], which contrasts those found in the carp fry of this research with the highest nutritional components in the prebiotic–treated fish. This discrepancy probably results from such factors as the type and concentration of prebiotic used the fish species, age, weight etc. The amount of carcass protein may be affected by diets containing prebiotic, although it seems that such are sponse may vary depending on the species tested^[21]. The present results on the analysis of carcass protein, fat and ash showed that there were no significant differences among treatments. By addition of MOS (2 and 4 g g/kg diet) in the sea bream Sparus aurata^[5-22], and through dietary MOS of 1.5, 3 and 4.5 g/kg in the Caspian lakekutum (Rutilus frisii kutum)^[23], were not observed significant differences between treatments in the analysis of carcass composition, which corroborate the current results. The results of this study further show that although protein levels were not significantly different between treatments compared with the control, carcass protein levels increased with rising dietary prebiotic Mito. Prebiotic Mito, like other prebiotics, can affect beneficial bacteria in the intestine of carp to increase their biomass and eventually elevate the digestibility. The increased carcass protein levels in the treatments of this study may also be related to the enhanced utilization of amino acids and digestibility of the diet^[4].

  In this study, the FCR values in treatments containing prebiotic Mito decreased especially at a level of 2 g per kg diet. It is likely that this prebiotic produces digestive enzymes (amylase, protease and lipase) as a result of the proliferation of probiotic bacteria and ultimately, reduces the amount of FCR in the host^[24]. These enzymes eventually render increased digestion of fats and proteins in the diet followed by marked rise in the nutrition efficiency and subsequent growth performance in the host^[25]. Further studies are needed to determine the effects of prebiotic Mito on the physiology of the digestive tract and digestive enzymes such as amylase, protease and lipase.

  The results of this study showed significant differences between the treated fry and control in the amounts of haematocrit, RBC, and hemoglobin showing the superiority of respiratory condition in treatments containing prebiotic Mito compared to the control fish. Hemoglobin and haematocrit volumes fluctuate as a function of changes in RBC values with a direct relation. Increased hemoglobin concentration affect the transmissibility of respiratory gases in blood, the efficiency of the heart, and fish weight gain^[26]. The juvenile carp fed 1.0 g/kg of MOS displayed higher quantities of hemoglobin, haematocrit, RBC, and WBC than those found at other treatments^[6], which with the exception of WBC counts, agree with our findings. WBC is one of the important indicators of health and immune system of an animal. Significant increases in the levels of WBC, RBC, and hemoglob in were reported in Labeo rohita, received dietary MOS in comparison with the control^[26]. On the other hand, researchers observed no significant effects of MOS on the WBC, RBC, haematocrit, and hemoglobin in channel catfish Ictalurus punctatus^[27], Nile tilapia O. niloticuss^[17], beluga Huso hus^[28], and sea bream Sparus auratus^[22], as opposed to control groups, all of which agree with our observation on WBC values.

Conclusion

  It can be concluded that the use of probiotic Mito at the levels examined here is capable of affecting the growth and nutritional performances; hence, it seems to be an appropriate dietary supplement for the fry of common carp.

References