MATERIALS AND METHODS

An over-winter 3600 juveniles common carp (20 g) were used in the present study, representing 4 treatments (Control, probiotic, prebiotic and acidifier) at three groups of fish for each were stoked in 12 earthen pond each (100 m³) At a density of 3 fish m^–3, at EL-Zawya fish farm, Kafr EL-shaikh Governorate, belonging to the general Authority for fish Resources Development (GAFRD), Ministry of Agriculture and land Reclamation, Egypt. The feeding experiment lasted for 6 months (183 days). The fish were acclimated on the culture system for 2 weeks. A random sample of 20 fish at the beginning and at the end of the experiment from each pond was taken, weight collectively and stored at -20°C for initial and final body composition analysis.

Water quality parameters: Including water Temperature (T), Dissolved Oxygen (DO) and pH were monitored weekly. The average values of these parameters throughout the study were: T = 26.5±1°C, DO = 7.3±1.2 mg L^–1 and pH = 7.75±0.20. They were appropriate for common carp cultivation during the whole experiment period.

Test diets and feeding regime: A commercial basal diet was used (control group) containing 25% crude protein, 5.79% ether extract, 6.70% crude fiber, 14.2% ash, 49.41% NFE and 3698 kcal kg^–1 GE supplemented with the different commercial growth promoters probiotic (Biogen^®) containing (Allicin, high-unit hydrolytic enzymes, Bacillus subtilis, amylase, protease, lipase and garlic powder) prebiotic (Garlen extra 4^®) containing (Garlic extract and blend of volatile oils) and acidifier (Galliacid^®) containing (Fumaric acid, calcium format, calcium propionate, potassium sorbate and hydrogenated vegetable oil) representing 4 treatments (control, probiotic, prebiotic and acidifier). They added in the diets at the recommend level of the producers (2, 0.2 and 0.6 kg t^–1 diet for probiotic, prebiotic and acidifier, respectively). Feeding level was 3% of the total biomass until the end of the experiment. The test diets were fed to the fish twice a day (9 am and 2 pm). Daily rations were readjusted each 15 days interval according to the new average weights until the end of the feeding period.

Viability test: The viability test of the probiotic (Biogen) was carried out before using according to the method outline by Martin et al.¹⁸. The viable contents was determined by containing Colony Forming Unit (CFU), which is considered an indication for the viability of the microorganisms present viable in this commercial probiotic and so represents its growth promoting effect determination showed the presences of (6×10⁶) CFU for the commercial probiotic (BIOGEN^©).

Immune response of fish as affected by treatments: At the end of the experiment a number of fish were subjected to the differential count of white blood cells. Blood films was staining by Giemsa’s stain method Sakai et al.¹⁹. Blood films were used to determine the number of leucocytes, being indicator for immune response for juveniles feeding on different types of natural growth promoters.

Chemical analysis of fish body: At the beginning and the end of the experiment, a sample of fish from each pond were performed on a pooled sample, which was weighed and frozen at -20°C for final body composition analysis. Moisture, protein, lipid and ash were performed according to the standard methods²⁰.

Calculations of fish performance: Growth performance and feed efficiency parameters were calculated as follows:

Weight Gain (WG) = Wf-Wi

where, Wi and Wf are initial and final weights (g), t is duration of experimental (days) and ln is the natural logarithm.

Statistical analysis: Statistical analysis was carried out using one-way analysis of variance using SPSS (version 16) for Windows (SPSS, Chicago, IL, USA)²¹. Differences between means were determined using Duncan’s multiple test (p<0.05)²².

RESULTS AND DISSCUSION

Average values of initial body weight, final body weight, weight gain and specific growth rate of common carp fed different growth promoters diets are given in Table 1. The data in Table 1 showed that initial weight was nearly similar in all treatment groups with no significant differences (p<0.05). The data showed also that there were significant differences (p>0.05) among all treatments in final body weights (FW), Weight Gain (WG) and Specific Growth Rate (SGR). Probiotic treatment recorded the highest final body weight, weight gain and specific growth rate (919.899 and 3.73), respectively followed by prebiotic (746, 726 and 3.61) then acidifier (664, 644 and 3.55). All tested diets were superior as compared with the control diet with no supplementation (552, 532 and 3.45 g). The present results were in agreement with the results obtained by many researchers. Renuka et al.²³ suggested that the incorporation of probiotic in common carp diets stimulated fish growth and digestion as micro biota colonization enzymes that hydrolyze complex molecules, facilitate better digestion and absorption of macronucleus resulting in higher protein and energy deposition in the body tissues. In these aspects²⁴, pointed out to the improvement of digestion and metabolism in the fish body due to the presence of the bacillus in the probiotic Biogen, moreover the prevention of pathogenic bacteria colonies in fish gut.

Table 1:	Growth performance parameters of common carp fed the tested diets
Values in the same column with different superscripts are significantly different at p<0.05

Faramarzi et al.²⁵ were in accordance with the results obtained in the present study, where they found that the addition of 0.1% probiotics (Bacillus subtilis c-3102 spores) in common carp fry diets improved fish growth and mitigated the effects of stress factors. In this particular, diets supplemented with Bacillus subtilis c-3102 spores resulted in improving growth performance of koi carp significantly than those fed the control basal diets.

The positive effect of probiotic was also observed in several kind of fish, in seabream (Sparus aurata L.)^26,27 and large croaker (Larimichthys crocea)²⁸. The high viability Bacillus subtilus may be considered as another reason for the positive and better effect of Biogen. This fact was proved by the studies carried out by Mohapatra et al.²⁹, who found that incorporation of live probiotic microorganisms (Lactobacilis lactis and Bacillus subtilus) resulted in maximum growth performance in rohu (Labeo rohita) fingerlings in comparison with some combinations of inactivated probiotics. Other similar results were also observed for Tilapia nilotica³⁰, L. rohita³¹ and Cyprinus carpio³².

The present results indicated that prebiotics had the second superior effect in the diet of common carp in growth performance parameters. The differences in these parameters between either the control diet or the probiotic diets were significant. The fish fed diet supplemented with prebiotic showed improvement in fish growth as compared with the control diet, but this improvement was leaser than that in probiotics diets. It was suggested that the improvement in growth parameters occurred with the prebiotic diets may be due to the fermentation of prebiotics in colon, which promote the growth of the bacterial populations associated with the healthy well functioning colon. Beneficial types of colonic bacteria have the ability of oligosaccharides fermentation which are not used effectively by potentially pathogenic bacteria species³³. As in the case of probiotics, the positive effect of prebiotics on growth was also found in different fish species such as Atlantic cod³⁴, Turbot larvae³⁵, Rainbow trout ^36,37 Atlantic salmon³⁸ and Hybrid tilapia³⁹.

Concerning acidifier, the results showed its superiority in increasing growth over the control diet. These results were in accordance with the results obtained by Ng et al.⁴⁰ who found that the dietary organic acids can exert strong anti-microbial effects and have the potential to exert beneficial effects on growth, nutrient utilization and disease resistance in tilapia when tested in hyprid tilapia (Oreochromis sp.) which may be a reflection of the reduction of pH in the digesta in the stomach and gut. This was indicated by the significant reduction of total bacteria per gram in the feaces of fish fed diets supplemented with organic acids. Other investigators studied the effect of organic acid salts or salts blend (calcium formate, calcium propionate, calcium lactate, calcium phosphate and citric acid) on growth of tilapia at different levels and found that these salts and blends may be especially useful during grow out period in tilapia culture. Baruah et al.⁴¹ observed that citric acid of microbial phytase have a synergistic effect on the bioavailability which was more pronounced in low-protein diets. Some researchers proved the ability of potassium diformate in controlling Vibrio anguillarum in tropical tilapia culture⁴². In this respect, higher cumulative mortality of fish fed diets was obtained with no organic acids as composed with those received diets supplemented with organic acid after 16 days past challenge with Streptococcus agalactiae⁴⁰.

Efficiency of feed and protein utilization: Feed and protein utilization parameters expressed as FCR, PER, PPV and ER were illustrated in Table 2. Data obtained showed that there were significant differences (p>0.05) among the different treatments in FCR. The best value (2.28) was recorded in probiotic diets and there was no significant difference between fish received prebiotic and acidifier diets. The differences between probiotic treatment and both the control or prebiotic and acidifier were significant.

Concerning PER, the results indicated a significant difference (p>0.05) between the control (1.79) and the other tested treatments, where the tested supplemented diets with probiotic, prebiotic or acidifier were significantly better than the control. On the other hand there were no significant differences among the tested diets (1.83, 1.83 and 1.83).

Protein productive value (PPV%) showed significant differences between the control diets and all the tested diets probiotic, prebiotic, acidifier (19.18, 32.81, 27.58 and 22.79) respectively except acidifier diets. PPV% was superior in probiotic diets than the other treatments. At energy retention (ER) there were significant differences (p>0.05) between all treatments and the control one.

Table 2:	Feed utilization parameters of common carp fed tested diets
Values in the same column with different superscripts are significantly different at p<0.05, FCR: Feed conversion ratio, PER: Protein efficiency ratio, PPV: Protein production value, ER: Energy retention

The highest value recorded for probiotic (18.63) then prebiotic (16.57) followed by acidifier (14.96), the lowest value recorded in control group (13.47).

The obtained data were in the same trend found in the previous studies about the efficiency of feed utilization, where several researchers recorded different degrees of improvement in feed and protein utilization parameters in diets supplemented with probiotic or growth promoters, which reflected the increasing growth rate^23,43. Faramarzi et al.²⁵ found also improvement in the feed utilization in common carp fed diet supplemented with 0.1% probiotics (Bacillus subtilis c-3102 spores). Similar positive effects in feed utilization were recorded in many fish species as mentioned previously during the discussion of growth performance.

From the economical stand point of view, the results declared that all diets treated with feed additives, commercial probiotic, prebiotic and acidifier which little costly compared with control basal diet. But the return in high growth performance and feed utilization parameters showed that probiotic treatment achieved the highest net financial return, followed by prebiotic, acidifier and control.

Body composition: Body composition of common carp fed different growth promoters are presented in Table 3. Statistical analysis of these data showed no significant difference (p<0.05) among all treatments at the end of the experiment in DM. Meanwhile the other parameters (CP, EE and ash) declared that there were significant differences (p>0.05) between all treatments. The highest CP found at carcass for probiotics treatment (62.09) then prebiotics (57.65). However, acidifier more superior (50.50) than the control (41.80) but it is lowest than the values at the beginning.

According to EE the final carcass there were significant differences between treatments the highest one recorded in acidifier (23.53) then probiotics (20.17) followed by control and prebiotics (22.20 and 21.87), respectively with no significant differences. The ash content of fish carcass was higher in fish treated with prebiotics and acidifier with no significant differences (10.70 and 10.12) and the same observed in control and probiotics (6.86 and 5.85).

The improvement in body composition of carp fed probiotic is a significant evidence of the improvement in general health condition of the reared fish. These positive effect in body composition of common carp may be due to improving of growth performance, enhance the metabolism and energy of fish body cells and raise the efficiency of feeds⁴⁴. The results of body composition in this study were in close agreement with Mohamed et al.⁴⁵ for tilapia. On the other hand Eid and Mohamed⁴⁶ found no statistical differences were observed in whole body moisture, crude protein, ether extract and ash for mono sex O. niloticus fingerlings fed diets containing different levels of commercial feed additives.

Immune response: Determining the immune response of experimental fish fed different diets containing natural growth promoters had been done by differential count of blood film. The objective of that is to assess the changes in white blood cells following spontaneous stress factors surrounding the common carp juveniles reared in earthen ponds in order to evaluate the serosity of the disease and other stress factors on the basis of blood alterations, which reflect the immune response are presented in Table 4.

The results found that there were significant differences in total count of white blood cells, the fish fed with control and acidifier group have highest total count (35.67 and 35.33) followed by prebiotic (33.33), the last one is probiotics (31.67).

By differentiation, no significant differences were observed in heterocytes, lymphocytes, monocytes, esenocytes and basophiles.

Table 3:	Body composition on dry matter basis of common carp fed the tested diets
Values in the same column with different superscripts are significantly different at p<0.05

Table 4:	Blood film analysis for common carp fed the tested diets
Values in the same column with different superscripts are significantly different at p<0.05, the tested diets

Haematological parameters are considered as proper indices for tracking health status of fish and their response to environmental stresses⁴⁷. The WBC (leucocytes) are one of the most important cells that can stimulate immune responses of fish and serves as one of the 1st line of body defence and their numbers increase sharply when infections arise⁴⁸. Also, these cells produce antibody and can perform macrophagus activity⁴⁹. The total WBC in this experiment indicated that all treatments were within the normal range which reflect the strength of immunity especially in the control and acidifier treatments.

CONCLUSION

This study indicates that common carp fed diets with different natural growth promoters may improve growth performance, feed utilization, body composition and net financial return in common carp juveniles diets than the control. But when comparing among the three sources the probiotic was the superior, followed by prebiotic then acidifier in practical filed.