All the experimental diets were readily accepted by the juvenile red seabreams at the start of the feeding trial and they fed aggressively during the 12 weeks of the feeding trial.
After 12 weeks of feeding trial, growth performance and feed utilization of the fish groups fed the KH and SH diets were significantly improved compared to those of fish fed the Con diet. Survival rate varied from 87 to 94% without significant differences among dietary treatments. No significant difference was observed in feed intake among the fish groups (Table 1-3).
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Table 1-3. Growth performance and feed utilization of red seabream (initial body weight, 29.0 ± 0.1 g) fed the four experimental diets for 12 weeks.
Diet
Con KH SH TH
Final body weight (g) 103±2.02b 113±4.55a 111±1.90a 106±2.46ab Specific growth rate (%)1 1.49±0.03b 1.60±0.05a 1.58±0.02a 1.53±0.03ab Feed conversion ratio2 1.80±0.05a 1.60±0.07b 1.61±0.03b 1.70±0.06ab Protein efficiency ratio3 1.34±0.03b 1.49±0.07a 1.49±0.03a 1.40±0.05ab Feed intake (g fish-1)4 133±1.05 135±1.98 131±0.65 131±2.47
Survival (%) 92.2±5.09 87.8±1.92 94.4±5.09 91.1±1.92
Values are mean of triplicate groups and presented as mean ± S.D. Values with different superscripts in the same row are significantly different (P<0.05). The lack of superscript letter indicates no significant differences among treatments.
1Specific growth rate (%) = [(loge final body weight - loge initial body weight)/days] × 100
2Feed conversion ratio = dry feed fed /wet weight gain
3Protein efficiency ratio = wet weight gain /total protein given
4Feed intake = dry feed consumed (g)/fish
17 3.1.2. Whole body composition
Whole body composition of red seabream after 12 weeks of feeding trial was not affected by dietary treatment (Table 1-4). However, slightly lower moisture and higher lipid contents were recorded in fish fed diets containing the tested hydrolysate. There seemed to be a trend of slightly higher protein and ash contents observed in fish fed the control diet.
Table 1-4. Whole-body composition of red seabream fed the four experimental diets for 12 weeks.
Diet
Con KH SH TH
Dry matter (%) 35.3±0.6 36.3±0.8 36.1±1.2 36.1±0.2
Protein (% DM) 51.4±1.3 49.8±0.8 50.3±3.0 49.9±1.2
Lipid (% DM) 33.8±0.4 35.3±2.1 35.2±0.8 35.0±0.4
Ash (% DM) 14.3±1.2 13.9±1.6 13.2±0.3 12.9±0.8
Values are mean of triplicate groups and presented as mean ± S.D. The lack of superscript letter indicates no significant differences among treatments.
18 3.1.3. Hematological parameters
After 12 weeks of feeding trial, hematological parameters of red seabream were not significantly influenced by dietary treatments (Table 1-5). However, numerically higher hematocrit and total protein levels were found in fish fed the diets containing the tested hydrolysates.
Table 1-5. Hematological parameters of red seabream fed the four experimental diets for 12 weeks.
Diet
Con KH SH TH
Hematocrit (%) 39.1±1.9 40.7±3.8 40.0±2.7 44.2±2.0
Hemoglobin (g dL-1) 7.67±1.20 7.43±0.61 7.09±0.35 7.37±0.57
Glucose (mg dL-1) 55.1±9.3 53.2±6.2 59.4±2.1 54.2±6.8
Total protein (g dL-1) 4.33±0.50 5.13±0.48 4.98±0.81 5.28±0.45 Total cholesterol (mg dL-1) 370±47.9 375±39.0 371±62.7 374±72.3 Triglyceride (mg dL-1) 386±89.4 394±39.7 384±56.5 383±56.5
Values are mean of triplicate groups and presented as mean ± S.D. The lack of superscript letter indicates no significant differences among treatments.
19 3.1.4. Non-specific immunity parameters
Non-specific immune responses of red seabream after 12 weeks of feeding trial including nitro blue tetrazolium (NBT), immunoglobulin, lysozyme, myeloperoxidase (MPO), superoxide dismutase (SOD) and antiprotease activities are provided in Table 1-6.
Dietary inclusion of the protein hydrolysates resulted in significantly higher Ig level compared to the Con diet. Significantly higher SOD and antiprotease activities were found in fish fed the KH diet than in fish fed the Con diet. Although, numerically higher lysozyme, MPO and respiratory burst activities (NBT) were detected in fish fed diets containing the hydrolysates, the differences were not significant.
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Table 1-6. Non-specific immune parameters of red seabream fed the four experimental diets for 12 weeks.
Diet
Con KH SH TH
NBT1 1.12±0.03 1.15±0.06 1.14±0.08 1.14±0.05
Ig2 15.4±1.39b 18.4±1.25a 18.6±0.67a 18.7±0.39a
Lysozyme3 10.9±0.70 12.3±0.37 12.0±0.55 11.7±0.69
MPO4 1.26±0.15 1.47±0.05 1.51±0.09 1.26±0.08
SOD5 30.6±4.25b 40.0±5.23a 37.3±2.32ab 32.8±0.70ab
Antiprotease6 28.7±1.93b 35.4±1.65a 32.4±2.57ab 34.9±3.16ab
Values are mean of triplicate groups and presented as mean ± SD. Values in the same row having different superscript letters are significantly different (P<0.05). The lack of superscript letter indicates no significant differences among treatments.
1Nitro blue tetrazolium activity (absorbance)
2Immunoglobulin (mg mL-1)
3Lysozyme activity (µg mL-1)
4Myeloperoxidase level (absorbance)
5Superoxide dismutase (% inhibition)
6Antiprotease (% inhibition)
21 3.1.5. Apparent digestibility coefficient
Incorporation of the tested hydrolysates in diets significantly enhanced apparent digestibility coefficient of protein and the highest value was obtained in fish fed the TH diet followed by the KH diet. Fish fed the SH diet showed remarkably higher apparent digestibility coefficient of dry matter than the others. However no significance was revealed (Fig. 1-1).
Figure 1-1. Apparent digestibility coefficient of dry matter (ADCd) and apparent digestibility coefficient of protein (ADCp) of fish fed the four experimental diets.
22 3.1.6. Challenge test
During the E.tarda challenge test, the first dramatic mortality was observed on the third day after injection where the fish fed the Con and SH diets showed lower disease resistance compared to those fed the other experimental diets (Fig.1-2). At the end of the challenge test the control group had the lowest survival rate (17.8%) and significantly differed from those of fish fed the KH and TH diets. Also, fish fed the SH diet exhibited numerically higher disease resistance than the control group but the difference was not significant.
Figure 1-2. Survival rate of red seabream fed the four experimental diets after
challenge with Edwardsiella tarda.
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4.1. DISCUSSION
In the present study dietary supplementation of KH and SH resulted in significant improvements in growth performance and feed utilization of red seabream. The positive effects of low molecular weight compounds in protein hydrolysates on fish growth and feed utilization have been well documented (Aksnes et al., 2006b; Zheng et al., 2012a). The protein hydrolysates used in this study comprise mainly small peptides with molecular weight below 5000 Da (Table 1). These results are in agreement with previous studies in which the inclusion of protein hydrolysates possessing low molecular weight peptides showed beneficial effects on growth performance and feed efficiency in fish. Refstie et al.
(2004) obtained faster growth rate of Atlantic salmon when 10-15% of fish meal was replaced by fish protein hydrolysate. Also, Zheng et al. (2012a) reported that ultra-filtered fish protein hydrolysate at dietary inclusion level of 3.7% can enhance growth and feed utilization of juvenile Japanese flounder. In contrast, Oliva-Teles et al. (1999) showed that partial replacement of fish meal with fish protein hydrolysate did not improve growth and feed utilization of juvenile turbot. The variations in the results of hydrolysate administration can be attributed to the changes in protein hydrolysate material, enzyme source and hydrolysis conditions (Klompong et al., 2009). Furthermore, several studies pointed out that dietary inclusion of enzymatic protein hydrolysate at a moderate level promotes growth performance and feed utilization of fish, while lower or higher levels can cause negative effects (Hevrøy et al., 2005; Espe et al., 1999). The results of this study may indicate that the inclusion level of the protein hydrolysates was moderate and their nutritional values were better than those of fish meal leading to better performance of red seabream.
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Improvement of diet palatability by protein hydrolysate supplementation has been suggested as one of the growth promoting effects of protein hydrolysates (Refstie et al., 2004;
Hevrøy et al., 2005). In a study by Kolkovski et al. (2000), coating of a commercial diet with liquid krill hydrolysate resulted in a significant improvement in ingestion rates of diet in yellow perch (P. flavescens) and lake whitefish (C. clupeaformis). In the present study, the fish were fed to apparent satiation and the results showed no significant effect of the hydrolysates on fish feed intake. Similar results were reported by Aksnes et al. (2006 a, b) on Atlantic cod and rainbow trout. This may indicate that improved performance of fish in this study was not related to the diet palatability. Such enhancement in fish growth by the hydrolysates was probably due to the fast assimilation nature of the hydrolysates leading to a fast passing and absorption of nutrients through intestinal membrane (Aksnes et al., 2006a;
Zheng et al., 2012a). The significant improvement in ADC of protein by the hydrolysate supplementation in the current study may support the general claim regarding to better absorbance of protein hydrolysates. This finding is in line with the results obtained by Zheng et al. (2012a) on Japanese flounder. However, Oliva-Teles et al. (1999) could not find any significant effect of dietary inclusion of fish protein hydrolysate on ADC of dietary protein for turbot.
In the current study, whole-body composition of red seabream was not significantly affected by hydrolysate incorporation. Similarly, Oliva-Teles et al. (1999) did not find any significant differences in whole-body proximate composition of turbot fed diets containing fish protein hydrolysate. Zheng et al. (2012b) examined the supplemental effects of ultra-filtered fish protein hydrolysate at two different levels as well as non-ultraultra-filtered hydrolysate on body composition of turbot. Their results showed significant increase in whole-body protein content when the fish were fed high concentration of the ultrafiltered protein hydrolysate compared to those fed fish meal based diet. The differences in the effects
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of protein hydrolysates on fish whole-body composition can be due to the different sources of hydrolysates and their inclusion levels as well as the fish species under study.
Hematological parameters are used as valuable biological indicators indicating physiological stress response as well as the general health condition of fish (Rey Vázquez and Guerrero, 2007; Siwicki et al., 1994). In this study, all the measured blood parameters ranged within normal reported ranges for juvenile red seabream (Ji et al., 2007; Abdul Kader et al., 2011), and no significant differences were found among dietary treatments. These results show that the fish were in a good nutritional and environmental condition in this study and the tested hydrolysates had no adverse effects on health condition of juvenile red seabream.
Several previous studies have reported the biological functions of protein hydrolysates by means of their bioactive peptides (Gildberg et al., 1996; BØgwald et al., 1996; Harnedy and FitzGerald, 2012). There have been increasing concerns on immune-stimulating and antibacterial properties of protein hydrolysates in aquaculture. Fish immune system can be trigged by protein hydrolysate supplementation (Kotzamanis et al., 2007;
Zheng et al., 2012b). Peptides with molecular weight from 500 to 3000 Da from fish protein hydrolysates have been reported to stimulate the activity of fish macrophages (Bogwald et al., 1996). However, there were varying degrees of success in previous studies due to the variation of bioactive peptide profiles of hydrolyzed protein depending on raw material, enzyme source and hydrolysis conditions (Klompong et al., 2009). Feeding Atlantic halibut with peptide-enriched live feed stimulated production of lysozyme and complements (Hermannsdottir et al., 2009). Similarly, Liang et al. (2006) found that supplementation of 15%
fish protein hydrolysate in Japanese sea bass diet could enhance the activities of lysozyme and complement. Tang et al. (2008) also demonstrated that inclusion of fish protein hydrolysate could increase lysozyme activity, serum complement and immunoglobulin M of
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the large yellow croaker. However, no positive effect of several fish protein hydrolysates was found on the immune system of coho salmon and turbot (Murray et al., 2003; Zheng et al., 2012b). In the current study, effects of the selected protein hydrolysates were examined on humoral parameters of non-specific immune response. The results showed significant increase of plasma Ig level in fish fed diets containing the hydrolysates. Significantly higher SOD and antiprotease activities were found in fish fed the KH diet.
The activation of non-specific immunity by immunostimulants is associated with increased protection against infectious disease (Sakai, 1999). The in vitro immunostimulatory effects of protein hydrolysates may also be effective in controlling disease-related loss among farmed fish (Murray et al., 2003). In this study, supplemental effects of the protein hydrolysates were examined in vivo by injection of E. tarda to the fish.
This bacterium is the causative agent of Edwardsiellosis in a wide range of commercially important fish species including red seabream (Zhang et al., 2012). Occurrence of antibiotic resistant strains of E. tarda in fish was reported (Aoki and Takahashi, 1987; Aoki et al., 1989). Therefore, prophylactic against E. tarda by the inclusion of immunostimulants in diets becomes more practical to implement in a fish farm. In the current study, fish fed the control and SH diets showed high mortality after a few days of bacterial challenge, while the groups fed the KH and TH supplemented diets showed significantly higher disease resistance.
Such enhancement in fish disease resistance can be partially due to the facilitated non-specific immune responses particularly in KH treated fish where significantly higher SOD and antiprotease activities as well as Ig level were obtained.
In conclusion, the findings in this study show that growth performance, feed utilization, and protein digestibility can be improved in red seabream juveniles by the dietary supplementation of krill hydrolysate concentrate or shrimp hydrolysate powder at moderate inclusion levels. Fish innate immunity and disease resistance can also be positively affected
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by the tested protein hydrolysates particularly krill hydrolysate concentrate. Further studies are needed to verify the optimum supplementation level of each protein hydrolysate in the fish or other fish species.
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