Abstract: Background and Objective: Fatty acids are considered one of the most important components in fish diets which fish need it for essential functions including growth, reproduction, immunity and maintenance of healthy tissues. Therefore, the present study was conducted to determine the appropriate levels of Aquafat-O® as a novel commercial source of lipid and fatty acids, through evaluation of the effects of Aquafat-O® on growth, physiological and immunity responses of hybrid red tilapia. Materials and Methods: Four isonitrogenous (331 g kg–1 protein) and isocaloric (19 MJ kg–1) diets were formulated to contain 0, 2, 4 and 6% Aquafat-O® kg–1. The diets were fed to triplicate groups of juvenile hybrid red tilapia (11.11 g average body weight), 2 times daily for 14 weeks. One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test is used for data analysis. Results: Growth performance and feed utilization parameters, haematological parameters, total antioxidant capacity and hepatic superoxide dismutase were significantly improved with increasing Aquafat-O® levels. Moreover, the dietary addition of 6% Aquafat-O® led to the reduction in lipid content of the whole fish body, serum glucose, total cholesterol, triglyceride and hepatic lipid peroxidase. Conclusion: Thus, the addition of 6% Aquafat-O® kg–1 diet had positive effects on growth performance, physiological and immune responses of hybrid red tilapia. Consequently, it could be concluded the practical using of Aquafat-O® at this level as a growth and physiological enhancer agent or an immunostimulant feed additive in fish farms.
INTRODUCTION
In commercial aquaculture, the cost of feed is the main problem as it constitutes up to 60% of the total cost of production1. Hence, many researchers examined the use of substitute protein and lipid sources, especially the vegetable sources to replace fish meal and fish oil (FO) that are the cheaper, leading to reduce the feed cost2,3. Lipid is considered one of the most important components in tilapia diets that provide energy and essential fatty acids. Fish also need lipids for essential functions including growth, development, reproduction and maintenance of healthy tissues and flesh quality4. In mammals including fish, cannot synthesis polyunsaturated fatty acids (PUFAs) like linolenic acid (LA), linoleic acid (LNA) and oleic acid (OA), thus they are termed essential fatty acids5. The availability of sufficient quantities of PUFAs is an important to meet the requirements for normal growth, development and maintenance of cellular structure and function. Partially, tilapia is unable to synthesize the 18-carbon PUFAs6. Furthermore, freshwater fish need LA and/or LNA as the essential fatty acids for normal growth and reproduction7-9.
The first hybrid red tilapia was produced in Taiwan in the late 1960s and it was a cross between ♂ Oreochromis niloticus×♀ Oreochromis mossambicus10. The red tilapia has several advantages such as the fast growth rate, good conversion, ability to grow in brackish and salt waters and low susceptibility to diseases11, which have promoted its spread. Randomly, some fish farmers feed tilapia fish on some commercial feed additives like Aquafat-O® as a growth and immune enhancer agent or as a precautionary measure against the environmental stressors. In addition, available information on the effect of PUFAs, especially 18-carbon PUFAs, on physiological and immunity of hybrid ♂O. niloticus×♀O. mossambicus are limited. Therefore, the present study was designed to determine the appropriate level of Aquafat-O® (a novel feed additive) for hybrid red tilapia, ♂O. niloticus×♀O. mossambicus fingerlings.
MATERIALS AND METHODS
Experimental diets: Aquafat-O® is calcium salt (powder) of PUFAs (OA, LA, LNA), which comprised moisture 3.5%, crude fat 84%, ashes 12.49% and butylated hydroxy toluene (BHT) 0.01%. It has been used as the source of fat and 18-carbon PUFAs in this study. Fatty acids profiles in Aquafat-O® were measured according to Kirk and Sawyer12, as shown in Table 1. All components of the basal diet were bought from the local market and the proximate chemical analysis was carried out according to AOAC13, as presented in Table 2.
| Table 1: | Fatty acid composition of the Aquafat-O® (% by weight of total fatty acids) |
The components were crushed then corn oil (6%, in the control treatment) was replaced by graded levels 0, 2, 4 and 6% of Aquafat-O® kg–1, referred to treatments number T1 (as a control group), T2, T3 and T4, respectively. All ingredients of the experimental diets were carefully milled and mixed, then pressed by manufacturing machine (pellets size 1 mm). Pellets were air-dried to less than 10% moisture and stored at 4°C until use.
Experimental design and conditions: This study was performed in Fish Research Laboratory, Faculty of Agriculture, Mansoura University, Al-Dakahlia Governorate, Egypt. The graded levels 0, 2, 4 and 6% Aquafat-O® kg–1 diet were examined, concerning their effects on growth performance, feed utilization, the chemical composition of the whole fish body, haematological and serum biochemical parameters and hepatic oxidative stress biomarkers of hybrid red tilapia fingerlings for 14 weeks. A total of 216 fingerlings of hybrid red tilapia (♂O. niloticus×♀O. mossambicus), with an average initial body weight 11.11±1.03 g. Fish were stocked into rearing tanks for 2 weeks as an adaptation period and fed a basal diet (the control diet). Then, fish were separately distributed into four experimental treatments (3 replicates/treatment), for 14 weeks. Fish were stocked at 18 fish/tank (500 L in volume). Each tank was supplied with an air stone connected to the electric compressor. Fish were hand-fed on tested diets to satiation 2 times/day (10.00 and 15.00). The amount of food was adjusted by weekly based on the actual fish body weight changes. Dechlorinated tap water was used to change 20% of the water volume in each tank 3 times a week.
Water quality parameters in each tank were measured 2 times weekly. The water temperature (via a thermometer) was 25±1.0°C and that was controlled through a thermostat controlled heater and pH-value determined by using Jenway Ltd., Model 350 pH meter, Staffordshire ST15 0SA, UK, which was average value of water pH 8.19±0.2.
| Table 2: | Feed composition (g kg–1) and proximate composition of the diets (air-dry basis, g kg–1) |
1Norel-Misr company, Egypt, 2Each 1 kg premix contains: Vit. A: 12000,000 IU, Vit. E: 10,000 mg, Vit. K3: 3000 mg, Vit. D3: 3000,000 IU, Vit. B1: 200 mg, Vit. B2: 5000 mg, Vit. B6: 3000 mg, Vit. B12: 15 mg, Vit. C: 2500 mg, Folic acid: 1000 mg, Nicotinic acid: 35000 mg, Biotin: 50 mg, Pantothenic acid: 10,000 mg, Mn: 80 g, Cu: 8.8 g, Zn: 70 g, Fe: 35 g, I 1g, Co: 0.15 g and Se: 0.3 g, 3NFE (Nitrogen free extract) =100-[%CP+%EE+%Ash+%crude fiber], 4Gross energy calculated based on 23.64, 39.54 and 17.57 MJ g–1 for protein, lipid and NFE, respectively |
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Dissolved oxygen (using Jenway Ltd., Model 970-dissolved oxygen meter, Staffordshire ST15 0SA, UK) was 7.21±0.3 mg L–1.
Fish sampling: At the end of the experiment, fish were stopped on feeding at 24 h before the sampling. Fish in each tank were weighed as a group to determine the final body weight. Also, fish weight (W) and the total length (TL) were individually measured (n = 8 of each treatment) for calculating the condition factor (K, %) according to the following equation:
At the start and at the end of the experiment, fish samples (n = 6 of each treatment) were collected and kept frozen (-20°C) until the chemical analysis of the whole fish body was done according to AOAC13.
Growth performance and feed utilization parameters: The growth performance parameters of fish such as total weight gain (TWG, g), average daily gain (ADG, g fish–1 day–1) and the specific growth rate (SGR, % day–1) as well as the survival (%) were calculated. Moreover, feed utilization parameters were computed such as feed conversion ratio (FCR), protein efficiency ratio (PER), protein productive value (PPV, %) and energy utilization (EU, %). These parameters were calculated per tank as followings:
where, Wi and Wf are initial and final weights (g), respectively and t is the time of the experiment (days):
Blood sample, haematological and serum biochemical analysis: Ten fish from each tank were transferred to a small plastic tank containing 10 L water supplemented with 3 mL pure clove oil (dissolved in 10 mL absolute ethanol) as a natural anesthetic material. Fresh whole blood samples (n = 5 of each treatment) were collected from the fish caudal peduncle in small plastic vials containing heparin. Then, the collected blood samples were used for the determination of haematological parameters. Other blood samples (n = 5 of each treatment) were assembled in small dry plastic vials and centrifuged at 3500 rpm for 15 min to obtain the blood serum. Serum samples were kept in the deep freezer (-20°C) until the biochemical analysis was carried out.
The pooled whole blood was used for identification of haematological parameters such as hemoglobin (Hb) by using commercial kits (Diamond Diagnostic, Egypt). Also, total red blood cells (RBCs) and total white blood cells (WBCs) were counted according to Dacie and Lewis14 on an Ao Bright-Line Hämocytometer model (Neubauerimroved, Precicolor HBG, Germany), as well as the packed cell volume (PCV, %) was measured according to Stoskopf15.The haematological indices of mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated using the total RBCs, Hb concentration and PCV according to Dacie and Lewis16. In addition, serum biochemical parameters such as glucose, total cholesterol and triglycerides were assessed according to Henry17, Ellefson and Caraway18 and McGowan et al.19, respectively. While, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were measured according to NCEP20.
Oxidative stress biomarkers: After taking the blood samples, two fish from each tank (n = 6 of each treatment) were randomly taken and sacrificed to obtain the liver. Then, the liver samples were kept at -80°C until analyzed. Liver samples were homogenized in the ice-cold buffer (5 mM potassium phosphate, pH 7.4 containing 0.9% sodium chloride and 0.1% glucose) using a glass homogenizer immersed in an ice-water bath to yield a 10% homogenate. After that, each homogenate was centrifuged at 4000 rpm for 15 min at 4°C. After centrifugation, the supernatant was collected and kept frozen at -20°C until analyzed. The hepatic oxidative stress biomarkers were determined as total antioxidant capacity (TAC) according to Pisoschi and Negulescu21, lipid peroxidase (LPO) and reduced glutathione (GSH) according to Rahman et al.22. Additionally, the activity of superoxide dismutase (SOD) was assessed using colorimetric kits of Randox Laboratories Company, UK, according to the method described by Nishikimi et al.23. While, activities of hepatic aspartate transaminase (AST) and alanine transaminase (ALT) were determined using kits of Diamond Diagnostic Company, Egypt according to the method of Reitman and Frankel24.
Statistical analysis: All data were statistically analyzed using SAS (version 9.2). Ratios and percentages were arcsine-transformed prior to statistical analysis to meet the assumption of normality. Growth performance, feed efficiency, the chemical composition of the whole fish body, haematological, serum biochemical parameters and hepatic oxidative stress biomarkers of hybrid red tilapia following different treatments (T1-T4) were subjected to one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Differences were considered statistically significant at p<0.05.
RESULTS
Growth performance and feed utilization parameters: Growth performance parameters (FW, TWG, ADG and SGR) significantly improved by increasing levels of dietary Aquafat-O®, where T4 recorded the highest values of these parameters, followed by T3 and T2 compared to the control group (T1) (p<0.05, Table 3). Also, fish fed 6% Aquafat-O® kg–1 diet showed the best significant values of FCR and PER and in the same time the lowest value of EU among other dietary levels of Aquafat-O® (p<0.05, Table 3). However, the differences in K-factor, survival rate %, FI and PPV among treatments were insignificant (p>0.05).
Chemical composition of the whole fish body: The effects of dietary Aquafat-O® on the chemical composition of hybrid red tilapia are shown in Table 4. No significant differences (p>0.05) in the body water content among all treatments were observed. The ash and protein contents increased and fat content (p<0.05) reduced with increasing levels of Aquafat-O®. Fish fed 6% Aquafat-O® kg–1 diet exhibited the highest (p<0.05) values of ash and protein contents and the lowest value of fat content compared to other treatments.
Haematological measurements: Haematological parameters (Hb and RBCs) and blood indices (MCV and MCHC) were significantly increased by increasing the levels of Aquafat-O® compared to the control group (p<0.05, Table 5). Fish in T4 followed by T3 gave the highest significant (p<0.05) values of Hb, RBCs, MCV and MCHC among other treatments.
| Table 3: | Effect of dietary Aquafat-O® levels on growth performance and feed utilization parameters of hybrid red tilapia |
Values are Mean±SE, Values in the same row with different superscript letters are significantly different (p<0.05), TWG: Total weight gain, ADG: Average daily gain, SGR: Specific growth rate, K: condition factor, FI: Feed intake, FCR: Feed conversion ratio, PER: Protein efficiency ratio, PPV: Protein productive value, EU: Energy utilization | |
| Table 4: | Chemical composition of the whole body (% on dry matter basis)of hybrid red tilapia fed different levels of Aquafat-O® |
Values are Mean±SE (n = 6), Values in the same row with different superscript letters are significantly different (p<0.05) | |
| Table 5: | Effect of dietary Aquafat-O® levels on haematological parameters of hybrid red tilapia |
Values are Mean±SE, Values in the same row with different superscript letters are significantly different (p<0.05), Hb: Hemoglobin, RBCs: Red blood cells, PCV: Packed cell volume, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, WBCs: White blood cells | |
While, no significant differences (p>0.05) of PCV, MCH and WBCs parameters were detected among all levels of Aquafat-O®.
Serum biochemical parameters: The gradual significant decrease of the serum glucose concentration by increasing levels of dietary Aquafat-O®, where T4 had the lowest significant (p<0.05) value of glucose compared to the other treatments as shown in the Fig. 1a. Also, the control group (T1) had the highest levels of serum total cholesterol, triglycerides and HDL among all treatments (p<0.05, Fig. 1b-d). Dietary 2% Aquafat-O® kg–1 recorded the lowest and the highest values of serum HDL and LDL, respectively compared to the other treatments (p<0.05, Fig. 1d, e).
Oxidative stress biomarkers: Significant (p<0.05) increases of the hepatic TAC by increased Aquafat-O® levels, especially at T4 compared to other treatments was displayed in Fig. 2a. In the same time, SOD activity increased and LPO diminished with higher Aquafat-O® levels (p<0.05, Fig. 2b, d). However, T2 had the lowest significant values of the hepatic GSH and AST activates among other treatments (Fig. 2c, e) (p<0.05). No significant differences (p>0.05) of the hepatic GSH between the control group and other treatments (T3 and T4) were detected. In the same trend, there are no significant differences between treatments of the hepatic ALT activity (Fig. 2f, p>0.05).
| Fig. 1(a-e): | Serum biochemical parameters analysis of hybrid red tilapia fed different levels of Aquafat-O®. Concentrations of (a) Serum glucose, (b) Total cholesterol, (c) Triglycerides, (d) High density lipoprotein (HDL) and (e) Low density lipoprotein (LDL) |
Data are shown as Mean±SE. Vertical bars indicate standard error, mean with different small letters indicate significant difference between treatments (p<0.05) |
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| Fig. 2(a-f): | Oxidative stress biomarkers analysis of hybrid red tilapia fed different levels of Aquafat-O®. (a) Hepatic total antioxidant capacity, (b) Superoxide dismutase, (c) Reduced glutathione, (d) Lipid peroxidase, (e) aspartate transaminase (AST) and (f) Alanine transaminase (ALT) |
Data are shown as Mean±SE. Vertical bars indicate standard error, mean with different small letters indicate significant difference between treatments (p<0.05) |
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DISCUSSION
Growth in fish associated with abundant food and appropriate environmental condition, as well as is a good indicator of fish health25. Results in this study exhibited the positive impact of the dietary inclusion of Aquafat-O® on growth performance and feed utilization of hybrid red tilapia, especially at level 6% Aquafat-O® kg–1 diet. This increase might be correlated to augmenting of haematological parameters as well as TAC and SOD, which indicate enhancing the immune responses of hybrid red tilapia. This improvement was possibly attributed to containing Aquafat-O® on PUFAs such as LA (18:2 n-6) and LNA (18:3 n-3). Freshwater fish required the dietary PUFAs as LA and LNA for optimum growth8,9. A similar response has been reported in other fish species such as Labeo rohita26, hybrid tilapia O. niloticus×O. aureus9, adult O. niloticus27 and O. niloticus fry28. On the other hand, the improvement of FCR and PER may be attributed to enhance of the fish palatability of feed by dietary addition of Aquafat-O®, which reflected on increased growth performance. The current findings are in agreement with those obtained by Khalil et al.27, who indicated that O. niloticus fed Aquafat-O® increment of their FI. However, Li et al.9 stated that addition 1% LA of hybrid tilapia O. niloticus×O. aureus does not affect feed efficiency.
The body composition parameters are good indicators of the physiological situation of a fish29. The chemical composition of fish is affected by fish feed. In the present study, the chemical composition of whole-body has improved, through increased protein content and reduced the fat content of hybrid red tilapia by increasing levels of dietary Aquafat-O®. The findings compatible with the decline of serum total cholesterol and triglyceride that obtained herein. Similar results obtained by Khalil et al.27 and Tan et al.30 on adult O. niloticus and Synechogobius hasta, respectively. However, Senadheera et al.31 found that no significant differences in chemical composition fillets by the addition of LNA/LA in diets of Murray cod.
Haematological parameters have been studied to evaluate the requirement of certain dietary micronutrients and the quality of feed or feeding strategies32. The data in the present study revealed the beneficial effects of dietary Aquafat-O® on haematological parameters, suggesting enhance the immunity and health status of fish. This enhancement could be attributed to contain Aquafat-O® on PUFAs like LNA, LN and OA. LNA plays an important constituent of the cell membrane of blood, capable of preserving the integrity of construction and functions of the cell membrane and an increase of lymphocyte33. Likewise, oleic acid plays a role in the activation of different pathways of immune competent34. The same trend observed in Nile tilapia by Al Samarae35 and Ferreira et al.36. Inversely, Li et al.9 found that addition of dietary LA and LNA did not effect on haematological measurements of hybrid tilapia, O. niloticus×O. aureus. The variations between the obtained results herein or those achieved by other researchers could be due to the fish species, age, physiological statues, type of dietary lipid or fatty acids and their levels.
A serum composition and biochemical constituent are the best indicators of the general and health status of the fish37. The reduction of serum glucose, total cholesterol, triglycerides and HDL of red tilapia fed different levels of Aquafat-O® observed in this study probably led to the decrease of crude fat in the whole fish body. In this respect, He et al.38 revealed that the serum total cholesterol and triglycerides contents reflected the absorption of lipid. Moreover, the reduction of serum total cholesterol by dietary Aquafat-O® might be due to the inclusion of highly percentages of PUFAs, which changed the cholesterol metabolism, including fecal secretion of steroids39 and the hepatic synthesis of cholesterol40. Similarly, Hui et al.41, who cleared the negative correlations between dietary lipid levels from one side and serum total cholesterol, triglycerides, LDL and VLDL-C of juvenile hybrid tilapia, O. niloticus×O. aureus from anther side. Likewise, Al Samarae35 suggested that adult O. niloticus males fed dietary Aquafat-O® up to 1% kg–1 diet led to reducing of serum total cholesterol, triglycerides and HDL, while these parameters were increased in the case of adult O. niloticus females. Additionally, the decline of glucose could be results of increase of glucose metabolism, which reflected to enhance the fish growth performance. Sanchez-Muros et al.42 also indicated that the growth promotion and protein sparing related to glucose, which is the preferred oxidative substrate for nervous tissue and blood cells.
Improvement of the antioxidant defense system recorded of the experimental fish as a result of feeding the graded levels of Aquafat-O®, subsequently the immune response of fish has been raised. Aquafat-O® led to increasing the hepatic TAO and SOD activities. TAO is an overall indicator of antioxidant defense against free radical. The SOD is a cytosolic enzyme that works on scavenging superoxide radicals and involved in protective mechanisms within tissue injury following the oxidative process43. This improvement was may be attributed to Aquafat-O® comprise the fatty acids as, LA and LNA, which play vital roles in triggers an immunological response to activating antigens44. At the same time, PUFAs affect immune cell structure44 and that might cause the elevation of the ability of the cells to resist the oxidative stress. Mourente et al.45 stated that dietary levels of lipids have been influence antioxidant defenses and oxidative status of fish.
Oxidative stress leads to occurrence lipid peroxidation that causes cellular injuries and damage46. The results of the present study showed the decrease of LPO, which perhaps resulting in decrease both of fat content in the whole fish body and serum total cholesterol, triglyceride and HDL. It has been reported that fish have several physiological ways to eliminate oxidative stress, including antioxidants enzymes like SOD and GSH47. Consequently, the increase of TAO and SOD may be caused to decrease of LPO. Similarly, Mourente et al.45 and Rueda-Jasso et al.48, who reported that dietary levels of lipids and the use of more saturated vegetable oils (such as palm oil) could be reducing oxidative stress and stimulated the liver antioxidant defense enzymes. Additionally, the results recorded a significant increase in the activities of the hepatic AST and ALT enzymes of red tilapia fed the graded levels of Aquafat-O®. Similar results were reported by Hui et al.41 and Al Samarae35. More recently, Lei et al.49 indicated that no significant differences of the serum AST and ALT concentrations of grass carp fed dietary addition of a-LA.
CONCLUSION
Finally, it could be concluded that dietary addition of Aquafat-O® at the highest level (6%) tested had positive effects on growth performance, feed utilization, physiological and immunity responses of hybrid red tilapia. Hence, using of dietary Aquafat-O® may be more usefulness agent in fish farming and hatcheries.
According to the physiological findings obtained, advanced studies are needed not only to determine the optimal level of dietary Aquafat-O® against different types of stressors such as the water cold stress, mycotoxins, high stocking density, water pollutants on fish species, but also for more understanding the relation between dietary lipid levels, types or sources and the antioxidant defense system of fish in different life stages.
SIGNIFICANCE STATEMENT
This study discovers the determination of the appropriate levels of Aquafat-O® (as a source of lipid and fatty acids), through evaluation the effects of Aquafat-O® on growth, physiological and immunity responses of hybrid red tilapia. That can be beneficial for avoiding the drastic effects of some stressors on fish and increasing the fish growth and production, improving the physiological responses, besides the expected economic efficiency or the environmental friendly effects. So, this study will help the researcher to uncover the critical areas of intensification of Aquaculture that many researchers were not able to explore. Thus a new theory on this useful feed additive (Aquafat-O®) only or possibly with other combinations by other feed additive (s) may be arrived at.