The production of rainbow trout depends on the responses of the consumer market. Consumers require fish with better flesh quality and appearance. Carotenoids, which belong to the natural constituents of salmonid fish feed, may help meet the latter requirement (Rehulka, 2000). It is important to use carotenoids in fish feeds. These give yellow, red and pink to the skin, flesh and eggs of fish ( Latscha, 1990). Pigments play an important role in the diet of animals and animal feed production industry. The carotenoids in the normal and abnormal environment fish are available and have positive effects on fish. Muscle coloration in the natural environment and fish that are fed with natural food, due to its accumulation in muscle is orange. (Zamanpor et al., 2005). Carotenoids are the main pigments of many aquatic animals. In salmonids, astaxanthin is responsible for the typical red colour of the flesh (Foss et al., 1984; Torrissen, 1986; Storebakken et al., 1987). As is the case with other, salmonids can not endogenously synthesize astaxanthin; therefore, it must be supplemented in the fish's ration. Research also indicates additional benefits from dietary carotenoids beyond the resulting coloration (Bell et al., 2000). Salmon meat staining depends to factors such as ration diet, growth rate, management conditions, amount and duration of use of pigments and etc. In addition it could be useful to creating the color of carotenoids in fish flesh, increase growth, boost the immune system by increasing antibody production, reduce stress, increase survival and fertilization, etc. (Meyers, 1977;Wouterset al., 2001; Guerin et al., 2003; Goodwin, 1984(. Astaxanthin, which is the most effective on pigmentation, exists densely in water organisms such as gammarus, copepods and starfish (Diler, 1997; Torrisen, 1989a; 1989b).One of the most important role of the pigments is biological functions related to growth, reproduction and tissue health in salmonids and shrimp, possibly due to the compound's strong antioxidant properties (Bell et al., 2000). It is not harmful to the human and could be anticancerogen and antioxidant (Schiedt, 1988). It provides salmon flesh with its characteristic rich pink-red color and accounts for more than 90% of the total carotenoid content found in the flesh of wild salmonids (salmon and trout). In the nature, salmonids absorb astaxanthin from the consume of crustaceans. The absorbed carotenoid is then transported in the blood to the muscles and skin where it is deposited (Bell et al., 2000). A number of studies on astaxanthin were published by Christiansen who examined the issue from the viewpoint of the effects of dietary astaxanthin supplementation on fertilization and egg survival in Atlantic salmon (Christiansen and Torrissen, 1997), growth and survival of Atlantic salmon juveniles (Christiansen and Torrissen, 1996), and first-feeding fry (Christiansen et al., 1995a), antioxidant status and immunity in Atlantic salmon (Christiansen et al., 1995b) and effects of astaxanthin and vitamin A on growth and survival during the first feeding of Atlantic salmon (Christiansen et al., 1994). Besides pigmentation, carotenoids are involved in certain physiological functions, as pointed out by (Nakano et al., 1995). who studied the biochemical characteristics of the liver and blood in rainbow trout fed a diet supplemented by red yeast (Phaffia rhodozyma). containing astaxanthin as its principal carotenoid pigment or synthetic astaxanthin. It follows from the literature survey that there is little knowledge of the effect of astaxanthin on blood factors and no investigations on growth indicators of the rainbow trout. The purpose of this study was investigation on effect of this pigment on the growth and coloration and blood indices of the rainbow trout.
2. MATERIALS AND METHODS
2.1. Feeding experiment
The experiment was conducted in pools of Mr.Sarshar farm that was located in the 3000 Road of Tonekabone region of Mazandaran province. Pools dimension in this experiment were (4mÃ-3mÃ-1m) and time period was 60 days in autumn season. The fish were fed 3 times on a daily basis. On days 0, 20, 40 and 60, all fish remaining in each cage were weighed. Throughout the experiment, the temperature, dissolved oxygen level and pH of the rearing water were8.64±0.965 °C, 9.12±0.379 mg1-1, 7.1±0.293, respectively. Water supply was river that originated from 3000 river.
The two different treatments used in the experiment were as follows:
Treatment A: fed with commercial normal diet (control group)
Treatment B: Diet supplemented with Astaxanthin 41 mg/kg
The commercial basal diet was supplied by Faradane company (Table 1) and synthetic astaxanthin was produced by Behparvar company. The experimental diet was made by adding the requisite amounts of the different pigment sources to a commercial diet. Food processor was done every tertian. For preparation ration, initially a small amounts of oil with pigments are well mixed and spray to pellets.Then the daily food of each pool was weight with a digital scale and packed.
Table 1. Nutritional composition of control diet (basal diet)
Chemical composition
Percentage (%)
Crude protein
48
Crude fat
12
Crude ash
12
Dry matter
10
300 pieces of rainbow trout with an average weight of (93±0.64 g) in a completely randomized design were compared for 8 weeks in the same breeding conditions. Fish were feeding three times a day (8, 12, 16) and feed required per day according to the weight of biomass in different time periods (usually after each bioassay) was calculated. Once every 20 days; randomly, 10 number of fish selected for biometry from each replicate, and then weighted with a digital scale with precision (0/01 g) and length was measured by millimeter ruler. In order to reduce stress in fish during the biometry, feeding was discontinued 12 hours before and after the biometry. Blood tests were done at the end of the research period that included RBC, WBC, HCT, Hb, IgM, Lym and Mono.The weight and length of fish in each pool, statistical calculations values of FCR, percentage of BWI, SGR, GR, CF and survival rate were calculated.
2.2. Colour measurements
During the experimental period, trout were sampled four times. The first sampling was done at the beginning of the experiment, and the subsequent samplings were carried out at 20, 40 and 60 days. In all of the samplings six trout were sacrificed. Each trial included three replicates. The total weight and total length of each trout was measured, and three fillets were obtained per trout in order to matching with SalmoFanTM.
2.3. Preparation of the blood samples
Blood samples were taken from 10 fish that caught randomize. The sampling was performed 20 hours after the last feeding during the forenoon, the samples were taken by puncturing the caudal veins immediately after catching and stunning amount of 2cc. Blood taken from the fish into micro test tubes containing a drop of heparin (anticoagulant) was poured and shake gently until completely mixed blood and heparin. To prepare serum hemolysis mentioned tubes were centrifuged at 3500 RPM for 15 min. Then, serum was removed by sampler tool of hemolysis tube and then was transferred into micro test tubes with a volume of 2cc (Jamalzadeh, 2001).
2.4. Statistical analysis
The data analyzed by SPSS 13 and Excel 2003 soft wares. For statistical analysis, the homogeneity of the control data, averages of blood factors were examined (Shapiro Wilk test) through Kruskal-Wallis and Man-Witney nonparametric test and the average weight and length measurements were calculated by ANOVA, the confidence level was %95 and Duncan Test were employed.
3. RESULTS
Water physicochemical parameters were measured including dissolved oxygen, temperature, pH its effect on feeding, breeding all the time these factors were carefully controlled (Table 2).
Results of water quality parameters were revealed no significant difference during the breeding period (p> 0.05).
Table 1. Water physicochemical parameters
Factor
average
Min.
Max.
Oxygen
9.12±0.379
8.14
9.91
Temperature
8.64±0.965
7.11
9.9
pH
7.1±0.293
6.6
7.8
Factor
average
Min.
Max.
Oxygen
9.12±0.379
8.14
9.91
Growth data of the fish on days 0, 20, 40 and 60 are summarized in Table 3. the first average weight was 93±0.64 g and mean total length was 18.1 ± 0.25 cm. Fish biometry performed on the basis of weight during the breeding period and revealed a significant difference between treatments (p<0.05), but there was no significant difference in terms of length (p>0.05) (Table 3 and 4). The lowest weight found in control group and the greatest weight was received, in astaxanthin group, but no significant difference was found in terms of performance parameters including growth and food consumption FCR ØŒSGRØŒ GRØŒ %BWI,CF and survival rate between different treatments was observed (P>0.05) (Table 5).The most SGR, BWI, GR, CF and survival rate was found related to astaxanthin and most FCR related to control group(Table 5).
Table 3. Mean weight (g) of the fish fed various diets.
Diets
0 day
20 day
40 day
60 day
Control
93±0.64 a
113.95±1.02 a
132.46±2.18 a
157.62±2.48 a
Astaxanthin
93±0.64 a
116/65±1/68ab
138/67±1/42 b
168/29±2/99 b
Each value is a mean ± s.e. (n = 3 replicates).Each replicate consists of measurements from 10 fish.The means with different letters in each column denote a significant difference (P < 0.05)
Table 4. Mean length (cm) of the fish fed various diets.
Treatments
Parameters
Control
Astaxanthin
Treatments
Parameters
RBC (Ã- 106/μL)
1006111±169314
1347000±253519
RBC (Ã- 106/μL)
WBC (/μL)
4133.3±715.9
9233.3±1019.8
WBC (/μL)
Hb (gr/dl)
6.51±1.27
8.68±1.65
Hb (gr/dl)
HCT (%)
37.11±7.32
43.89±7.39
HCT (%)
Lym (%)
62.44±12.3
96.11±2.85
Lym (%)
Each value is a mean ± s.e. (n = 3 replicates). Each replicate consists of measurements from 10 fish.The means with different letters in each column denote a significant difference (P < 0.05)
Table 5. Mean growth parameters of the fish fed various diets.
Diets
0 day
20 day
40 day
Control
18.1±0.25 a
18.83±0.17 a
22.54±0.45 a
Astaxanthin
18.1±0.25 a
19.33±0.44 ab
22.66±0.8 ab
Diets
0 day
20 day
40 day
Control
18.1±0.25 a
18.83±0.17 a
22.54±0.45 a
Astaxanthin
18.1±0.25 a
19.33±0.44 ab
22.66±0.8 ab
Each value is a mean ± s.e. (n = 3 replicates).Each replicate consists of measurements from 10 fish.The means with different letters in each column no significant difference (P > 0.05)
Table 6. Mean blood factorsof the fish fed various diets.
Diets
FCR
SGR
BWI%
GR
CF
survival
Diets
Control
4.55±0.28
35.28±0.22
19.02±1.29
1.26±0.06
1.13±0.03
91.33±2.31
Control
Each value is a mean ± s.e. (n = 3 replicates). Each replicate consists of measurements from 10 fish.
*( On the basis of NaparametricKruskal-Wallis and manwitny tests, the separate character is not used (a,b) )
Results revealed that coloration of the fish was fed with astaxanthin showed significant change color than the control group was created. According to the SalmoFanTM, degree of color was 28. In this experiment, blood factors including RBC, WBC, Hematocrit, Hemoglobin, Lymphocytes and IgM revealed significant differences between treatments (p<0.05) but Monocytes and Neutrophils, were showed no significant difference between treatments (p>0.05) (Table 6). Most RBC, WBC, Hb, HCT, Neu,Mono, Lym and IgM were associated with astaxanthin.
4. Discussion
So the carotenoids should be used as a food supplement. Obviously, the carotenoid pigments in salmon biology are a sign of normal matter (Wozanik, 1996).In the present study, growth of rainbow trout was affected by the addition of synthetic carotenoids into their diet, because at the end of the feeding trial high increase their initial weight was achieved. Rations used in these experiments revealed significant differences in relation to weight and blood factors of the twenty-second day (p<0/05) and treatment astaxanthin provided better conditions than the control group for the fish. Much research in this area used in aquaculture. However, conflicting results have been reported. The results of this experiment with studies in Atlantic salmon fed astaxanthin (Christiansen et al., 1997; 1995)ØŒRodeus uyekii fed astaxanthin (Kim etal., 1999) and penaeus monodon fed with Dunaliella extract (Supamattaya, 2005) Is consistent. The results with research in rainbow trout fed with synthetic astaxanthin (Nakano et al., 1995; Amar et al., 2004) and penaeus monodon and penaeus japonicus using astaxanthin, beta-carotene and cantaxanthin ( Yamada et al., 1990; Chein et al., 1992 & Boonyaratpalin et al., 2001; Buyukcapar, et al., 2007 ) Been done is contradictory. This paradox may react differently to the cultured species or different life stages it is associated. Among the possible causes of weight gain can be a positive effect astaxanthin on metabolism, Accelerate and increase the efficiency of digestion and absorption of nutrients (Amar et al., 2001; Tacon, 1981), sufficient duration of the pigment (Supamattaya, 2005), suitability of fish weights considered in this study and most importantly, the optimal diet for maximum growth irritable and fish growth in the use of astaxanthin noted. In studies on the survival of penaeus monodon fed with astaxanthin (Gocer etal. 2006) is similar. astaxanthin coloration due to Salmo FanTM was 28° that with findings similar to those of (Mehrabi et al., 2010; Yanar et al., 2007; Zarinfar and Mollaei, 2005; Ghyasvand et al., 2009) and with (Bjerkeng et al., 2000; Erdem, 2009; Ando et al., 1986) is contradicted. This difference may be due to rearing conditions and age of the fish is feeding time.Pigments also raise the immune system and increase the resistance which with findings of (Amaninejadetal., 2009; Bjerkenget al., 2000; Torrisenet al., 1996) was similar. Amaninejad announced that carotene in algae due to increased resistance of immune system. Treatments in this study, which were fed with astaxanthin had increased immune system compared to control diet. Most RBC, WBC, Hb, HCT, Lym, Neu and IgM treated to astaxanthin.(Faghani and coworkers, 2009) reported the level of Hct 21/1±0/65, HB 5/23±0/64 and Lym 82±5/1. Rehulka and coworkers in 2009, examined effect of astaxanthin on blood factors on rainbow trout and level of RBC(1.06 vs. 1.15 T/l)ØŒHB) 71.8 vs. 76.5 gr/l), Hct(0.386 vs. 0.422) announced. Mccarthy and colleagues (1973) number of RBC (1.7 -1.2)Ã-106 per cubic millimeter count, hemoglobin 9-7 g dl and hematocrit 32-45% reported. (Haley & Weiser, 1985) reported the average number of RBC (1.5 ± 0.16)Ã-106 per cubic millimeter count in rainbow trout. Astaxanthin was found to be more effective than control group. Although salmonids absorb astaxanthin 10-20 times as other pigments (Torrisen et al., 1989). This development is important for commercial aquaculture and thus be market-friendly.
5. Conclusion
In conclusion, treatment that fed with astaxanthin in terms of coloration, growth and immune resistance were in better condition than the control group According to studies and information gaps can be use of animal sources contain carotenoids due to expensive synthetic carotenoid pigments and purification by knowledge and technology of biological pigments, use appropriate levels of carotenoids in the diet of male and female productive aquatic, generation to provide better quality for future studies were suggested.
Acknowledgment
Authors appreciate from Mr. Belash Sarshar, Fish breeder cellar, Mr. Erfanshahkar, Mr. Farshad Mahisefat, Mr. Ravin Shojaei and all those who Participated in the financial affaires for their financial support of the experiment.
