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Gibson GR & Roberfroid MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 125,. 1401-1412.

Lepage, G. and Roy, C.C., 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res., Vol. 25,

pp. 1391-1396.

Patterson J.A., Burkholder M.K., Prebiotic feed additives: rationale and use in pigs. Proceedings of 9th International Symposium on Digestive Physiology in Pigs,

Banff. Canada.(2003): 319-331.

Sorgeloos P., Lavens P., Leger Ph., Tackaert W. & Versichle D. (1986) Manual for the culture and use of brine shrimp Artemia in aquculture. Laboratory of brine shrimp Artemia in aquaculture, State University of Ghent, Belgium pp: 319.

Watanabe, T., Kitajima, C., Fujita, S. 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture 34, 115-143.

Effect of Dietary Vitamin C Supplementation on Growth Performance and Body Composition of Caspian brown trout (Salmo trutta caspius Kessler, 1877) Fry

Maryam Khajavi1., Abdolmohammad Abedian-Kenari1*

1Department of Aquaculture, Marine Science Faculty, Tarbiat Modares University,

Noor, Mazandaran, Iran.

* E-Mail; aabedian@yahoo.co.uk

Abstract

This study was conducted to determine the effects of different levels of dietary vitamin C on the growth performance and body composition of Salmo trutta caspius fry (mean initial weight of 100±10mg) for 6 weeks experimental period. Five purified diets contained 0, 100, 500, 1000 and 2000 mg ascorbic acid (LAPP) per kg diet were formulated The results showed that the growth performance of fish was affected by the ascorbic acid content of different diets. The highest survival, body weight gain and SGR were obtained from diet four (1000 mg LAPP per kg diet). No significant difference was observed in the whole-body proximate composition of S. trutta caspius but fat content showed a significant difference. These findings suggest that vitamin C could be added as a dietary supplement for improving physiological performance and optimal growth of S. trutta caspius.

Keywords; Salmo trutta caspius; Vitamin C; Survival; Body Composition

Vitamin C plays an important role in growth and immunity of fish (Lin and Shiau, 2005). It is the most important water-soluble antioxidant and performs as a co-factor in many hydroxylating reactions (Kumari and sahoo, 2005). The most of teleost fish are unable to synthesize ascorbic acid due to the lack of the last enzyme of the biosynthetic pathway: L-gulonolactone oxidase. Therefore, Vitamin C must be supplied via the feed to obtain optimal growth, immunity, reproduction and survival. (Verlhac, 1999; Dobrowski, 2001). The studies also have been shown importance of relationship between vitamin C and properties such as growth, survival and body composition in aquatic animals. (Ai et al, 2006; Bae et al, 2012; Xiao et al 2010; Mai et al, 1998; Falahatkar et al, 2006; Misra et al, 2007) There are a few studies on the nutrition requirements of S. trutta caspius but no information is reported on its requirement of vitamin C. The aim of present study is to determine the effects of different levels of dietary vitamin C on survival, growth and body composition of this fish.

Material and Methods

At the beginning a batch of Caspian brown trout eggs were obtained from the breeding and cultivation center of Gezel Cheshme Kowsar (Tonekabon, Iran). The eggs were transferred to the hatchery of marine sciences faculty of Tarbiat Modares University (Noor, Mazandaran, Iran) and incubated in a rectangular trough with recirculation water. After yolk sac absorption and start of exogenous feeding, some fries were choosing for the experiment. Fries were reared in five groups contained different levels of vitamin C (as mentioned in below) for 6 weeks. Each group was stocked in 90 liter tank in triplicate and each tank was contained 70 fish with initial weigh of 100 ± 10 mg. Fry were fed five times daily and all groups of fish were fed their respective diet by hand to visual satiety (visual observation of first feed refusal)

The formulation of experimental diets was performed by Lindo software at concentrations of 0 or control (D1), 100 (D2), 500 (D3), 1000

(D4) and 2000(D5) mg vitamin C (LAPP) Kg-1 diet according to Table 1.

Tablel: Ingredient (g kg-1) and proximate composition of experimental diet

Ingridients

D1

Diets (mgVit.C Kg1 diet) D2                D3 D4

 

D5

(g Kg"1 food)

0

100

500

1000

2000

Casein1

490

490

490

490

490

Gelatin

135

135

135

135

135

Dextrin

177.3

177.3

177.3

177.3

177.3

Fish Oil2

65

65

65

65

65

Soybean Oil3

65

65

65

65

65

Lecithin3

10

10

10

10

10

Antioxidant3

0.2

0.2

0.2

0.2

0.2

Antifungal3

30

30

30

30

30

Mineral premix3

20

20

20

20

20

Vitamin premix (without vitamin c) 4

2.5

2.5

2.5

2.5

2.5

Vitamin c5

0

0.1

0.5

1

2

Cellulose

5

4.9

4.5

4

3

Proximate composition (g 100g-1 in

Dry Matter)

 

 

 

 

Fat

13.5

12

13.5

12.16

12.6

Ash

8.29

7.84

7.96

8.11

7.91

Protein

51.8

53.29

54.3

53.15

52.56

Moisture

8.26

7.16

7.18

7.83

7.92

1 Fluka (France); 2 MANAQUA (Iran); 3 Ghonche (Iran); 4 Arasbazar (Iran); 5 F.Haffman-La Roch (Sweden)

Growth indices such as: SGR, BWG and survival rate were measured in experimental groups after 6 weeks according to the below formula:

Specific growth rate (SGR) = (Ln final weight - Ln initial weight /duration) *100

Body Weight gain (BWG) = (final weight - initial weight /initial weigh)*100

Survival Rate (%)= The number of fish in first period/ the number of fish in final period*100

The proximate composition of experimental diets and fish whole body were performed using the standard methods of Association of Official Analytical Chemists (AOAC, 1995). Dry matter, crude protein, crude lipid and ash were measured by drying at 105 0C for 24 h, Kjeldahl method, Soxhlet method and combustion at 550 0C, respectively.

All data were subjected to One-way ANOVA analysis by using SPSS 17.0. Differences between the means were determined by Duncan test. The level of significance chosen was P<0.05 and the results are presented as means ± standard deviation (SD).

Results and Discussion

Some common deficiency signs, including scoliosis and lordosis, poor growth, higher mortality and lower SGR were observed in the fish of the control group (without vitamin c). The growth performance of fish was affected by the ascorbic acid content of different diets. As dietary ascorbic acid increased from 0 to 1000 mg kg-1 diet, the SGR increased from 2.55 to 4.06 % day-1 and survival rate increased from 53.33 to 84%. (Table 2). The fish fed the control diet significantly had a lower survival than other groups. Such results were reported from other species (Bae et al, 2012; Xiao et al 2010; Misra et al, 2007 and Ai et al, 2006). But in compare with some species which are able to Vit. C de novo synthesis, results are different. (Falahatkar et al, 2006; Mai, 1998). The metabolic rate is the primary factor regulating the AA requirements. Therefore, fish larva, displaying relatively a faster growth and metabolism than juveniles and adults, might need higher dietary AA levels to sustain optimal growth and physiological condition (Merchie et al, 1997).

No significant difference was observed in the whole-body proximate composition of S. trutta caspius but fat amount showed a significant difference in different groups. Same results on body composition were obtained in other studies (Bae et al, 2012; Xiao et al 2010). Dietary vitamins can affect on metabolism and body composition(Hwang and Lim,2002). The results showed that fat level was high in D4 and it could be due to more metabolism in fat synthesis. These findings were observed in other report (Falahatkar et al, 2006 ).

Table 2: Growth performance and body composition of Caspian brown trout fed different levels of Vit. C

Parameters

 

Diets (mgVit.C Kg-1 diet)

 

 

 

 

0

100

500

1000 2000

Survival%

53.33±17b

71.55±5a

76.07±9a

83.03±4 a

82±6a

BWG(%)

204.81±49c

300.67±25 bc

337.6±71 abc

460.98±121 a

366.66±57 ab

SGR

2.55±00.5 b

3.3±0.15 a

3.49±0.39 a

4.06±0.51 a

3.65±0.3 a

Body composition

 

 

 

 

Ash(%)

1.63±0.08

1.94±0.6

1.56±0.2

1.76±0.4

1.71±0.12

Moisture (%)

85.11±0.6

81.23±5.6

84.53±0.86

82.33±4.2

84.019±0.3

Protein(%)

11.85±0.48

13.79±3.5

11.77±0.79

13.27±3.3

11.99±0.28

Fat(%)

1.19±0.19c

1.74±0.33bc

1.85±0.17b

2.47±0.59 a

1.89±0.04b

Data are presented as mean ± SD of triplicate groups. Values within the same line with different letters represent significant difference (P<0.05)

In conclusion the present study suggests that vitamin C could be added to food as a dietary supplement for improving physiological performance and optimal growth of S. trutta caspius.

References

Ai, Q., Mai, K., Zhang, C., Xu, W., Duan, Q., Tan, B., Liufu, Z. (2004). Effects of dietary vitamin C on growth and immune response of Japanese seabass,. Aquaculture, 242(1-4), 489-500.

AOAC., 2005: Official Method Of Analysis 17th (end), Washington. DC: Association of Official Analytical Chemists.

Bae J.-young, Park G.-hyun, Yoo K.-yeol, Lee J.-yeol, Kim D.-jung, and Bai S. C. (2012). Re-evaluation of the Optimum Dietary Vitamin C Requirement in Juvenile Eel , Anguilla japonica by Using L-ascorbyl-2-monophosphate. Aquaculture, 25, 98 - 103.

Dabrowski K. ( 2001 ). Ascorbic Acid in Aquatic Organisms—Status and Perspectives. CRC Press, Boca Raton, 255- 277.

Falahatkar B B., Soltani, M., Abtahi, B., Kalbassi, M. R., and Pourkazemi, M. (2006). Effects of dietary vitamin C supplementation on performance , tissue chemical composition and alkaline phosphatase activity in great sturgeon ( Huso huso ). Sciences-New York, 22, 283-286.

Hwang D F and Lim T K. (2002). Effect of temperature on dietary vitamin C requirement and lipid in common carp.Comparative Biochemistry and Physiology, Biochemical and Molecular Biology,131,1-7.

Kumari, J., & Sahoo, P. K. (2005). High dietary vitamin C affects growth, non-specific immune responses and disease resistance in Asian catfish, Clarias batrachus. Molecular and cellular biochemistry, 280, 25-33.

Lin M., and Shiau S. (2005). Dietary ascorbic acid affects growth, nonspecific immune responses and disease resistance in juvenile grouper,. Aquaculture, 244, 215-221.

Mai K. (1998). Comparative studies on the nutrition of two species of abalone , Haliotis tuberculata L . and Haliotis discus hannai Ino . Effects of dietary vitamin C on survival , growth and tissue concentration of ascorbic acid. Aquaculture, 161,

383-392.

Misra C. K., Das B. K., Mukherjee S. C., and Pradhan J. (2007). Effects of dietary

vitamin C on immunity, growth and survival of Indian major carp Labeo rohita, fingerlings. Aquaculture Nutrition.

Merchie G., Lavens P., and Sorgeloos P. (1997). Optimization of dietary vitamin C in fish and crustacean larvae: a review. Aquaculture, 155, 165-181.

Verlhac V and Gabaudan J., (1999). The effect of vitamin C on fish health.Centre for Research in Animal Nutrition, Soci6t6 Chimique Roche, BP 170, 68305 Saint-Louis Cedex, France: P. 33.

Xiao L D., Mai K S., Ai Q H., Xu W., Wang X J., Zhang, W B., Liufu Z. (2010).

Dietary ascorbic acid requirement of cobia, Rachycentron canadum Linneaus. Aquaculture Nutrition, 16, 582-589.

Reduce Oxidative Damage in Caspian Brown Trout (Salmo trutta caspius Kessler, 1877) Fry by Using Different Levels of Dietary Vitamin C

Maryam Khajavi1., Abdolmohammad Abedian-Kenari1

1Department of Aquaculture, Marine Science Faculty, Tarbiat Modares University, Noor, Mazandaran, Iran. E-Mail; aabedian@yahoo.co.uk

Abstract

The present study was intended to determine the effects of different levels of dietary vitamin C on two antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) from Salmo trutta caspius fry during 6 weeks. Five purified diets contained 0, 100, 500, 1000 and 2000 mg ascorbic acid (LAPP) per kg diet were formulated for mentioned study. The findings showed that the CAT activity was significantly affected by the ascorbic acid content of different diets, while no significant difference was observed in SOD activity among dietary vitamin C treats. The highest activity of both enzymes was obtained from diet without vitamin C that reveals fry is in oxidative condition and more sensitive.

Keywords: Salmo trutta caspius, Superoxide dismutase, Catalase, Dietary vitamin C

Introduction

Physiological antioxidant protection involves both enzymatic system such as free radical scavenging enzymes, including superoxide dismutase (SOD), and catalase and non-enzymatic system (exogenous dietary micronutrients) such as vitamins (Puangkaew et al., 2005 ; Tocher et al.,

2003). Vitamin C is an important dietary antioxidant, it significantly decreases the adverse effect of free radicals such as reactive oxygen and nitrogen species that can cause oxidative damage to macromolecules such as lipids, DNA and proteins. In addition, ascorbic acid can regenerate other antioxidants such as a-tocopheroxyl, urate and P-carotene radical cation from their radical species. Thus, ascorbic acid acts as co-antioxidant for a-tocopherol by converting a-tocopheroxyl radical to a-tocopherol and helps to prevent the a-tocopheroxyl radical mediated peroxidation reactions (Nuida, 2003). Several studies have examined the influence of nutritional variations on antioxidant enzymes in fish (Lygren., et al 2000; Tocher., 2002; Mourente., 2002; Puangkaew et al 2005). The aim of this study was to examine the impact of dietary vitamin C supplementation on SOD and CAT activity.

Material and Methods

At the beginning a batch of Caspian brown trout eggs were obtained from the breeding and cultivation center of Gezel Cheshme Kowsar (Tonekabon, Iran). The eggs were transferred to the hatchery of marine sciences faculty of Tarbiat Modares University (Noor, Mazandaran, Iran) and incubated in a rectangular trough with recirculation water. After yolk sac absorption and start of exogenous feeding, some fries were choosing for the experiment. Fries were reared in five groups contained different levels of vitamin C (as mentioned in below) for 6 weeks. Each group was stocked in 90 liter tank in triplicate and each tank was contained 70 fish with initial weigh of 100 ± 10 mg. Fry were fed five times daily. The formulation of experimental diets was performed by Lindo software at concentrations of 0 (or control; D1), 100 (D2), 500 (D3), 1000 (D4) and 2000 (D5) mg vitamin C (LAPP) Kg-1 diet according to (Table 1).

End of the experiment amount of 4 fish from each replicate were sampled for the analysis. The muscle were homogenized (1:10, w/v) in homogenization buffer containing 100 mM potassium phosphate buffer (pH 7.4), 100 mM KCl and 1 mM EDTA for 1.5 min. Homogenates were centrifuged at 10,000 g for 30 min (+4 ° C). Supernatants were used as enzyme source (Atli and Canli, 2010).

Tablel: Ingredient (g kg-1) and proximate composition of experimental diets

Ingridients D1

 

Diets (mgVit.C Kg-1 diet) D2                D3 D4

D5

(g Kg-1 food)

0

100

500

1000

2000

Casein1

490

490

490

490

490

Gelatin

135

135

135

135

135

Dextrin

177.3

177.3

177.3

177.3

177.3

Fish Oil2

65

65

65

65

65

Soybean Oil3

65

65

65

65

65

Lecithin3

10

10

10

10

10

Antioxidant3

0.2

0.2

0.2

0.2

0.2

Antifungal3

30

30

30

30

30

Mineral premix3

20

20

20

20

20

Vitamin premix (without vitamin c) 4

2.5

2.5

2.5

2.5

2.5

Vitamin c5

0

0.1

0.5

1

2

Cellulose

5

4.9

4.5

4

3

Proximate composition (g 100g-1 in Dry Matter)

 

 

 

 

Fat

13.5

12

13.5

12.16

12.6

Ash

8.29

7.84

7.96

8.11

7.91

Protein

51.8

53.29

54.3

53.15

52.56

Moisture

8.26

7.16

7.18

7.83

7.92

1 Fluka (France); 2 MANAQUA (Iran); 3 Ghonche (Iran); 4 Arasbazar (Iran); 5 F.Haffman-La Roch (Sweden)

The activity of Superoxide dismutase (SOD, EC 1.15.1.1) was assayed based on the ability of SOD to inhibit the reduction of NBT (Nitroblue tetrazolium) by superoxide anion (Winterbourn et al, 1975; Worthington Enzyme Manual 1993). One unit is defined as the amount of enzyme causing half the maximum inhibition of NBT reduction. Different volumes of extracts were added to cuvettes containing 0.2 mL of 0.1M EDTA, 0.1 mL of 1.5 mM NBT and reached to 3ml with 67 mM phosphate buffer (pH 7.8). Then, 0.05 mL of 0.12 mM riboflavin was added at zero time and at timed intervals. All cuvettes were incubated in a light box for 12 min and absorbance at 560 nm was read at timed intervals by a spectrophotometer (PerkinElmer, Lambda25; USA). The amount of enzyme resulting in 50% of maximum inhibition of NBT reduction was determined. Catalase activity (CAT, EC 1.15.1.1) was determined according to Aebi (1984) by following the decrease in absorbance at 240 nm due to hydrogen peroxide (H2O2) consumption. The difference in absorbance per unit time (60 s in our assay) was used as a measure of CAT activity. Total soluble protein was measured by

the Bradford (1976) method using bovine serum albumin as a standard. Enzyme activities were expressed as specific activity (Unit / mg protein).

Mean values of enzyme activities were compared among the different groups using one-way analysis of variance (ANOVA). Differences between the means were determined by Duncan test. The level of significance chosen was P<0.05 and the results are presented as means ± standard deviation (SD).

Result and Discussion

Catalase activities were significantly decreased in S. trutta caspiuss fed with diet C5 compared with control group and no significant difference was observed in SOD activity among dietary vitamin C treats but SOD activity was lower in diets with higher amount of vitamin C (Table 2). Similary Mourente et al., 2002 repoted the both of catalase and SOD activities at Sparus aurata were reduced by dietary vitamin E as antioxidant after 30 days feeding.

In another study, the level of dietary vitamin E showed some significant effects on the activities of the enzymes of the liver antioxidant defense system of juvenile turbot (Scophthalmus maximu.), halibut (Hippoglossus hippoglossus) and sea bream (Sparus aurata) (Tocher et tal., 2002). No interactions were observed between dietary vitamin E and antioxidant enzyme activities in atlantic salmon but liver SOD and GPX activities were significantly reduced in hyperoxygenated fish although catalase activity was unaffected (Lygren et al. 2000). CAT is a scavenger of H2O2, therefore its high activity in control group could indicate the presence of a large amount of H2O2 in the system (Puangkaew et al., 2005). Decreased production and non-availability of the substrate (O2-) in response to antioxidant may be a reason for decreased SOD activities. (Janssens et al., 2000).

Table 2. SOD and CAT enzyme activity associated with different levels of dietary vitamin C.

Parameters

 

 

Diets (mgVit.C Kg-1 diet)

 

 

0

100

500 1000

2000

SOD

0.13+0.05

0.109+0.02

0.12+0.02 0.09+0.03

0.06+0.04

CAT

0.188±0.01a

0.13+0.08ab

0.12+0.03ab 0.086+0.03b

0.08+0.04b

Data are presented as mean + SD of triplicate groups. Values within the same line with different letters represent significant difference (P<0.05). CAT, SOD; ^mol/min/mg protein

Refrences

Atli G and Canli, M. (2010). Response of antioxidant system of freshwater fish Oreochromis niloticus to acute and chronic metal ( Cd , Cu , Cr , Zn , Fe ) exposures. Ecotoxicology and Environmental Safety, 73, 1884-1889.

Bradford M M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analitical Biochemistry 72, 248-25.

Janssens B J., Childress J J., Baguet, F., Rees J F. (2000). Reduced enzymatic antioxidantive defense in deep sea fish. Experimental Biology, 3725, 3717-3725.

Lygren B., Hamre K., and Waagbe R. (2000). Effect of induced hyperoxia on the antioxidant status of Atlantic salmon Salmo salar L . fed three different levels of dietary vitamin E. Aquaculture Research, 31, 401-407.

Mourente G., Dfaz-Salvago E., Bell, J. G., and Tocher, D. R. (2002). Increased activities of hepatic antioxidant defence enzymes in juvenile gilthead sea bream ( Sparus aurata L) fed dietary oxidised oil: attenuation by dietary vitamin E. Aquaculture, 214, 343 - 361.

Naidu K A. (2003). Vitamin C in human health and disease is still a mystery, An overview. Nutrition Journal, 2, 7.

Puangkaew, J., Kiron V., Satoh S., and Watanabe T. (2005). Antioxidant defense of rainbow trout (Oncorhynchus mykiss ) in relation to dietary n-3 highly unsaturated fatty acids and vitamin E contents. Comparative Biochemistry and Physiology, 140, 187 - 196.

Tocher D. R., Mourente G., Eecken A. V., Evjemo J O., Diaz E., Bell J G., Geurden., Lavens P and Olsen Y. (2002). Effects of dietary vitamin E on antioxidant defence mechanisms of juvenile turbot (Scophthalmus maximus L .), halibut ( Hippoglossus hippoglossus L .) and sea bream ( Sparus aurata .). Aquaculture

Nutrition, 8, 195-207.

Worthington Enzyme Manual. (1993) Superoxide Dismutase. Worthington Biochemical

Corp., Freehold, NJ, pp. 368-369.

Winterbourn C., Hawkins R.E., Brian M. and Correll R.W. (1975) The estimation of red cell superoxide dismutase activity. Laboratory and Clinical Medicine 85, 337­341.

Ingestion Rate of Ornamental Fish Larvae Mayan Cichlid (Cichlasoma urophthalmus) Fed on Live Food Freshwater Cladoceran, Ceriodaphnia quadrangula

Mohammad Hossein Khanjani1*, Omidvar Farhadian2

1 PhD Student of Propagation and Cultivation Aquatics, University of Hormozgan, Iran

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