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Gangadharappa, H.V, Pramod, K.T.M and Shiva, K.H.G. (2007) Gastric floating drug delivery systems: a review. Indian J. Pharm. Ed. Res. 41, 295-305..

Shang, Y. C. and Tisdell, C. A. (1997). Economic decision making in sustainable aquaculture development. In:

Stickney, R. R. and McVey, J. P. (2002). Responsible Marine Aquaculture, New York, CABI Publishing.

Mokhtari-Abkenari, A. ,Chizeri, M. & Salehi, H.(2007). The Study of the Attitude of

the Experts of Iran's Fisheries towards Sustainable Aquaculture, Journal of Agricultural Extension and Education, Iran, Vol. 2. No 2.

Udoto, M. and Flowers, J. (2001). Perceptions of agricultural education teachers toward sustainable agricultural practices. 28th annual national agricultural education research conference, p.433.

Williams, D. L. and Wise, K. L. (1997). Perceptions of Iowa secondary school

agricultural education teachers and students regarding sustainable agriculture. Journal of Agricultural Education, 38(2), 15-20.

Study of amino acids profile in Persian sturgeon larvae (Acipenser persicus) fed Artemia and daphnia

Seyedeh Sedigheh Babaei1*, Abdolmohammad Abedian Kenari1, Rajabmohammad Nazari 2

1Fisheries Department, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, P.O.Box 64414-356. Noor, Mazandaran, Iran. Sedigheh.babaei@yahoo.com.

2 Shahid Rajaee Sturgeon Hatchery Center, Sari, P.O. Box 833, Sari, Mazandaran, Iran.

Abstract

The aim of this study is comparison essential amino acids in Persian sturgeon larvae fed with Artemia and daphnia. The larvae were obtained from artificial propagation. After yolk sac absorption (9-10 days post hatching (dph)), fish larvae were fed with Artemia urmiana until day 14 and then fed with Daphnia sp. to 40 dph, according to standard hatchery procedures. The sampling were obtained at 14 and 40 dph. Results showed Arginine, Leucine and Methionine were highigest value and Histidine was lowest value (0.9 %) among indispensable amino acid (IAA) profiles in Persian sturgeon larvae. While Methionine and Arginine were highest value in Artemia and Daphnia. Histidine (0.79 %) and Phenylalanine (0.8%) were lowest value in Artemia and Daphnia, respectively. The comparison between amino acid profiles in larvae and live foods showed Phenylalanine was limiting amino acid in 14 day (-65.5 %) and 40 day (-73.4 %), therefore, it should be add to diets.

Key words: Amino acid, Persian sturgeon, Larvae, live food.

Persian sturgeon lives in the southern part of the Caspian Sea and it is one of the vulnerable fish species (kiabi et al. 1999). Therefore, the artificial propagation of these species is essential for restocking. The lack of nutritionally balanced diets, during early life stages, is the main bottleneck for rearing technology of this species. During early developmental stages, amino acids are important fuel molecules and major substrates for the synthesis a large number of bioactive molecules and proteins. The main objective of the present study was to provide the data of amino acids profile in Persian sturgeon larvae and their foods, in order to obtain data about amino acid requirements.

Materials and methods

This study was carried out at Iranian governmental sturgeon hatchery, Shahid Rajaei (Sari, Iran). The larvae were obtained from artificial propagation and transferred to 3 culture tanks. The larvae were obtained from artificial propagation of one female (26.33 kg) and one male (19 kg) and transferred to 3 culture tanks. The water temperature 17-18°C, pH 8 and oxygen saturation 5.5- 7 mg/l were measured during the culture period. After yolk sac absorption (9-10 dph), fish larvae were fed with Anemia urmiana (Artemia Research Center, Uromieh, Iran) until day 14 and then fed with Daphnia sp. to 40 dph, according to standard hatchery procedures.

The sampling was obtained at 14 and 40 dph. Larvae and live foods (Artemia urmiana and Daphnia sp.) were washed and stored at -80°C for analysis. For determination of amino acid contents of larvae, the samples were hydrolysed in 6 N HCl for 24 h at 110 °C in glass vials replaced with nitrogen. Derivatization of amino acids in the samples, was used by o-phthaldialdehyde (OPA) as a pre-column derivatization reagent, followed by high pressure liquid chromatography with fluorescence detection and spherical type column, all parts were from Knauer Corporation, Berlin, Germany, using the method of Lindroth and Mopper (1979) as modified by Flynn (1988). Indispensable amino acids (IAA)

data are expressed in weight percentage of the IAA pool ((weight of one IAA) x (weight of all IAA)-1 x100) (Saavedra et al., 2007). The first limiting amino acid was determined by the formula: (IAA diet - IAA larvae)*100*(IAA larvae)-1 (Conceicao et al., 2003).

Result and Discussion

The predominant indispensable amino acid observed in Persian sturgeon larvae were Arginine, Leucine and Methionine whereas Histidine presented the lowest relative IAA levels. Histidine and Phenyl-alanine were lowest value in Artemia and Daphnia, respectively (Table 1). The comparison between amino acid profiles in larvae and live foods showed was limiting amino acid in 14 day and 40 day (Table 2). This means larvae must spend more time catching live preys to compensate the dietary amino acid losses (Aragao et al., 2004). Since Persian sturgeon is one of the most economically important species in aquaculture and restocking purposes of Caspian Sea, Phenyl-alanine and other essential amino acids (histidine and lysine) supplementation in Persian sturgeon diets seemed necessary for optimal growth.

Table 1. The Essential amino acids profile in Persian sturgeon and live foods (g 100gprotein-1)

 

 

Amino acid

 

Daphnia

 

14 DPH

40 DPH

Atemia

 

Arg

8.05±0.34

9.57±0.14

8.58±0.62

9.39±0.44

His

0.95±0.05

0.9±0.02

0.79±0.8

0.82±0.03

Ile

5.89±0.48

5.23±0.12

6.95±0.65

6.72±1.01

Leu

8.65±0.36

8.13±0.20

8.08±0.63

8.79±0.56

Lys

2.32±0.14

3±0.23

2.2±0.08

2.02±0.13

Phe

3.01±0.02

2.9±0.08

1±0.02

0.8±0.01

Thr

6.18±0.18

6.08±0.14

6.47±0.23

6.01±0.45

Val

3.52±0.2

2.14±0.11

3.59±0.18

3.76±0.12

Met

8.14±0.87

11.02±1.03

8.9±0.28

9.57±0.46

TEAA

46.71±1.04

48.97±1.29

46.56±1.13

47.88±0.59

Table 2. Relative differences between IAA from the live foods (Artemia and Daphnia) and Acipenser persicus (limiting AA is marked in bold)

EAA

Larvae age 14 DPH

40 DPH

Arg

-10.34

16.64

His

-12.22

-13.68

Ile

32.88

14.09

Leu

-0.61

1.61

Lys

-26.66

-12.93

Phe

-65.51

-73.42

Thr

6.41

-2.75

Val

67.75

6.81

Met

-19.23

17.56

References

Aragao, C.; Conceigao, L.E.C.; Fyhn, H.J. and Dinis, M.T., 2004. Estimated amino acid requirements during early ontogeny in fish with different life styles: gilthead seabream (Sparus aurata) and Senegale sole (Solea senegalensis). Aquaculture, Vol. 242, pp.589-605.

Conceigao, L.E.C., Grasdalen, H., R0nnestad, I., 2003. Amino acid requirements of fish larvae and post-larvae: new tools and recent findings. Aquaculture. 227: 221­232. DOI:10.1016/S0044-8486(03)00505-2.

Flynn, K.J., 1988. Some practical aspects of measurements of dissolved free amino acids in natural waters and within microalgae by the use of HPLC. Chemistry and Ecology. 3: 269-293. DOI: 10.1080/02757548808070848.

Kiabi, B.H.; Abdoli, A. and Naderi, M., 1999. Status of fish fauna in the south Caspian basin of Iran. Zoology in the Middle East, Vol. 18, pp.57-65.

Lindroth P., Mopper K., 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Analyt Chem, Vol. 51: 1667-74. DOI: 10.1021/ac50047a01.

Saavedra, M., Beltran, M., Pousao-Ferreira, P., Blasco, J., Dinis, M.T., Conceigao, L.E.C., 2007. Evaluation of bioavailability of individual amino acids in Diplodus puntazzo larvae: Towards the ideal dietary amino acid profile. Aquaculture. 263: 192-198. DOI: 10.1016/j.aquaculture.2006.10.027.

The effects of dietary administration of Selenium enriched yeast on growth performance and disease resistance to Yersinia ruckeri of the rainbow trout, (Oncorhynchus mykiss)

Amir Tukmechi1, Raheleh Shahraki 21

1 Department of Pathobiology and Quality Control, Artemia and Aquatic Animals Research Institute, Urmia University, Urmia, Iran.

2 Department of Biology, Faculty of Science, Urmia University, Urmia, Iran.

Introduction

The trace element, selenium (Se), has been well recognized as being essential in humans and animals such as fish because of its significant role in the activity of glutathione peroxidase enzyme, which protects cell membranes from damage (Miezeliene et al, 2011). In higher organisms Se plays a critical role in maintaining oxidative and immune status, and is thus essential for the preservation of optimal health (Brown and Arthur, 2001). In salmonids Se deficiencies lead to ataxia, muscular dystrophy, lipid peroxidation and decreased plasma and hepatic glutathione peroxidase activity (Poston et al.,1976). Studies in catfish show that sub-optimal dietary Se decreases disease resistance and organic Se is more potent than selenite in restoring immune defences (Wang et al., 1997). In farmed fish, requirements for Se may be elevated due to the low availability of Se from diets containing fishmeal and the effects of various physical and environmental stressors (Rider et al., 2009).

Selenium enriched yeast frequently taken as a dietary supplement or is applied in relatively large doses as a preventive or therapeutic (Behne et al., 2009). Se utilization may increase during stress due to an increased demand for antioxidant enzymes, including GSH-Px and Trx-R. Indeed trout fed practical diets and subjected to confinement stress require additional Se for the maintenance of optimal oxidative status (Kucukbay et al., 2009). Recent studies in rainbow trout embryos reported that SeMe treatment resulted in the formation of superoxide and the subsequent consumption of reduced glutathione (Palace et al.,2004). It is generally

believed that the ingestion of the organic Se compounds is better and safer than that of the inorganic Se. Hence, the safe use of selenium yeast is permitted as a source of selenium in animal dietary by the Food and Drug Administration (Wang et al2010). The aim of this study was to assess the effects of dietary administration of Selenium enriched yeast on growth performance and disease resistance to Yersinia ruckeri of the rainbow trout.

Materials and methods

Rainbow trout of 50±5 g average weight were obtained from a commercial fish farm in Urmia, Iran. Fish were allowed to acclimate themselves to the laboratory conditions for 10 days before starting the experiment. During 60 days,600 rainbow trout were fed diets supplemented with different dosages of selenium enriched yeast at 1x 106, 1x107, 1x108 CFU g-1 or a control diet. Sampling was scheduled on days of 0, 30 and 60 for biometery. After the feeding trial, remaining fish of each treatment were challenged by pathogenic Yersinia ruckeri and kept under observation for 14 days to record clinical signs and daily mortality rate. Fish were fed with commercial pelleted diet (Faradaneh, Iran) at 3% of body weight daily.

Results

The results showed that diet supplemented with selenium enriched yeast in all treatment groups significantly promoted the growth parameters compared to control group (Table 1). In addition, the lowest fish mortality was obtained in 1 x 108 CFU g-1 treatment.

Table 1. Growth performance of rainbow trout fed diets with selenium enriched yeast. Each value (X± SD) is the average performance of 30 fish/treatment for growth performance for a period of 60 days.

Items

Control

1x106 CFU g1

1x107 CFU g1

1x108 CFU g1

Final weight (g)

92.38±7.01a

99.9±3.5b

106.64±8.7c

105.53±2.16c

Weight gain (%)

217.87±16.5a

240.6±7.1b

260.19±14.9c

255.47±7.7c

Condition factor

1.248±0.01a

1.245±0.01a

1.261±0.02a

1.262±0.02a

Specific growth rate (%)

3.22±0.5a

3.23±0.2b

3.354±0.5c

3.353±0.1c

The same superscript alphabets in the same row are not significantly different at P < 0.05.

Discussion and conclusion

Selenium is an essential component of the antioxidant enzyme and is needed for proper functioning of the immune system and is an essential micronutrient for fish, birds, humans, and many microorganisms (Fujishima et al., 2011). Kucukbay et al (2009) showed that An increased requirement of Se for optimal oxidative status as a result of confinement stress has recently been reported in trout fed practical diets. Also, Supplementation with Se improved growth and antioxidant status of fish and the effects of selenomethionine were relatively greater than sodium selenite. As reported by several authors (Cotter et al., 2008; Lin and Shiau, 2005; Wang et al., 2007) selenium deficiency might result in growth depression, and Se supplemented diet can improve growth performances of fish.

Our results contrary to the findings Lorentzen et al (1994) and Monteiro et al (2007) found that diets supplemented with either inorganic or organic selenium forms from 1 to 2 mg kg-1 levels did not affect the growth of fish species. Our experiment indicated that selenium enriched yeast influenced the increase of the growth performance and immunity, and it is appropriate for supplementation in the diet of rainbow trout.

Behne, D., Albe,r D., Kyriakopoulos, A. 2009. Effects of long-term selenium yeast

supplementation on selenium status studied in the rat. Journal of Trace Elements in Medicine and Biology, 23: 258-264.

Brown, K.M., Arthur, J.R., 2001. Selenium, selenoproteins and human health: a review.

channel catfish fed organic and inorganic sources of selenium. Public Health Nutr. 4

(2B), 593-599.

Cotter, P.A, Craig, S.R., McLean, E., 2008. Hyper accumulation of selenium in hybrid bass: a functional food for aquaculture? Aquacult. Nutr. 14: 215-222.

Fujishima, Y., Ohsawa, M., Itai, K., Kato, K., Tanno, K., Turin, T.C., Onoda T, Endo, S., Okayama, A., Fujioka,T. 2011. Serum selenium levels are inversely associated with death risk among hemodialysis patients. Nephrol Dial

Transplant, 26: 3331-3338.

Kucukbay, F.Z., Yalak, H., Karaca, I., Sahin, N., Tuzcu, M., Cakmak, M.N. 2009. The effect of dietary organic or inorganic selenium in arainbow trout (Oncorhynchus mykiss) under crowding conditions, Aquaculture Nutrition, 15: 569-576.

Lin, Y.H., Shiau, S.Y., 2005. Dietary selenium requirements of juvenile grouper, Epinephelus malabaricus. Aquaculture 250, 356-363.

Lorentzen, M., Maage, A., Julshamn, K., 1994. Effects of dietary selenite or selenomethionine on tissue selenium levels of Atlantic salmon (Salmo solar).

Aquaculture 121, 359-367.

Miezeliene, A., Alencikiene, G., Gruzauskas, R., Barstys, T. 2011. The effect of dietary selenium supplementation on meat quality of broiler chickens. Biotechnology Agronomy Society Environment, 15: 61-69.

Monteiro, D.A., Rantin, F.T., Kalinin, A.L., 2007. Use of selenium in matrinx~ a feed, Brycon cephalus. Braz. J. Anim. Health Prod. 8, 32-47.

Palace, V.P., Sapllholz, J.E., Holm, J., Wautier, K., Evans, R.E., Baron, C.L. 2004. Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss) embryos can generate oxidative stress. Ecotoxicol Environ Saf , 58:17-21.

Poston, H.A., Combs Jr, G.F., Leibovitz, L., 1976. Vitamin E and selenium interrelations in the diet of Atlantic salmon (Salmo salar): gross, histological and biochemical deficiency signs. J. Nutr. 106, 892-904.

Rider, S.A., Davies,S.J., Jha, A.N., Fisher, A.A., Knight, J., Sweetman, J.W. 2009.

Supra-nutritional dietary intake of selenite and selenium yeast in normal and stressed rainbow trout (Oncorhynchus mykiss): Implications on selenium status and health responses. Aquaculture, 295: 282-291.

Vidal, D., Bay, S.M., Schlenk, D. 2005. Effects of Dietary Selenomethionine on Larval Rainbow Trout (Oncorhynchus mykiss). Arch. Environ. Contam. Toxicol, 49: 71-75.

Wang, C., Lovell, R.T., Klesius, P.H., 1997. Response to Edwardsiella ictaluri challenge by Channel Catfish Fed Organic and Inorganic Sources of Selenium. Journal of Aquatic Animal Health, 9: 172-179.

Wang, Y., Hanb, J., Lia, W., Xua, Z.. 2007. Effect of different selenium source on growth performances, glutathione peroxidase activities, muscle composition and selenium concentration of allogynogenetic crucian carp (Carassius auratus gibelio). Anim. Feed Sci. Tech. 134, 243-251.

Wang, Z., Zhang, L., Tan, T. 2010. High cell density fermentation of Saccharomyces cerevisiae GS2 for selenium-enriched yeast production. Korean Journal of Chemical and Engineering, 17(6): 1836-1840.

Effect of different stocking density on catalase and

superoxide desmutase during ontogeny of rainbow trout (Oncorhynchus mykiss)

Samira Sharifi1, Amir Parviz Salati1, Peyman Asadian2, Einolah Gorjipor3

1 Department of Fisheries, Faculty of Marine Natural Resources, Khoramshahr University of Marine Science and Technology

2 Department of Veterinary Medicine, Faculty of Veterinary Medicine, University of Lorestan

3 Iranian Fisheries Research Organization

Abstract

The objective of this study was to determine the effects of stocking density on the activity of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) in eggs and larvae of Rainbow trout (Onchorhynchus mykiss). Three levels of stocking, 200, 400 and 600 egg/lit were used in this study. Activity of SOD and CAT was measured during fertilization, cleavage, organ generation, eyeing, hatching and feeding activity. Activity of mentioned enzymes did not show a significant difference between treatments. Our results showed that stocking density had no effect on enzymes activity during ontogeny in O. mykiss.

Key word: egg, larvae, superoxide dismutase, catalase, Oncorhynchus mykiss

Introduction

The egg and larval stage of fish life are the most sensitive and important stages because fishes in these stages are especially vulnerable to changes associated to environmental stress such as temperature, oxygen and pollution (Von Westernhagen, 1988). Generation of ROS is

an inescapable part of aerobic life. All aerobic organisms have evolved a diverse array of mechanisms to minimize impacts due to ROS. The mechanism for scavenger of ROS are include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) and by non- enzymatic defenses such as glutathione, vitamin C, vitamin E, P-carotenes and ubiquinones (Sanz et al., 2010). The stocking density is known as a biological stress factor in aquaculture. Environmental conditions including stocking density influence genetic components, some stress factors, enzyme activities and oxygen consumption (Aksakal et al., 2011). A rise in the energy demands, imposed by crowding is offset by metabolic adjustments (van der Boon et al., 1991;Wedemeyer, 1996; Vijayan et al., 1990), and this may enhance the generation of ROS.

The aim of the present study was to determine the effects of density on activity of superoxide dismutase and catalase in egg and larvae of O. mykiss.

Materials and methods

Brood stocks of O. mykiss were obtained from a 4 year flock from Research and Breeding center of Coldwater fishes of Shahid Motahari in Yasooj.

Adult fish selection was carried out based on physical health and gamet quality of fishes. After fertilization, the dead eggs were removed and healthy eggs were distributed into three densities including normal (400 egg/lit), low (200 egg/lit) and high (600 egg/lit) density with water temperature of 10.8'C.

Samples were taken on 1, 3, 8, 16, 31, and 48 dpf stages. These stages correspond respectively to fertilization, cleavage, organ generation, eyeing, hatching and feeding activity. Samples were frozen in liquid N2 and stored at -80'C until analysis.

The whole embryos or larvae samples were homogenized in 7 volumes of ice-cold 20mM-phosphate buffer (pH 7-4) containing 1mM-EDTA using ultra-turrax. The homogenates were centrifuged at 1000g for 10min

at 4'C to remove debris and frozen in liquid N2 and stored at -08'C until analysis. The activity of CAT was assayed using Goth (1991) method and activity of CAT was assayed using Maklund (1974) method.

Result

there was no significant difference in superoxide dismutase and catalase activity with increasing density on 1, 3, 8, 16, 31, and 48 dpf stages (p>0.05) (fig 1-1 and 2-1).

■ 15

II

& low density normal density 'j/r M high density

0

1 3        /dpf   16       31 4/

Fig 1. Changes in the activities of SOD of each different days of sampling (1,3,8,16,31 and 48 dpf) between densities (low, normal and high)

ri

/

6

4

1 3        /   dpf 16       31 4/

Fig 2. Changes in the activities of CAT of each different days of sampling (1, 3, 8, 16, 31 and 48 dpf) between densities (low, normal and high).

There is not enough Information about the effects of density on catalase and superoxide dismutase activities in egg and fish larvae. Some researchers reported effect of density on activity of Antioxidant enzymes in other stage in fishes and Crustaceans. Results of present study did not indicate significant difference in superoxide dismutase and catalase activity with increasing density in 1, 3, 8, 16, 31, and 48 dpf stages (p>0.05), that agree with results that obtained from study of Trenzado et al., (2009) in rainbow trout which considered catalase enzyme and also agree with results of study Li et al., (2006) on Fenneropenaeus chinensis which considered superoxide dismutase enzyme. Aksakal et al., (2011) reprted that increasing increase in stocking density attenuates activity of antioxidant enzymes in O. mykiss.

There is not enough Information about the effects of density on catalase and superoxide dismutase activities in egg and fish larvae. Some researchers reported effect of density on activity of Antioxidant enzymes in other stage in fishes and Crustaceans. Results of present study did not indicate significant difference in superoxide dismutase and catalase activity with increasing density in 1, 3, 8, 16, 31, and 48 dpf stages (p>0.05), that agree with results that obtained from study of Trenzado et al., (2009) in rainbow trout which considered catalase enzyme and also agree with results of study Li et al., (2006) on Fenneropenaeus chinensis which considered superoxide dismutase enzyme. Aksakal et al., (2011) reprted that increasing increase in stocking density attenuates activity of antioxidant enzymes in O. mykiss.

References

Aksakal, E., Ekinci, D., Erdogan, O., Beydemir, S., Alim, Z. and Ceyhun, S.B., 2011. Increasing stocking density causes inhibition of metabolic-antioxidant enzymes and elevates mRNA levels of heat shock protein 70 in rainbow trout. Livestock

Science, 141: 69-75.

Goth L., 1991. A simple method fpor determination of serum catalase activity and revision of reference range.ClinicaChimica Acta,196:143-152.

Li, Y., Li, J. and Wang, Q., 2006. The effects of dissolved oxygen concentration and stocking density on growth and non-specific immunity factors in Chinese shrimp, Fenneropenaeuschinensis. Aquaculture, 256: 608-616.

Maklund, S., 1974.Involvement of superoxide anion radical in autoxidation of pyrogallol and a convenient assay of superoxide dismutase. European Journal of Biochemistry, 46: 469-474.

Sanz, A., Dfaz, M.E., Furn6, M., Trenzado, C.E., Garcfa-Gallego, M. and Domezain, A., 2010. Antioxidant defences in the fi rst life phases of the sturgeon Acipensernaccarii. Aquaculture, 307: 123-129.

Trenzado, C.E., Morales, A.E., Palma, J.M. and Higuera, M., 2009.Blood antioxidant defenses and hematological adjustments in crowded/uncrowded rainbow trout (Oncorhynchusmykiss) fed on diets with different levels of antioxidant vitamins and HUFA. Comparative Biochemistry and Physiology, 149: 440-447.

Wedemeyer, G.A., 1996. Physiology of fish in intensive culture systems.Northwest Biological Science Center, National Biological Service.U.S. Department of the Interior, Chapman and Hall, International Thompson Publishing.

van der Boon, J., van den Thillart, G.E.E.J.M. and Addink, A.D.F., 1991. The effects of cortisol on intermediary metabolism in teleost fish. Comparative Biochemistry and Physiology, 100A: 47-53.

Vijayan, M.M., Ballantine, J.S. and Leatherland, J.F., 1990. High stocking density alters the energy metabolism of brook charr, Salvelinusfontinalis. Aquaculture, 88: 371-381.

Von Westernhagen, H., 1988. Sublethal effects of pollutants on fish eggs and larvae. In: W.S. Hoar. and D.J. Randall, eds. Fish Physiology. Vol, 11. The Physiology ofDevelopingFish, Part A, Eggs and Larvae. Academic Press Inc Book Department in San Diego, pp. 253-346.

Effects of replacement fish oil by vegetable oils on histology and micromorphometry of duodenum of rainbow trout, Oncorhynchus mykiss

Rasoul Shahrooz 1, Naser Agh 2, Nasim Jafari 3, Ali Kalantari4, Reza Jalili5' Forouzan Bagherzadeh Lakani 6

1 Department of Anatomical Sciences, School of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Fisheries, Artemia and Aquatic Animals Research Institute, Urmia University, Iran

3' 5' 6 Department of Fisheries, Faculty of Natural Resources, Urmia University, Iran 4 Graduated from School of Veterinary Medicine, Urmia University, Urmia, Iran.

Abstract

This study was performed to examine the effect of replacing fish oil with vegetable oils on histology, micromorphometry and histochemical structure of intestine in rainbow trout. Five experimental diets containing different vegetable oil were tested: 1), Kilka oil (Clupeonella sp.) 2), canola oil (CO) 3) safflower oil (SO) 4) linseed oil (LO) 5) a mixture of canola oil (40%), linseed oil (30%) and safflower oil (30%)(CSLO). Rainbow trout with a mean initial weight of 15±0.2 g were fed experimental diets for 60 days. Samples of intestine from six fish per tank were taken at the end of the experiment, fixed in 10% buffered formalin, paraffin sections of 6 |im thickness were prepared and stained with PAS and H&E and observed by light microscopy. Histomorphometrical study of duodenum revealed that mean diameter and length of folds, height of epithelium, mean distribution of goblet cells (except in safflower oil), in response to replacement of vegetable oils increased significantly (P<0.05).Whereas, thickness of submucosal layer and tunica muscularis significantly decreased (P<0.05). These data revealed a positive role of vegetable oils on absorbing features of duodenum.

Key words: Rainbow trout, Duodenum, Histomorphometry, Fish oil, Vegetable oil.

As world food-grade fisheries have reached sustainable limits, the demands upon the aquaculture industry are increasing, therefore salmonid farming has expanded drastically and so has the need for a reliable feed supply. Aquaculture is based upon feeding fish appropriately under adequate conditions and it needs to develop nutritionally balanced diets in particular concerning protein and lipid (Olsen et al., 2003; Wassef et al., 2007).

Fish meal and fish oil have historically been the dominant raw materials in the production of fish feeds and supply the major portions of protein and lipid in commercial rations of fish in semi-intensive and intensive culture systems (Caballero et al., 2002; Wassef et al., 2007). Plant oils stand out as the most likely candidates to partly substitute for fish oils in fish feeds because of the limited availability of fish oil and fish meal, together with the increase in aquaculture (SOFIA, 2008). The inclusion of vegetable oils (VOs) in the diets of carnivorous fish yields growth rates similar to those obtained under FO diets (Caballero et al., 2002; Glencross et al., 2003; Mourente et al., 2005). A number of studies have shown that they can replace significant parts of the fish oil in diets for salmonids without compromising growth, feed efficiency or reproduction (Dosanjh et al., 1984; Hardy et al., 1987; Thomassen and Rosjo, 1989; Greene and Selivonchick, 1990; Labbe et al., 1993; Guillou et al., 1995). However, digestive and absorptive processes can be affected and recent data have shown that salmonid gastrointestinal (GI) tract may be damaged by high levels of vegetable oils (Olsen et al., 2003), so this study considers effects of replacement fish oil by vegetable oils on histology and micromorphometrical features of duodenum of rainbow trout, Oncorhynchus mykiss.

Material and methods

Fish were purchased from a local trout farm and acclimated for 2 weeks during which they were fed commercial diet. Forty fish with average weight of 15±0.2 g were stocked in 15 polyethylene tanks (300

L) supplied with freshwater at a flow rate of 7.5 L min-1. Light/dark cycle was 12 L:12 D. Water quality parameters were monitored daily for each tank and pH, temperature and dissolved oxygen were maintained at 7.3-7.7, 14-15°C and 6.8-7.5 mg L-1, respectively.

Five experimental diets with similar protein, lipid and energy content were formulated to contain different vegetable oil sources to replace fish oil (Table 1). The first diet contained only the Kilka oil (Clupeonella sp.) was the primary sources of oil in the control diet (FO). The second diet supplemented with canola oil (CO) containing high levels of oleic acid 18:1n-9. The third diet contained safflower oil (SO) containing high levels of linoleic acid 18:2n-6. The fourth diet supplemented with linseed oil (LO) containing high levels of linolenic acid 18:3n-3. The fifth diet supplemented with a mixture of canola oil (40%), linseed oil (30%) and safflower oil (30%) was designed to provide moderate amount of 18:1n-9, 18:2n-6 and 18:3n-3 fatty acids (CSLO).

Fish meal was defatted three times using a 2:1 mixture of hexane and ethanol (400 mL100 g-1 fish meal) the primary sources of protein in experimental diets (Miller et al. 2007). Briefly, all dry ingredients were thoroughly mixed in a mixer. Oil was added and thoroughly mixed for 5 min and then moistened by adding cold distilled water until stiff dough yielded. The wet dough was grinded and converted to strands (3 mm in diameter) using a meat grinder. The strands were dried at 50°C for 8 h using an oven. Afterwards, they were manually crumbled into appropriate size and sieved. Pellets were stored at 4°C during the experiment. Fish were fed three times per day at 3% body weight for 8 weeks. Samples of intestine from six fish per tank were taken at the end of the experiment, fixed in 10% buffered formalin, dehydrated in a graded ethanol series and embedded in paraffin. Sections (6 |m) were stained with PAS and haematoxylin and eosin (H&E) and observed at light microscopy. The results were analysed using analysis of variance, one way ANOVA, for which the homogeneity of variances and the normal distribution were tested according to the Levene and Shapiro_Wilk tests, and comparison among the means was made using Bonferroni's test. All statistical analyses were conducted using SPSS

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