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1 Department of Fishery, Faculty of Natural Resources, Urmia University.
2 Department of Pathobiology, Artemia and Aquatic Animals Research Institute, Urmia University.
* Corresponding authors: rezvanehjenabi@Yahoo.com
Prebiotics are favorable live microorganisms and, along with their metabolites, are used commonly as feed additives in the rearing of farm animals. The aim of this study was to evaluate the effect of prebiotic Mannan oligosaccharides (0, 2.5 and 5 g/kg of diet)on the growth performance and survival in rainbow trout (Oncorhynchus mykiss). For this purpose, fish were fed with diet containing mentioned concentrations as a prebiotic for 60 days. At the end of the feeding experiment, 10 fish per each replicate were sampled randomly and anaesthetized with Clove powder (200 mg ml-1). Then the total length and total weight were measured for each fish, separately and growth performance parameters were calculated. The obtained results were showed that the use of prebiotic (2.5 and 5 gr) significantly (P<0.05) improved the growth performance of rainbow trout than the control group. Based on this result, we conclude that the use of natural and safe substances instead of antibiotic application for growth enhancing could improve the growth performance of rainbow trout.
Key words: Rainbow trout, Manan®, Growth parameters and survival.
Today, with the recent ban on the use of antibiotic growth promoters in aquafeeds within the EU (Regulation, 2005) alternative nutraceutical products to enhance production and health status is a topic of concerted interest. Prebiotics, such as mannan oligosaccharides (MOS) have proved to be effective at enhancing health and growth performance of fish (Staykov et al., 2007; Torrecillas et al., 2007; Burr et al., 2008), improve gut morphology (Salze et al., 2008; Dimitroglou et al., 2009) and modulate the intestinal microbiota (Dimitroglou etal., 2009). Prebiotics are nondigestible carbohydrates (NDCs) that selectively stimulate the growth and metabolism of health-promoting bacteria already present in the host gut (Ahmadifar et al., 2004). The uses of dietary supplements that are involved in growth and enhance the immune system are strategies that can be helpful in improving health, resistance to the stress and pathogens. Prebiotic beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, which can improve the host health (Gibson and Roberfroid, 1995). Criteria which allow the classification of a food ingredient as a prebiotic include: (1) it must be neither hydrolyzed nor absorbed in the upper part of the gastrointestinal tract, (2) selective fermentation by one or a limited number of potentially beneficial bacteria in the colon, and (3) alteration of the composition of the colonic microbiota toward a healthier composition (Fooks and Gibson, 2002). Among the established prebiotics such as fructooligosaccharide, transgalactooligosaccharide, inulin and mannan oligosaccharide (MOS), MOS is most commonly used as the dietary supplementation for fish and crustacean species (Sang and Fotedar, 2010). MOS is a glucomannoprotein complex derived from the cell wall of yeast (Saccharomyces cerevisiae) (Sang and Fotedar, 2010). Some studies were conducted to investigate the effects of MOS on the growth and immune response of fish and crustaceans. For example, MOS has been shown to Enhances growth in cultured common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), sea bass (Dicentrarchus labrax)
and Jian carp (Cyprinus carpio Var. Jian) (Zhou and Li, 2004; Staykov etal., 2007). Staykov etal (2007) showed Growth, feed efficiency and survival were improved in two experiments with rainbow trout that were fed a diet containing 2 g.kg-1 Mannan oligosaccharide (MOS) compared with those fed the basal diet. Although specified beneficial effects of prebiotics, very little research has been done in this field (Mahhious and Ollevier, 2005). The aim of this study was to evaluate the effect of Manan oligosaccharide (a commerical prebiotic) on the growth parameters and survival in rainbow trout (Oncorhynchus mykiss).
Materials and Methods
Fish and experimental design:
For hundared rainbow trout (10± 1.2 g mean weight) were prepared from a local fish farm, Urmia, Iran. Immediately fish were transferred to the laboratory with oxygen supply and adapted to the laboratory conditions for 10 days, Then, they were divided into three groups with triplicate included: control, fed with commercial pellet (Faradaneh, Iran), group 2 (2.5 grams of MOS per kilogram of diet) and group 3 (5 grams of MOS per kilogram of diet). Fish culture was conducted in 300 liter PVC tanks and forty fish located in each tank. Trial was conducted for 60 days and water quality parameters such as dissolved oxygen (10.3 mg/l), temperature (14.1±2 °C), and pH value (7.5 ± 0.4) were measured every day during the experimental period. Desired amount of MOS (2.5 and 5 gr) dissolved in 10 ml of sterile normal saline and sprayed with 5 ml sunflower oil on diet. Then, allowed to diet dried at the room temperature and at a clean place. Control group only received commercial pellet diet that sprayed with normal saline and sunflower oil and also growth parameters were evaluated on days 0 and 45.
Calculated growth factors
At the end of the trial, ten fish per each replicate were sampled randomly and anaesthetized with Clove powder (200 mg ml-1). Then the total length and total weight were measured for each fish, separately and
growth performance parameters such as Feed conversion ratio, specific growth rate and condition factor were calculated. Growth performance and feed utilization were calculated according to the following formulae: Weight Increase (BWI) = Wt - W0, (FCR) = Wet weight gain in g / dry feed fed in g, (SGR % / d) = (Ln Wt - LnW0) x 100/t and (CF) (g/cm3) = 100 x Wt/Lt3.
The results were subjected to analysis of variance (ANOVA) followed by Tukey test to compare different treatments using the SPSS 15. Correlation coefficients were significant with P< 0.05. Determination of the LD50 was conducted using probit analysis program.
Results and Discussion
The aim of this study was MOS add to the rainbow trout,s diet to increase the economic growth of this species. Environmental protection in terms of the legal, social and ethical is a necessity. MOS supplement can bind to certain gram - negative bacteria and prevent intestinal cloning, so it is a bacteria removal mechanism from gut (Spring etal.,
After 60 days, the results showed: Mortality rates were not statistically significant (P<0.05) among the treatment (2.5 and 5 grams of MOS per kilogram of diet) and control groups. In the third group because of the high level of MOS, fish were compared to a control group and the second group has better conditions.
Table 1. The results of biometrics from fish fed with MOS for 45 days.
16.30 ± 0.31a
17.30 ± 0.50bc
18.43 ± 1.75c
51.19 ± 1.57a
55.75 ± 1.38b
59.99 ± 1.75c
1.27 ± 0.06a
0.94 ± 0.08b
0.91 ± 0.02b
1.16 ± 0.24a
1.26 ± 0.01b
1.31 ± 0.02c
1.17 ± 0.03a
1.13 ± 0.07ab
0.95 ± 0.04b
These data are shown in the Standard Deviation ± Mean.
MOS has been reported to increase growth in some terrestrial vertebrate animals (Savage etal., 1997) and crustacean (Daniels 2006; Genc4 etal., 2007; Hai and Fotedar, 2009).The results of the current study indicate that dietary supplementation with 2.5 and 5 g.kg-1 Mannan oligosaccharide had significant effects (p <0.05) on rainbow trout growth\ performance, and feeding parameters such as, body weight increase, length increase, specific growth rate, feed conversion ratio except from the group treated with 5 g.kg-1 MOS treatment that showed a significant differences condition factor, compared with control and seconde groups. Staykov etal. (2007) used 2 g.kg1 MOS to evaluate the effect on growth performance and immune status of rainbow trout (Oncorhynchus mykiss) and found significantly improved growth performance, weight gain, reduced feed conversion ratio, reduced mortality, and improved indicators of immune status compared with controls, and also like the rainbow trout (Yilmaz etal., 2007). By the way, in many studies, it seems improvements in growth performance are reported due to higher MOS supplement in marine fish species; for example, using two levels of MOS (2 and 4%) in diet of juvenile sea bass (Dicentrarchus labrax) makes 10% improvement in growth and specific growth rates (Torrecillas etal., 2007). In contrast to positive effects of MOS, studies by Pryor etal., (2003) on Gulf sturgeon (Acipenseroxyrinchus desotoi) that were fed with 0 and 3 g.kg-1 Mannan oligosaccharide and Genc4 etal., (2006) on African catfish (Clarias gariepinus) that treated with different levels of dietary Mannan oligosaccharide (1, 2 and 3%) for 80 day. The experiment indicated that the prebiotic Mannan oligosaccharide could be used in rainbow trout diets as a growth promoter. MOS is an interesting prospect for replacement of growth-promoting in the aquaculture industry and could be a useful tool in the rearing of delicate early stages of certain marine species and we conclude that the use of natural and safe substances instead of antibiotic application for growth enhancing could improve the growth performance of rainbow trout. On the other hand, the use of them is suitable and has benefits effects in environmental pollutions.
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Burr, G., Hume, M., Neill, W.H., Gatlin III, D.M., (2008). Effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by red drum Sciaenops ocellatus (Linnaeus). Aquac. Res. 39, 1680-1686.
Daniels, C., (2006) Developing and understanding the use of Bio-Mos in critical stage of European lobster (Homarus gammarus) culture. The National Lobster Hatchery, UK.
Dimitroglou, A., Merrifield, D.L., Moate, R etal., (2009). Dietary mannan oligosaccharide supplementation modulates intestinal microbial ecology and improves gut morphology of rainbow trout, (Oncorhynchus mykiss). Am Soc Anim Sci87:3226-3234.
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Genc, M.A., Aktas, M., Genc, E., Yilmaz, E., (2007). Effects of dietary mannan oligosaccharide on growth, body composition and hepatopancreas histology of Penaeus semisulcatus (de Haan 1844). Aquac. Nutr. 13, 156-161.
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Hai, N.V., Fotedar, R., (2009) Comparison of the effects of the prebiotics (Bio-Mos and b-1, 3-D-glucan) and the customised probiotics (Pseudomonas synxantha and P. aeruginosa) on the culture of juvenile western king prawns (Penaeus latisulcatus Kishinouye, 1896). Aquaculture 289:310-316.
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Salze, G., McLean, E., Schwarz, M.H., Craig, S.R., (2008). Dietary mannan oligosaccharide enhances salinity tolerance and gut development of larval cobia. Aquaculture 274, 148-152.
Savage, T.F., Zakrzewsla, E.I, Andreasen, JR., (1997). The effect of feeding mannan oligosaccharide supplemented diets to poult on performance and morphology of the small intestine. Poult Sci 76:139.
Spring, P., Wenk, C., Dawson K.A et al., (2000). The effects of dietary mannan oligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella- challenged broiler chicks. Poult Sci 79:205-211.
Staykov, Y., Spring, P,. Denev, S et al (2007). Effect of a mannan oligosaccharide on the growth performance and immune status of rainbow trout (Oncorhynchus mykiss). Aquacult Inter 15:153-161.
Torrecillas, S., Makol, A., Caballero, M.J., Montero, D., Robaina, L., Real, F., Sweetman, J., Tort, L., Izquierdo, M.S., (2007). Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides. Fish Shellfish Immunol. 23, 969-981.
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Fish meal replacement by plant protein and the problem of anti-nutritional factors
A. Keramat Amirkolaie
Department of fisheries, Faculty of animal science and fisheries, Sari Agricultural and natural resources university, Km 9 Darya Boulvard, P.O.Box: 578, Sari, Iran
Replacement of fish meal by plant protein has been introduced as an alternative to alleviate the problem of high cost and availability of fishmeal. However, the full substitution of fishmeal by plant ingredient is almost impossible due to anti-nutritional factors. The main goal of this study is to investigate the most important anti-nutritional factors in plant ingredients and introduce a solution to reduce the impact of anti-nutritional factors on aquaculture industry. Protease inhibitors are widespread in many oil seed meals such as soybean meal which reduce the digestibility of protein in gastro intestinal tract. Heating process and purification can reduce the negative impact on growth and digestibility. Glucosinolates in canola and mustard meal are associated with thyroid dysfunction and liver haemorrhage in fish. Saponins can increase permeability of small intestine thereby inhabit active nutrient transport. Non-starch polysaccharides which are present in oils seed meals, change physico-chemical properties of digesta. This condition has a negative impact on the nutrients digestibility and intestinal morphology.
Key words: plant ingredients, fishmeal, aqua-feed, canola, non-starch polysaccharide
Aquaculture has shown a rapid growth during past three decades and accounted for 45.7 percent of the world's fish food production for human consumption in 2008 (Turchini et al.,
2009; FAO, 2010). This growth rate is faster than any other terrestrial animal meat production sector (poultry and livestock: FAO, 2008 and 2010). The sustainable growth in aquaculture depends largely on external feed input so as meet the demand (Tacon and Metian, 2008). The fin fish and crustacean aquaculture sector is still largely dependent upon marine capture fisheries for supplying key dietary nutrient inputs, including fish meal and fish oil. Fishmeal as a major element of aqua-feed is an expensive commodity and also has an uncertain future (Tacon and Metian, 2008). Replacement of fish meal by plant protein mainly oil seed has been introduced as an alternative to alleviate this problem (Thiessen et al., 2004; Gatlin et al., 2007). However, the full substitution of fishmeal by plant ingredient is almost impossible due to anti-nutritional factors. These factors can reduce nutrients digestibility (Francis et al., 2001) and have a negative impact on gut health (Iwashita et al 2008 and 2009). Therefore, the main goal of this research is to investigate the most important anti-nutritional factors in oil seeds and introduce a solution to reduce the impact of anti-nutritional factors on aquaculture industry.
Anti-nutritional factors (ANF)
ANF can be defined as substances which interfere with food utilization and affect the health and production of animals. ANF may be divided in a number of groups according to their function.
1. factors affecting protein utilization and digestion, such as protease inhibitors, tannins, lectins
2. factors affecting mineral utilization, such as phytates, gossypol pigments, oxalates, glucosinolates
4. miscellaneous substances such as mycotoxins, mimosine, cyanogens, alkaloids, saponins and phytoestrogens.
Protease inhibitors are widespread in many oil seed meals such as soybean meal which reduce the digestibility of protein in gastro intestinal tract (Francis et al., 2001). Protein inhibitors are protein molecules, which have the ability to inhibit the activity of proteolytic enzymes within the alimentary tract. They act through binding to digestive enzymes and make them unavailable for protein hydrolysis which in turn will reduce the digestibility of protein and availability of protein for growth. It seems that below the 5 mg/g level, most cultured fish are able to compensate for the presence of trypsin inhibitors by increasing trypsin production. Heating process and purification can reduce the negative impact on growth and digestibility.
Phytate is as a major anti-nutritional factor in canola, soybean meal and sesame meal. Growth in commonly cultured fish species, such as carp, tilapia, trout and salmon, is negatively affected by inclusion of phytate containing ingredients in the diet (Francis et al. 2001). This substance depresses bio-availability of minerals especially phosphorous and also impaired protein digestibility caused by formation of phytic acid-protein complexes. Phytates, particularly in cereals, are concentrated in the outer endosperm. Milling to remove the outer layer of seeds therefore reduces the phytate content of the seed considerably. Addition of phytase enzyme and/or degradation lead to a lower level of phytate.
Glucosinolates are the main ANF present in rapeseed meal and mustard oil cake, which are potentially attractive protein sources in fish feeds. The presence of intact glucosinolates has been correlated with the occurrence of liver haemorrhage. Thyroid abnormalities (increased thyroid activity acitivity) have been observed in carp, tilapia, salmon and
rainbow trout when fed diets containing glucosinolates (Francis et al. 2001). Over the long term, thyroid dysfunction induced by continuous consumption of glucosinolate containing feed is certain to affect metabolism and growth in fish. Heat treatment is effective in reducing the glucosinolate content of feed materials. Extracting with water was found to be a cost-effective method of removing glucosinolates from full-fat and fat-free Moringa oleifera kernels (Makkar and Becker, 1997).
Saponins are steroid or triterpenoid glycosides found in many of the potential, alternate plant-derived feed ingredients for fish, like soybean meal, lupin seed meal, pea seed meal and sun flower oil cake. The negative effects of saponins could be caused by the effects of these surface-active components on biological membranes. Saponins can increase permeability of small intestine thereby inhabit active nutrient transport. Because of the high solubility of most saponins in water, aqueous extraction would remove most saponins from feed ingredients.
Non-starch polysaccharides (NSP)
NSP are polymers of monosaccharides joined through glycosidic linkages. NSP form the major part of dietary fiber. Dietary fiber can be physiologically defined as "the dietary components resistant to degradation by mammalian enzymes" or chemically as "the sum of NSP and lignin (Bach Knudsen and Jorgensen 2001). NSP are generally resistant to mammalian digestive enzymes (Iji, 1999). On basis of their reaction with water, NSP are classified as either soluble or insoluble. Non-starch polysaccharides which are present in oils seed meals, change physico-chemical properties of digesta. This condition has a negative impact on the nutrients digestibility and intestinal morphology. While Inclusion of low levels of purified cellulose in the diet of several fish species can have positive effects on fish performance, Very high inclusion levels of insoluble NSP have negative effects in tilapia. Soluble NSPs have been shown to reduce growth performance in several fish
species. Digesta viscosity and moisture increased with increasing concentration of soluble NSP which is associated with a depressed fat and protein digestibility. The use of NSP degrading enzymes has been successful in eliminating anti-nutritive effects of soluble NSP present in wheat, barley and other cereal based diets. Pre-fermentation of plant ingredients has also been attempted in order to reduce NSP contents and therefore improve fish performance (Skrede et al. 2002).
In future, fish diet will have a larger content of plant ingredients thereby more problems associated to ANF. It is necessary to design experiments in order to determine the impact of ANF on feed efficiency and gut heath in commercial species. In addition, measuring concentration of ANFs and their interaction effects in component feed is very important. Such studies would provide useful data for designing optimum inclusion levels of plant ingredients and treatment methods that would neutralise the negative effects of the anti-nutritional factors.
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Gatlin, D.M., Barrows, F.T., Brown, P., et al.,2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture Research 38, 551579.
Iwashita, Y., Yamamoto, T., Furuita, H., Sugita, T., Suzuki, N., 2008. Influence of certain soybean antinutritional factors supplemented to a casein-based semipurified diet on intestinal and liver morphology in fingerling rainbow trout Oncorhynchus mykiss. Fish. Sci. 74, 1075-1082.
Iwashita, Y., Suzuki, N., Matsunari, H., Sugita, T., Yamamoto, T., 2009. Influence of soya saponin, soya lectin, and cholyltaurine supplemented to a casein-based semipurified diet on intestinal morphology and biliary bile status in fingerling rainbow trout Oncorhynchus mykiss. Fish. Sci. 75, 1307-1315.
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Effects of dietary xylo oligosaccharide on fatty acid profile of Artemia urmiana
Behzad Khafaji Rad1*, Naser Agh2, Mehran Javaheri Baboli3, Khadije Najafi1
1* Department of fisheries, Science and Research Branch, Islamic Azad University, Khouzestan Iran
2 Artemia and Aquatics Research Institute, Urmia University, Urmia, Iran
3 Department of Fisheries Science, Faculty of Agriculture and Natural Resources, Ahvaz
Branch, Islamic Azad University, Khouzestan, Iran
Live food has proved to be important in Larviculture of fish and shellfish from many aspects. Larvae of many species cannot utilize commercial diet either due to small mouth size, particle size, and immobility of the particle. Commercial feed at many instances cannot even fulfill the nutritional requirements of the fish larvae. Live food is more acceptable by many fish and shellfish larvae because it acts as natural prey organism for them and has specific advantages over particulate diet. Moreover, live food may be enriched by different nutrients such as fatty acids, amino acids, vitamins, antibiotics, vaccines, probiotics and prebiotics to act as a carrier of these nutrients to the larvae. This characteristic of Artemia nauplii has added to its importance in Larviculture (Bengeston et al., 1991; Watanabe et al., 1983, Gatesoupe, 1994).
Prebiotics are defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, which can improve the host health" (Gibson and Roberfroid 1995). They are not digested by enzymes of the upper gastrointestinal tract, but instead selectively fermented by some types of intestinal bacteria in the large intestine of humans (Gibson & Roberfroid 1995). or animals (Flickinger & Fahey 2002; Patterson & Burkholder 2003). Prebiotics can stimulate growth and/or metabolic activity of beneficial bacteria and suppress potentially deleterious ones, thus modifying the composition of the intestinal microflora (Crittenden 1999). In this work we used xylo
oligosaccharide to study its probable effects on improving fatty acid profile of Artemia urmiana.
Meterials and Method
Cysts of Artemia were hatched according to standard method (Sorgeloos, 1986). 500 newly hatched nauplii were transferred to each cylindro-conical containers containing 1 liter of 80 ppt brine water. The containers were placed inside an aquarium incubator at 28°C. Four feeding treatments each in 4 replicates were tested: Treatment (1) control diet consisting wheat bran powder and Dunaliella salina, Treatments (2, 3 and 4) control diet + 10, 30 and 60 mg xylo oligosaccharide/day respectively. The Artemia were cultured for 30 days. All Artemia were harvested and analyzed for fatty acid using standard protocol by a gas chromatography (Lepage and Roy, 1984). The results were finally analyzed statistically using SPSS-ANOVA.
The results of fatty acid analysis is shown in Table 1 and Fig.1.
The results clearly showed that supplementation of xylo oligosaccharide causes a significant reduction in the amount of total SFA and MUFA fatty acids. But supplementation of 10 mg prebiotic/L/day significantly improves concentration of total n-3 PUFA, total n-3 HUFA and sum of HUFA fatty acids. However, supplementation of 60 mg/L/day prebiotic resulted in non significant increase in n-6 PUFA fatty acids. Increase in different fatty acid groups could be as a result of stimulatory effect of prebiotic on some intestibal probiotics and their role in metabolism of different fatty acids. However a more detailed study is required for understanding this phenomenon.
Iran-Larvi, 11-12 December 2012-Karaj
mg fatty acid/g tissue
CON ■ lOppm 30ppm 60ppm
Fig. 1. Major Fatty acid groups of Artemia urmiana fed different experimental diets
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