May | June 2011 Feature title: Preliminary effects of β-glucans on Nile tilapia health and growth performance

International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2009 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

The International magazine for the aquaculture feed industry

F: β-glucans
Table 2: Cyanobacteria as a potential source of fermentable carbohydrates (Vargas et al. 1998, J. Phycol. 34, 812)

F:F: β-glucans Microalgae
Experimental design
required to achieve this ambitious goal, but also the combination of this process with a realistic Strategic and Business Plan. The experiment was undertaken at the Aquaculture and Fish Nutrition Research Aquarium, University of Plymouth, UK. Nile tilapia (Oreochromis niloticus) (6.8 ± 0.2g) were randomly distributed 1 into 12 1: AlgaEnergy’s CO2BIOCAP Figure x 150-l fibreglass tanks containing well-aerated recirculated mobile laboratory freshwater. Fish were fed to apparent satiation 3 times a day for 70 kind, days. Fish incorporate four types on which will were batch weighed of photobioreactors (PBR): columns, tubular a weekly basis following a 24 hr reactors, semi-open and and reared at 28 starvation period in a second stage, raceways. Thewith a will be entirely auto± 1ºC plant 12:12 hr light:dark mated and controlled by specially designed photoperiod. software, which manages all38% crude proTwo isonitrogenous (ca. the cultivation parameters. Its goal(ca. 12%research lipid) tein) and isolipidic is to crude and develop new PBR processes and The basal diets were formulated (Table 2). technologies in this field. For this reason, the plant diet served as a control diet (diet A). will have the flexibility and capacitythe grow Experimental diet B consisted of to basal simultaneously different species of at 310 diet supplemented with β- glucan microalgae in diet. The glucan source was a blend mg kg1 different growing conditions, using indoor andand (1,6) chained glucan. Each of β-(1,3) outdoor PBR. The cultivation area was be initially by mechanically m2 and diet will produced of about 1,000 stirring the ingredients into uphomogenous mixture culture volume a to 72,000 l. AlgaEnergy’s plant will using a Hobart food mixer.be visited during theWarm water wasEurope congress that 3rd Algae World added to reach a Carbohydrates (% of dry weight)
Anabaena sp. ATCC 33047 Anabaena variabilis Anabaenopsis sp. 28.0 ± 2.0 22.3 ± 2.5 16.3 ± 1.5

β-glucans on Nile tilapia
health and growth performance
by M D Rawling and H Kühlwein, Aquaculture Nutrition and Health Research Group, School of Biomedical and Biological Sciences, University of Plymouth, UK

Preliminary effects of

Strain

2

006 marked a turning point in European aquaculture, when the European Union ratified a ban on the non-medical use of antibiotics in the regulation on feed additives for use in animal nutrition (EC № 1831/2003). This put a statutory stop to the use of all antibiotics and ionophore anticoccidials as growth promoters in intensive aquaculture practice and alternatives have received much attention (Bricknell and Dalmo, 2005; Merrifield et al., 2010; Dimitroglou et al., 2011). Such measures may help to facilitate consumer perceptions of biosecurity and eco-friendly fish farming. In this context much attention has been focused towards the development of immunomodulatory compounds such as β-glucans.

β-(1,6)-linked D-glucopyranosyl side chains, in which case it provides a comb-like structure (Bohn and BeMiller, 1995). The most abundant source of natural β-glucans with highly immunomodulating properties are yeasts, where research effort has focused in particular on β-(1,3) (1,6)-D-glucans, extracted from the baker’s yeast Saccharomyces cerevisiae. The β-glucan layer in the middle of the three-layered yeast cell wall gives strength and rigidity to the cell wall, forming a microfibrillar network. There are other β-(1,3)-glucans from different sources available (Table 1). One of the first studies conducted in 1969 by Chihara et al., showed an inhibiting effect of the fungal β-glucan lentinan on tumour growth in transplanted mice tumours after systemic infection. Lentinan and schizophyllan are nowadays used clinically in cancer therapy in Japan (Kaneko et al., 1989).

Pathogens exhibit evolutionary conserved pathogen-associated molecular patterns (PAMPs), which are recognised by host immune cells via contact with specific receptors such as pattern recognition receptors (PRRs) (Medzhitov and Janeway, 2000; Didierlaurent et al., 2005). It is recognised that PRRs for β-glucans are present in all vertebrates as well as invertebrates (Raa, 2000) and in addition are important for the recognition of fungal pathogens. As a result it has been well documented that β-glucans have positive effects on the immune cells of both fish and shrimp. Indeed it has been reported that β-glucans increase the activity of phagocytic cells (for example, macrophages) and the production of signal molecules such as cytokines, which results in the generation of new immune cells (Raa, 2000).

Immunomodulatory mechanisms of action of β-(1,3)(1,6)-D-glucans

β-glucans are widely distributed in nature and can be found in the cell walls of yeasts, cereal grains, algae, bacteria, fungi and mushrooms. β-glucans belong to the group of polysaccharides consisting of repeating β-(1,3)-linked D-glucose monomers that can be linear or branched with randomly distributed single

Sources and chemical structure

Table 1: Overview of other available beta (1,3)-D-glucan sources (adapted from Soltanian et al, 2009) Origin Lentinus edodus (Shiitake) Fungi

β-glucan
Lentinan

Branching frequency 2/5 1/3 1/3 1.0-1.3 1/10 0.05 0.005-0.009 unbranched unbranched

Reference Wenner et al., 2008

Sclerotium glucanicum & sclerotiorum Scleroglucan, SSG 1/3, highly branched Rice et al., 2005 Schizophyllum commune Schizophyllan Grifola frondosa (Maitake) Grifolan Poria cocos Wolf Pachyman Seaweed Algae Bacteria Lichen Laminaria digitata Laminarin Laminaria hyperborea Laminaran Euglena gracilis Paramylon Chaetoceros mülleri Chrysolaminaran Alcaligenes faecalis Curdlan Umbilicaris pustulata Pustulan
20 | InternatIonal AquAFeed | May-June 2011 24

Kubala et al., 2003 Tada et al., 2009 Wang et al., 2004 Osmond et al., 2001 Nagaoka et al., 2000 Skjermo et al., 2006 Bäumer et al., 2001 Kataoka et al., 2002 Yiannikouris et al., 2004

a realistic Figure 2: Total circulatory Figure 1: Growth performance Business Plan leukocyte levels of fish Nodularia sp. (Chucula) of Nile tilapia after 10 weeks 16.9 ± 2.6 after 70 days of is developing a AlgaEnergy feeding on of feeding on experimental 37.6 ± 2.5 Nostoc commune experimental diets diets. Data expressed as means responsible scientific agenda Nostoc paludosum 26.6 ± 1.9 ± Standard deviations aimed at achieving the commerNostoc sp. (Albufera) 26.8 ± 4.0 cially viable production of biofuels However, there is from data regarding The derived little microalgae. the The use Nostoc sp. (Caquena) diets for ± 1.7 of glucans in practical 23.3 immune response and growthprovided for R&D programmes performance fish such as turbot (Scophthalmus maximus, ± 2.0 Nostoc sp. (Chile) 23.3 of tilapia that purpose apparentthe selection when fed to include satiation on Debaulney et al., 1996), rainbow trout Nostoc sp. (Chucula) 15.7 ± 1.8 diets containing β-glucans (Whittington on and genetic engineering work et (Oncorhynchus mykiss, Peddie et al., 2002), 20.2 al., Atlantic salmonNostoc sp. (Llaita) (Salmo salar, Salinas et al., ± 1.5 2005).various types of microalgae, which carry substantial quantities current 2004), EuropeanNostoc bass(Loa) sea sp. (Dicentrarchus ± 1.2 Consequently, the aim of the of lipids 32.1 investigationcarbohydrates (some of which or was to assess dietary inclulabrax, Bagni et al., 2005) is well documented. sion of a commercial are patented), the development of and for transport), and the flue gases from β-glucan on the growth new photobioreactors more efficient and conventional energy generation facilities with lower and the establishment of canTable 2: Formulation of experimental diets.of therefore be used as a 1source Each ingredient costs, performance, feed utilisacomponent is expressed as g kg per diet tion, and innate immune a suitable and scalable production process. CO2 for large-scale microalgae cultivation DietsAt present, biofuels produced tilapia response of Nile from installations. Ingredients A B (Oreochromis competitive microalgae are not financially niloticus). The production of liquid biofuels for with the firstvehicles (biodiesel and bioethanol), is a very generation biopromising alternative Herring meal LT921 300.00 300.00 fuels obtained A series of factors must be taken into 18th Annual Practical Short Course on 2 365.01 365.01 Corn starch from convenaccount when selecting microalgae as a Aquaculture Feed Extrusion, 164.74 164.74 Lysamine pea protein3 tional agricultursource of biofuel precursors, such as: high 3 100.00 Glutalys (maize) Nutrition, & Feed Management al crops, and bioproductivity, temperature tolerance,100.00 tolermass30.00 production ance tooil4 high performance in fermentapH, 30.00 Fish September 25-30, 2011 and 17.75 processing ble Soybean oil carbohydrates for ethanol production 17.75 o 30+ lectures over a wide must therefore or in fatty acids transformable to biodiesel, 20.00 20.00 PNP Vitamin premix5 variety of aquaculture be substantially for example. Barox plus liquid (antioxidant) 0.500 0.500 industry topics improved so that We also need to establish the most β-glucan6 1.00 the price of the suitable type of cultivation system to -be o one-on-one interaction with product can be used (open, closed or mixed), and the most qualified industry experts Proximate analysis (% dry matter Dry Extruder reduced by an favourable operating conditionsbasis) (batch, o at the internationally order of magnisemi-continuous, continuous, number of recognized Food Protein R&D Center on the campus tude94.2 at least. phases, matter (%) Dry etc.). 93.7 of Texas A&M University in College Station, Texas A39.3 n e r g y lgaE Tables Protein 3 below show some examCrude 2 and (%) 37.8 o discussion and live equipment is currently ples of cyanobacteria as potential sources Crude lipid (%) 8.6 8.6 demonstrations following lectures engaged in the of fermentable carbohydrates for ethanol on four major types of extruders Ash (%) 6.9 6.8 construction of production and the lipid content of some -1) 19.5 Gross energy (MJ kg o various shaping dies (sinking, its first plant, a microalgae for biodiesel production. 20.4 floating, high fat), coating (surface The expectations control diet, microalgae diet. c h n o l o g i c a l raised by B = β-glucan Te Dietary codes: A = vs vacuum), nutrition, feed as 1 Fish meal: of second-generation bio- Scotland, UK. for a source United fish products, Aberdeen, Platform formulation, and MUCH MORE! Experimentation fuels have led to the creation of a large 2 Corn starch: Sigma Aldrich Ltd, UK. with Microalgae number of companies, some of which have Twin Screw Extruder 3 significant investment. Our Frêres, France. P T E M ) , ( madeLysamine pea protein: Roquette company located at the AlgaEnergy isSevenseas Ltd, UK. in the near 4 Epanoil: convinced that For more information, visit International future microalgae will be able to provide http://foodprotein.tamu.edu/extrusion 5 Vitamin premix: each 1kg of premix contains: 12.1% Airport of us with these forms of Vit A 1.000 µg/kg, Vit D3 0.100 clean energy so or contact calcium, Ash 78.7%, Madrid-Barajas. necessary for Ethe alpha tocopherol acetate) 7000.0 mg/kg, sustainable economic Dr. Mian N. Riaz µg/kg, Vit (as development(as cupricsocieties. Not only mg/kg,This is intended Copper of our sulphate) 250.000 is Magnesium mnriaz@tamu.edu 1.56%, Phosphorus development the to be a model constant research and 0.52% 979-845-2774 Full Fat Soy Demonstration basisβ-glucan: a blend of innovation (1,6) chainedplatform of its 6 for a continuous β-(1,3) and process glucan.
May-June 2011 | InternatIonal AquAFeed | 21 25

R&D and

F: β-glucans
consistency suitable for cold extrusion to form 1 mm pellets. twice per day, as opposed to three times per day in the present study. Efthimiou (1996) reported no improvements of dentex (Dentex dentex) growth performance Results and discussion when diets were supplemented with 0.5% β-(1,3) (1,6)-D-glucans every second week Growth, feed utilisation for two months. and carcass analysis However, similar to the present study This study endeavoured to determine dietary β-glucans have been reported to the growth performance and health effects improve fish growth performance, where of including β-glucan in diets for Nile Cook et al. (2003) fed a commercial tilapia. Growth performance and feed β-glucan preparation to snapper (Pagrus utilisation of tilapia after 10 weeks feedauratus) at a dose of 0.1% of diet weight ing on experimental diets is presented for 84 days. in Table 3 and Figure 1. A high growth In a similar investigation Misra et al. performance was observed in both groups; (2006) fed a β-glucan extracted from barley fish biomass increased by over 900% with to rohu (Labeo rohita) fingerlings at a dose feed conversion ratio (FCR) ≤ 1.0 and ranging from 0-500 mg β–glucan kg of diet specific growth rate (SGR) > 3.5. SGR for 56 days. improved significantly from 3.5 ± 0.06% In the present study after 70 days of in the control fed fish (group A) to 4.1 ± feeding on the experimental diets feed 0.15% in the β-glucan fed fish (group B; intake of fish fed β-glucan (36.6g kg-1 BW-1 P = 0.005). Mean final weight gain of the β-glucan fed fish (72.1g fish-1, P = 0.004) day-1) was considerably higher (35.5 – 38.6g was significantly greater than control fish kg-1 BW-1 day-1) than control fed fish (28.3g (50.9 g fish-1). kg-1 BW-1 day-1); however, this was not significant due to high variance. This trend is at Table 3: Growth performance of Nile tilapia after 10 weeks least suggestive toward of feeding on experimental diets. Values expressed as means and pooled standard error. Dietary codes: A = control diet, B = increased absolute mean β-glucan diet feed intake of fish fed on β-glucan to satiation Diets three times a day; furParameters A B ther research is required to evaluate appetite -1) response and optimise Initial body weight (g fish 6.9 6.7 β-glucan concentration. 57.8a 78.8b Final Body weight (g fish-1) Despite increased growth Weight gain (g fish-1) 50.9a 72.1b compared to control fed Food consumption (g kg-1 BW-1 day-1) 28.3 36.6 fish, the supplementation 1.99a Condition factor (k) 1.82a of β-glucan had no effect on feed utilisation and Net protein utilisation (NPU) 49.0 50.4 carcass analysis Protein efficiency ratio (PER) 2.59 2.59
Specific growth rate (SGR) Feed conversion ratio (FCR) superscript letters 3.5a 1.0 4.1b 0.9

F: β-glucans
(Thompson et al., 1995) and Asian catfish (Clarias batrachus; Kumari and Sahoo, 2006a). Contrary to these investigations, the result of the present study showed that after 70 days of feeding fish on diets containing β-glucan had no observable effect on serum lysozyme activity (567.1U. ml-1, P > 0.05) when compared to control fed fish (693.8U. ml-1). Despite not being significantly different the activity was considerably less than the control, which may be explained by the high dietary glucan supplementation; previously, Whittington et al., (2005) found that tilapia serum lysozyme activity significantly decreased (P < 0.05) when fed dietary β-glucan at 200 mg kg-1. Similarly Anderson (1992) and Couso et al. (2003) found negative effects towards fish immune responses and disease resistance when fed dietary β-glucan at 10 g kg-1 for periods of up to 40 days. Compared to control fed fish (1.61 x 104 µl-1) total leukocyte levels were significantly elevated in fish fed β-glucan diets (3.53 x 104 µl-1, P < 0.001) (Figure 2). This result is consistent with data reported for various fish species including: Atlantic salmon (Robertsen et al., 1994), channel catfish (Ictalurus punctatus; Duncan and Klesius, 1996), common carp (Cyprinus carpio; Selvaraj et al., 2005) and rohu (Misra et al., 2006). The data from the present study suggests that the inclusion β-glucan at the dietary levels used had no detrimental effects towards the measured fish health parameters.
Bricknell, I. and Dalmo, R.A.: Fish & Shellfish Immunology, 2005. 19: pp. 457-472. Chihara, G. et al. Nature, 1969. 222: pp. 687-688. Cook, M.T. et al. Fish & Shellfish Immunology, 2003. 14: pp. 333-345. Couso, N. et al. Aquaculture, 2003. 219: pp. 99-109. Dalmo, R.A. and Bogwald, J.: Fish & Shellfish Immunology, 2008. 25: pp. 384-396. deBaulny, M.O. et al.: Diseases of Aquatic Organisms, 1996. 26: pp. 139-147. Didierlaurent, A. et al.: Cellular and Molecular Life Sciences, 2005. 62: pp. 1285-1287. Dimitroglou, A. et al.: Fish and Shellfish Immunology, 2011. 30: pp. 1-16. Duncan, P.L. and Klesius, P.H.: Journal of Aquatic Animal Health, 1996. 8: pp. 241-248. Efthimiou, S.: Journal of Applied IchthyologyZeitschrift für Angewandte Ichthyologie, 1996. 12: pp. 1-7. Engstad, R.E. et al. Fish & Shellfish Immunology, 1992. 2: pp. 287-297. The European Parliament and the Council of the European Union: Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition, 2003. Kaneko, Y. et al. International Journal of Immunotherapy, 1989. 5: pp. 203-213. Kataoka, K. et al. Journal of Biological Chemistry, 2002. 277: pp. 36825-36831. Kubala, L. et al. Carbohydrate Research, 2003. 338: pp. 2835-2840. Kumari, J. and Sahoo, P.K.: Diseases of Aquatic Organisms, 2006.70: pp. 63-70. Medzhitov, R. and Janeway, C. Jr.: Immunological Reviews, 2000. 173: pp. 89-97. Merrifield, D.L. et al. Aquaculture, 2010. 302: pp. 1-18. Misra, C.K. et al. Aquaculture, 2006. 255: pp. 82-94. Nagaoka, H. et al. Hepatogastroenterolgy, 1999. 46: pp. 2662-2668. Osmond, R.I. et al. European Journal of Biochemistry, 2001. 268: pp. 4190-4199. Peddie, S. et al. Veterinary Immunology and Immunopathology, 2002. 86: pp. 101-113. Raa, J.: In: Avances en Nutricion Acuicola V. Merida, Yucatan, Mexico: Memorias del V Simposium Internacionale Nutricion Acuicola. 2000. Rice, P.J. et al. The Journal of Pharmacology and Experimental Therapeutics, 314: pp. 1079-1086. Robertsen, B. et al., in: Stolen J. and Fletcher T.C., Editors, Modulators of Fish Immune Responses, SOS, Fair Haven, 1994. pp. 83-99. Salinas, I. et al. Fish & Shellfish Immunology, 2004. 17: pp. 159-170. Selvaraj, V. et al. Fish & Shellfish Immunology, 2005. 19: pp. 293-306. Skjermo, J. et al. Aquaculture, 2006. 261: pp. 10881101. Soltanian, S. et al. Critical Reviews in Microbiology, 2009. 35: pp. 109-138. Tada, R. et al. Carbohydrate Research, 2009. 344: pp. 400- 404. Thompson, K.D. et al. Diseases in Asian aquaculture, 1995. 11: pp. 433–439. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Wang, Y. et al. Carbohydrate Research, 2004. 339: pp. 2567-2574. Wenner, C. A. et al. Planta Medica, 2008. 74: pp. 909-910. Whittington, R. et al. Aquaculture, 2005. 248: pp. 217-225. Yiannikouris, A. et al. Journal of Food Protection, 2004. 67: pp. 2741-2746.

Conclusion
The present study demonstrated that β-glucan fed to Nile tilapia at 310mg β-glucan kg-1 for 10 weeks had a positive effect on growth with no apparent detrimental effects towards carcass composition or health status. Although there was no significant difference in the feed intake it was apparent that feed intake of fish fed β-glucan was considerably improved. Feed utilisation was not significantly affected further indicating that improved growth may have been due to improved appetite of fish fed diets containing β-glucan.

abSignificant differences between groups are indicated by

Haematology and immunology

Contrary to the findings of the present study, an investigation by Whittington et al., (2005) reported that a yeast β-glucan at dietary levels of 50, 100 & 200 mg β–glucan kg did not significantly affect weight gain of tilapia after 84 days of feeding. The current study used a commercial product at 310 mg β-glucan kg-1. The differences of growth performance may be explained by the higher β-glucan level in the current study or the fact that Whittington et al., (2005) fed to apparent satiation only

Biochemical and haematological analysis can often provide vital information for health and management assessment of cultured fish. In the present study haematocrit, haemoglobin and erthyrocyte levels were not affected by the inclusion of β-glucans (data not shown). Serum lysozyme activity also remained unaffected. Research has demonstrated that β-glucans can enhance the non-specific immune response of fish (Dalmo and Bogwald, 2008). Indeed, yeast glucans have been reported to enhance lysozyme activity in Atlantic salmon (Engstad et al., 1992), rainbow trout

References
Anderson, D.P.: Annual Review of Fish Diseases, 1992. 2: pp. 281-307. Bagni, M. et al. Fish & Shellfish Immunology, 2005. 18: pp. 311-325. Bäumer, D. et al. Journal of Phycology, 2001. 37: pp. 38-46.

22 | InternatIonal AquAFeed | May-June 2011

May-June 2011 | InternatIonal AquAFeed | 23

This digital re-print is part of the May | June 2011 edition of International Aquafeed magazine. Content from the magazine is available to view free-of-charge, both as a full online magazine on our website, and as an archive of individual features on the docstoc website. Please click here to view our other publications on www.docstoc.com.

LINKS

VO L U M E 1 4 I S S U E 3 2 0 1 1

• See the full issue
Aquaculture:

Natural ingredients for sustainable aquaculture

• • •

Visit the International Aquafeed website Contact the International Aquafeed Team Subscribe to International Aquafeed

Maturation diets:
diets for shrimp – Is there alternative to natural food? Preliminary effects of β-glucans on Nile tilapia health and growth performance

β-glucans:

Microalgae
Microalgae and cyanobacteria

THE INTERNATIONAL MAGAZINE FOR THE AQUACULTURE FEED INDUSTRY
IAF11.03.indd 1 04/05/2011 09:07

To purchase a paper copy of the magazine, or to subscribe to the paper edition please contact our Circulation and Subscriptions Manager on the link above.

INFORMATION FOR ADVERTISERS - CLICK HERE

www.aquafeed.co.uk

Sign up to vote on this title
UsefulNot useful