May | June 2011

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

The International magazine for the aquaculture feed industry

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2006 marked a turning point in European aquac-ulture, 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 anticoc-cidials 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 facili-tate consumer perceptions of bio-security and eco-friendly fish farming. In this context much attention has been focused towards the development of immunomodulatory compounds such as β-glucans.

Sources and chemical structure

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

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 immunomodulat-ing 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 rigid-ity 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 inhibit-ing effect of the fungal β-glucan len-tinan 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).

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

Pathogens exhibit evolutionary con-served pathogen-associated molecular patterns (PAMPs), which are recognised by host immune cells via contact with spe-cific 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 docu-mented 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).

Preliminary effects of

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

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

Origin β-glucan Branching frequency Reference

Fungi

Lentinus edodus (Shiitake) Lentinan 2/5 Wenner et al., 2008

Sclerotium glucanicum & sclerotiorum Scleroglucan, SSG 1/3, highly branched Rice et al., 2005

Schizophyllum commune Schizophyllan 1/3 Kubala et al., 2003

Grifola frondosa (Maitake) Grifolan 1/3 Tada et al., 2009

Poria cocos Wolf Pachyman 1.0-1.3 Wang et al., 2004

SeaweedLaminaria digitata Laminarin 1/10 Osmond et al., 2001

Laminaria hyperborea Laminaran 0.05 Nagaoka et al., 2000

AlgaeEuglena gracilis Paramylon - Skjermo et al., 2006

Chaetoceros mülleri Chrysolaminaran 0.005-0.009 Bäumer et al., 2001

Bacteria Alcaligenes faecalis Curdlan unbranched Kataoka et al., 2002

Lichen Umbilicaris pustulata Pustulan unbranched Yiannikouris et al., 2004

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The use of glucans in practical diets for fish such as turbot (Scophthalmus maximus, Debaulney et al., 1996), rainbow trout (Oncorhynchus mykiss, Peddie et al., 2002), Atlantic salmon (Salmo salar, Salinas et al., 2004), European sea bass (Dicentrarchus labrax, Bagni et al., 2005) is well documented.

However, there is little data regarding the immune response and growth performance of tilapia when fed to apparent satiation on diets containing β-glucans (Whittington et al., 2005).

Consequently, the aim of the current investigation was to assess dietary inclu-

sion of a commercial β-glucan on the growth performance, feed utilisa-tion, and innate immune response of Nile tilapia (Oreochromis niloticus).

Experimental designThe experiment was under-

taken at the Aquaculture and Fish Nutrition Research Aquarium, University of Plymouth, UK. Nile tilapia (Oreochromis niloticus) (6.8 ± 0.2g) were randomly distributed into 12 x 150-l1 fibreglass tanks containing well-aerated recirculated freshwater. Fish were fed to appar-ent satiation 3 times a day for 70 days. Fish were batch weighed on a weekly basis following a 24 hr starvation period and reared at 28 ± 1ºC with a 12:12 hr light:dark photoperiod.

Two isonitrogenous (ca. 38% crude pro-tein) and isolipidic (ca. 12% crude lipid) diets were formulated (Table 2). The basal diet served as a control diet (diet A). Experimental diet B consisted of the basal diet supplemented with β- glucan at 310 mg kg1 diet. The glucan source was a blend of β-(1,3) and (1,6) chained glucan. Each diet was produced by mechanically stirring the ingredients into a homogenous mixture using a Hobart food mixer.

Warm water was added to reach a

Table 2: Formulation of experimental diets. Each ingredient component is expressed as g kg1 per diet

Diets

Ingredients A B

Herring meal LT921 300.00 300.00

Corn starch2 365.01 365.01

Lysamine pea protein3 164.74 164.74

Glutalys (maize)3 100.00 100.00

Fish oil4 30.00 30.00

Soybean oil 17.75 17.75

PNP Vitamin premix5 20.00 20.00

Barox plus liquid (antioxidant) 0.500 0.500

β-glucan6 - 1.00

Proximate analysis (% dry matter basis)

Dry matter (%) 93.7 94.2

Crude Protein (%) 37.8 39.3

Crude lipid (%) 8.6 8.6

Ash (%) 6.9 6.8

Gross energy (MJ kg -1) 20.4 19.5

Dietary codes: A = control diet, B = β-glucan diet. 1 Fish meal: United fish products, Aberdeen, Scotland, UK.2 Corn starch: Sigma Aldrich Ltd, UK. 3 Lysamine pea protein: Roquette Frêres, France. 4 Epanoil: Sevenseas Ltd, UK.5 Vitamin premix: each 1kg of premix contains: 12.1%

calcium, Ash 78.7%, Vit A 1.000 µg/kg, Vit D3 0.100 µg/ kg, Vit E (as alpha tocopherol acetate) 7000.0 mg/kg, Copper (as cupric sulphate) 250.000 mg/kg, Magnesium 1.56%, Phosphorus 0.52%

6β-glucan: a blend of β-(1,3) and (1,6) chained glucan.

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F: β-glucans

Figure 1: Growth performance of Nile tilapia after 10 weeks of feeding on experimental diets. Data expressed as means ± Standard deviations

Figure 2: Total circulatory leukocyte levels of fish after 70 days of feeding on experimental diets

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and for transport), and the flue gases from conventional energy generation facilities can therefore be used as a source of CO2 for large-scale microalgae cultivation installations.

The production of liquid biofuels for vehicles (biodiesel and bioethanol), is a very promising alternative

A series of factors must be taken into account when selecting microalgae as a source of biofuel precursors, such as: high productivity, temperature tolerance, toler-ance to pH, high performance in fermenta-ble carbohydrates for ethanol production or in fatty acids transformable to biodiesel, for example.

We also need to establish the most suitable type of cultivation system to be used (open, closed or mixed), and the most favourable operating conditions (batch, semi-continuous, continuous, number of phases, etc.).

Tables 2 and 3 below show some exam-ples of cyanobacteria as potential sources of fermentable carbohydrates for ethanol production and the lipid content of some microalgae for biodiesel production.

The expectations raised by microalgae as a source of second-generation bio-fuels have led to the creation of a large number of companies, some of which have made significant investment. Our company AlgaEnergy is convinced that in the near future microalgae will be able to provide us with these forms of clean energy so necessary for the sustainable economic development of our societies. Not only is constant research and development the basis for a continuous innovation process

required to achieve this ambitious goal, but also the combination of this process with a realistic Strategic and Business Plan.

R&D and a realistic Business Plan

AlgaEnergy is developing a responsible scientific agenda aimed at achieving the commer-cially viable production of biofuels derived from microalgae. The R&D programmes provided for that purpose include the selection and genetic engineering work on various types of microalgae, which carry substantial quantities of lipids or carbohydrates (some of which are patented), the development of

new photobioreactors more efficient and with lower costs, and the establishment of a suitable and scalable production process.

At present, biofuels produced from microalgae are not financially competitive with the first-generation bio-fuels obtained from conven-tional agricultur-al crops, and bio-mass production and processing must therefore be substantially improved so that the price of the product can be reduced by an order of magni-tude at least.

A l gaEnerg y is currently engaged in the construction of its first plant, a Techno log i ca l Platform for Experimentation with Microalgae ( P T E M ) , located at the I n t e r n a t i o n a l Airport of Madrid-Barajas. This is intended to be a model platform of its

kind, which will incorporate four types of photobioreactors (PBR): columns, tubular reactors, semi-open and in a second stage, raceways. The plant will be entirely auto-mated and controlled by specially designed software, which manages all the cultivation parameters. Its goal is to research and develop new PBR processes and technolo-gies in this field. For this reason, the plant will have the flexibility and capacity to grow simultaneously different species of micro-algae in different growing conditions, using indoor and outdoor PBR. The cultivation area will be initially of about 1,000 m2 and the culture volume up to 72,000 l.

AlgaEnergy’s plant will be visited during the 3rd Algae World Europe congress that

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

StrainCarbohydrates

(% of dry weight)

Anabaena sp. ATCC 33047 28.0 ± 2.0

Anabaena variabilis 22.3 ± 2.5

Anabaenopsis sp. 16.3 ± 1.5

Nodularia sp. (Chucula) 16.9 ± 2.6

Nostoc commune 37.6 ± 2.5

Nostoc paludosum 26.6 ± 1.9

Nostoc sp. (Albufera) 26.8 ± 4.0

Nostoc sp. (Caquena) 23.3 ± 1.7

Nostoc sp. (Chile) 23.3 ± 2.0

Nostoc sp. (Chucula) 15.7 ± 1.8

Nostoc sp. (Llaita) 20.2 ± 1.5

Nostoc sp. (Loa) 32.1 ± 1.2

Figure 1: AlgaEnergy’s CO2BIOCAP mobile laboratory

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(Thompson et al., 1995) and Asian cat-fish (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; previ-ously, 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 nega-tive 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 vari-ous 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.

ConclusionThe 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 detri-mental 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.

ReferencesAnderson, 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.

twice per day, as opposed to three times per day in the present study. Efthimiou (1996) reported no improvements of den-tex (Dentex dentex) growth performance when diets were supplemented with 0.5% β-(1,3) (1,6)-D-glucans every second week for two months.

However, similar to the present study dietary β-glucans have been reported to improve fish growth performance, where Cook et al. (2003) fed a commercial β-glucan preparation to snapper (Pagrus auratus) at a dose of 0.1% of diet weight for 84 days.

In a similar investigation Misra et al. (2006) fed a β-glucan extracted from barley to rohu (Labeo rohita) fingerlings at a dose ranging from 0-500 mg β–glucan kg of diet for 56 days.

In the present study after 70 days of feeding on the experimental diets feed intake of fish fed β-glucan (36.6g kg-1 BW-1 day-1) was considerably higher (35.5 – 38.6g kg-1 BW-1 day-1) than control fed fish (28.3g kg-1 BW-1 day-1); however, this was not

significant due to high variance. This trend is at least suggestive toward increased absolute mean feed intake of fish fed on β-glucan to satiation three times a day; fur-ther research is required to evaluate appetite response and optimise β-glucan concentration. Despite increased growth compared to control fed fish, the supplementation of β-glucan had no effect on feed utilisation and carcass analysis

Haematology and immunology

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

consistency suitable for cold extrusion to form 1 mm pellets.

Results and discussion

Growth, feed utilisation and carcass analysis

This study endeavoured to determine the growth performance and health effects of including β-glucan in diets for Nile tilapia. Growth performance and feed utilisation of tilapia after 10 weeks feed-ing on experimental diets is presented in Table 3 and Figure 1. A high growth performance was observed in both groups; fish biomass increased by over 900% with feed conversion ratio (FCR) ≤ 1.0 and specific growth rate (SGR) > 3.5. SGR improved significantly from 3.5 ± 0.06% in the control fed fish (group A) to 4.1 ± 0.15% in the β-glucan fed fish (group B; P = 0.005). Mean final weight gain of the β-glucan fed fish (72.1g fish-1, P = 0.004) was significantly greater than control fish (50.9 g fish-1).

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 dif-ferences 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

Table 3: Growth performance of Nile tilapia after 10 weeks of feeding on experimental diets. Values expressed as means and pooled standard error. Dietary codes: A = control diet, B = β-glucan diet

Diets

Parameters A B

Initial body weight (g fish-1) 6.9 6.7

Final Body weight (g fish-1) 57.8a 78.8b

Weight gain (g fish-1) 50.9a 72.1b

Food consumption (g kg-1 BW-1 day-1) 28.3 36.6

Condition factor (k) 1.82a 1.99a

Net protein utilisation (NPU) 49.0 50.4

Protein efficiency ratio (PER) 2.59 2.59

Specific growth rate (SGR) 3.5a 4.1b

Feed conversion ratio (FCR) 1.0 0.9abSignificant differences between groups are indicated by superscript letters

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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. 1088-1101.

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.

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

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 Ichthyology-Zeitschrift 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

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VOLUME 14 I S SUE 3 2 011

THE INTERNATIONAL MAGAZINE FOR THE AQUACULTURE FEED INDUSTRY

Aquaculture: Natural ingredients for sustainable

aquaculture

Maturation diets:diets for shrimp – Is there alternative to

natural food?

β-glucans:Preliminary effects of β-glucans on Nile tilapia

health and growth performance

Microalgae Microalgae and cyanobacteria

IAF11.03.indd 1 04/05/2011 09:07

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