I N C O R P O R AT I N G f i s h far m ing t e c h no l og y

May | June 2013 Controlling mycotoxins with binders

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FEATURE

Controlling mycotoxins with binders
by Adrien Louyer, aquaculture supervisor, Olmix Asia Pacific, Marie Gallissot, technical supervisor, Olmix SA, Dr Nguyen Van Nguyen, director of The Research Center for Fish Nutrition and Fishery Postharvest Technology - RIA2.

F

eed ingredients and feed prices are increasing; it is becoming harder to maintain the nutritional balance of the feed without increasing too much the feed price. Now, the use of ingredients from less stringent quality is likely to increase. Though plant materials are usually more reasonable in price than animal products, they can present problems through the presence of naturally occurring contaminants. Indeed, contamination of feed commodities by microorganisms and mycotoxins is the first negative factor impacting animal feed quality. Numerous researches have studied the decrease of performances with contaminated feeds.

making it difficult to diagnose mycotoxicosis. Even if extensive studies are done in this field, many mycotoxins effects remain unknown.

Prevention
Because of their effect on the immune system and fish performances, the presence of toxins impairs the farm economic perform-

ances. Strategies of prevention and control exist. In order to avoid deleterious effects of mycotoxins on fish, the best is to avoid contamination of the plants with moulds through adapted cultural practices. During harvest and storage, mycotoxins production must be prevented by reducing mould stress condi-

Table 1: Formulated diets with different Mt.X+ doses (values are expressed as a % on an as fed basis) Feed Ingredients D0 (0% Mt.X+) D0.05 (0.05% Mt.X+) 17.00 28.00 18.75 35.00 0.25 0.30 0.50 0.10 0.05 0.050 100.00 89.22 10.78 28.55 5.48 5.86 8.84 40.34 3.63 D0.15 (0.15% Mt.X+) 17.00 28.00 18.75 35.00 0.15 0.30 0.50 0.10 0.05 0.15 100.00 89.22 10.78 28.55 5.48 5.86 8.84 40.34 3.63

Fish meal 65% Soybean meal Cassava meal rice bran DCP Premix- M-V Fish oil lysine (lys) Methionine (Met) Mt.X+ total Proximate composition (% as fed basis) Dry matter Moisture Crude protein Crude fat Crude fibre Crude ash nitrogen free extract Gross energy (kcal.g-1)

17.00 28.00 18.75 35.00 0.3 0.30 0.50 0.10 0.05 0.00 100.00 89.22 10.78 28.55 5.48 5.86 8.84 40.34 3.63

Lipopolysaccharides (LPS), also known as endotoxins, are present in the cell membrane of gram negative bacteria. They are a structural component of the cell wall and are continuously released in the environment at cell death and during cell growth or division. Therefore, endotoxins are omnipresent in feed, water and fish gut which has shown to be an important bacterial reservoir. Endotoxins act as neurotoxic compounds and have immunosuppressive effect on fish. Mycotoxins are a diverse group of potential toxic metabolites produced by a variety of fungal species that often contaminate feedstuffs and consequently fish diets. Mycotoxins can vary in shape and size. They are heat stable and resist to extrusion process. For the ones that have been identified, it is known that a few parts per billion (ppb) already impact animal growth performances. Mycotoxins effects are specie dependent; cross contamination of different mycotoxins increases the damage caused (synergy) and results in uncharacteristic symptoms, thus

Fishmeal 65 percent (Vietnam, Kien Giang); Soybean meal 47 percent (India); Fish Oil (Chile fish oil), cassava meal (Vietnam, Tay Ninh), Rice bran (Vietnam, Tien Giang), Lysine and Methionine (Japan)
16 | InternatIonal AquAFeed | May-June 2013

FEATURE
Table 2: Contamination level of the feed for the most common mycotoxins MYCotoXIn t-2 toxin Deoxynivalenol (Don) Zearalenone Fumonisins (B1+B2) aflatoxins ochratoxin α leVel < 0.01ppm <0.01 ppm <0.01 ppm 0.025 ppm (B1:0.015+ B2:0.010) <0.004 ppm (aFB1:<0.001) <0.001 ppm

tions such as quick temperature or humidity change. Unfortunately, even with the best management procedure, it is extremely difficult to totally avoid mycotoxin contamination. Additionally, it is very hard to manage endotoxin ingestion by the fish. Endotoxins found in the fish intestine are brought by contaminated food and water or can be liberated from intestinal gram-negative bacteria. One of the best solutions to control these toxins is to use a wide spectrum binder in the feed. Olmix, a French company, has developed a patented hybrid material called Amadeite® (Figure 1): a clay which interlayer space has been extended by the insertion of algae polysaccharides (ulvans). The adsorption of toxins in this material is a complex mechanism involving the surface area of montmorillonite, the polyanionic structure of ulvans and the scaffold structure formed in the interlayer space. Based on this unique ingredient, a wide spectrum toxin binder, MT.X+ was created.

Experimental study in the Mekong Delta
The objective of this study was to evaluate the effects of MT.X+ on growth performances and feed utilization for tra catfish juveniles.

Material and Methods
Location and set up The experiment was done in a commercial farm in the Mekong Delta during two months. 1,080 healthy catfish fingerlings (initial weight around 30 g) obtained from a local supplier were used for the test. They were raised in floating cages (hapas), of 2x2x2 metres, in which pangasius were randomly allocated (120 fish per hapas). The cages were in the same pond to avoid water difference. Daily water exchange was done with a tidal system. Fish were acclimated for a week before the beginning of the trial. Experimental design Three iso-nitrogenous and iso-energetic diets were formulated (Table 1). Control diet, Experimental diet 1 and Experimental diet 2 respectively contained 0, 0.05 and 0.15% of MT.X+. The diets were produced by RIA2 feedmill, using extrusion process with pellet size of 5 ± 1 mm. The diets were randomly allocated to cages. Three replicates

per diet were done. Fish were fed ad libitum twice a day and excess feed was removed from the cages 20 minutes after feeding. To check the mycotoxin contamination of the feed used, mycotoxin analysis has been done by HPLC MS/ MS method in an independent laboratory, LDA 22, in France. Water quality was watched by recording daily dissolved oxygen (DO), temperature (T°C), pH, nitrites (NO2) and ammonia (NH3). DO, NO2 and NH3 were analysed by commercial aquaria test kit. Proximate composition of the diets was analysed according to the AOAC procedures. The parameters used to evaluate growth performance and feed utilization were expressed as Daily Weight Gain (DWG), Feed Conversion Rate (FCR) and Survival Rate (SUR). Data from each treatment were subjected to one-way ANOVA (differences were considered significant at p < 0.05) and to a Duncan multiple range of tests by using R software.
May-June 2013 | InternatIonal AquAFeed | 17

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FEATURE
Table 3: average water quality parameters during the time of the experiment Parameters level Maximum limit [10] 7-9 28-300 C ≤ 0,3 0.01 -1 ≥ 2,0

pH toC nH3 (mg/l) no2 (mg/l) Do (mg/l)

6± 1 28 ± 1 2 ±0 0-2 ±1 4-6 ±1

Results
Mycotoxin analysis Among the 44 different mycotoxins that were tested, levels of the most common mycotoxins are displayed in Table 2. The contamination level was very low for this sample. Water quality analysis Water quality parameters are displayed in Table 3. During all the time of the experiment ammonia (NH3) was higher than the Vietnamese accepted limit (2 mg/l instead of <0.3 mg/l). NO2 was higher than the Vietnamese limit during the last month of the experiment (2 mg/l instead of <1 mg/L). Moreover, it was observed that the fish density outside the hapas was very high. The water was probably heavily loaded with pathogens.

Figure 1: The interlayer space of Montmorillonite is multiplied by 10 thanks to the intercalation of green algae polysaccharides, the ulvans. The interlayer space is enlarged from 0,3-0,4 nm to 3-4 nm allowing to capture 2 nm molecules such as Trichothecenes or fumonisins

+18% in final weight). The supplementation with 0.05% of MT.X+ did not impact the performances in comparison of control group. Several factors can explain the obtained results: Mycotoxin contamination was very low in this experiment. We cannot exclude the possibility that the real contamination was higher than measured, due to uncertainty linked with the analysis (sampling method, unknown toxins). However, as we observe

susceptibility”, Infection and Immunity journal, 57(4): 1054-1058. Nayak S. K., et al 2008; “Effect of endotoxin on the immunity of Indian major carp, Labeorohita’, Fish and Shellfish Immunology, 24(4): 394-399. NRC (National Research Council) (2011), Nutrient requirements of fish and shrimp, National Academy Press, Washington, D.C., pp.233-247. Rodriguez M. A., et al 2012: “Mycotoxin Detoxification: Science vs Marketing, All About Feed, Mycotoxin special p 24-26. Tapia-Salazar, M. et al. 2010. Mycotoxins in aquaculture: Occurrence in feeds components and impact on animal performance. En: Cruz-Suarez, L.E., RicqueMarie, D., Tapia-Salazar, M., Nieto-López, M.G., VillarrealCavazos, D. A., Gamboa-Delgado, J. (Eds), Avances en Nutrición Acuícola X - Memorias del Décimo Simposio Internacional de Nutrición Acuícola, 8-10 de Noviembre, San Nicolás de los Garza, N. L., México. ISBN 978-607-433-546-0.Universidad Autónoma de Nuevo León, Monterrey, México, pp. 514-546. Spring et al 2005. Mycotoxin a rising threat to aquaculture, Nutritional Biotechnology in the Feed and Food Industries p 323-331 Havenaar R et al, 2006:” Efficacy of sequestrant/ chelatorAmadeite, in the binding of mycotoxins during transit through a dynamic gastrointestinal model (TIM) simulating the GI conditions of pigs; The world mycotoxin forum- The fourth conference, November 6-8 2006, Cincinnati, Ohio, USA. AOAC (1992) :AOAC Official Method 992.15Crude Protein in Meat and Meat Products Including Pet Foods Combustion Method First Action 1992 http:// www.aoac.org/omarev1/992_15.pdf http://thuvienphapluat.vn/archive/Thong-tu/Thongtu-45-2010-TT-BNNPTNT-dieu-kien-co-so-vungnuoi-ca-tra-tham-canh-vb109053t23.aspx

Table 4: Zootechnical performances of pangasius (Pangasius hypophthalmus) fed with diets containing different levels of Mt.X+ D0 Survival rate (%) Final body weight (g/fish) Daily weight gain (g/day) Feed intake (g/fish) FCr 88.7 a ± 7.6 70.05a ± 7.6 0.64b ± 0.15 101a ± 9.8 2.57 ab ± 0.38 D0.05 88.8a ± 4.5 70.86ab ± 4.5 0.66ab ± 0.05 106a ± 3 2.62a ± 0.27 D0.15 88.3a ± 5.4 82.66b ± 5.4 0.85a ± 0.08 106a ± 8.1 2.01b ± 0.04

Figures are presented as mean ± SD, values in the same row with different superscript letters are significantly different (p< 0.05)

Zootechnical performances Growth performances, feed efficiency and survival rate are presented in Table 4. After 60 days of feeding period, no significant difference was observed on survival rate (± 88%) and feed intake (± 104 g/fish). However, the final body weight was significantly different between fish fed 0.15% MT.X+ and fish fed control diet (82.66 and 70.05 g/fish, respectively). As a consequence, FCR was significantly lower for fish fed D0.15 compared to fish fed D0.05 or D0 (2.01, 2.62 and 2.57, respectively).

Discussion
Fish fed either control diet, MT.X+ 0.05% or MT.X+ 0.15% had similar survival rate and feed intake. However, Feed Conversion Ratio, Daily Weight Gain and Final Weight were significantly different among diets. Fish fed MT.X+ 0.15% had significantly better performances than fish fed control diet (-0.5 point in FCR,

a dose-dependent effect of MT.X+ in this experiment, this possibility is likely dismissed. On the other hand, water quality and pond management showed to be poor. In a context of excess ammonia and nitrite concentrations, frequent and important water exchange and probable high pathogen load, fish undergo stress and immunosuppression. MT.X+, by binding endotoxins and supporting the immune system helps fish to cope with these stressors. Better protected, fish fed MT.X+ better valorize the feed and show improved growth performances. MT.X+: improve protection, improve performances.

References
Halver, J. E. and Hardy, R.W. (2002), Fish Nutrition, Elsevier Science, pp.601-618. Roeder D.J., 1989: “Endotoxic-lipopolysaccharidespecific binding proteins on lymphoid cells of various animal species: association with endotoxin
18 | InternatIonal AquAFeed | May-June 2013

This article was first published on www.aquafeed.com

More InforMatIon:
Adrien Louyer, alouyer@olmix.com Marie Gallissot, mgallissot@olmix.com Dr Nguyen Van Nguyen, nguyenria2@gmail.com

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They are what they eat
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Controlling mycotoxins with binders Niacin
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