Fermentation properties and nutritional quality of selected aquatic macrophytes as alternative fish feed in rural areas of the Neotropics

Y Cruz, C Kijora, E Wedler*, J Danier** and C Schulz***

Animal Breeding in the Tropics and Subtropics, Humboldt University of Berlin.
Philippstraße 13. D-10115, Berlin, Germany
yorcelisc@gmail.com
* Instituto de Investigaciones Tropicales de la Universidad Del Magdalena (INTROPIC).
Carrera 32 No 22 – 08. A.P.:2-1-21630. Santa Marta, Colombia
** BioanalytikWeihenstephan, Nutrition and Food Research Center, Technical University of Munich, AlteAkademie 10, D-85350 Freising, Germany
*** Institute for Animal Husbandry, Christian-Albrechts-University of Kiel. Olshausenstraße 40 D-24098 Kiel, Germany
*** GMA – Gesellschaft für Marine Aquakultur GmbH, Büsum, Germany

Abstract

In order to facilitate fish cultivation in rural areas of the Neotropics, the potential of locally available aquatic macrophytes from northern Colombia (Lemna minor, Spirodela polyrhiza, Azolla filiculoides and Eichhornia crassipes) as alternative fish feedwas studied. Considering the importance of fermentation in the improvement of nutritional value of non-conventional feeds, the fermentation properties (Trial I)andthe effects of anaerobic fermentation on the nutritional quality of the selected aquatic plants (Trial II) were evaluated.

Results of the first trial indicated that although the fermentability coefficients (FC) of the selected aquatic macrophytes revealed a hardly fermentable material (FC< 35), the use of bacteria inoculants (Lactobacillus plantarum) and molasses (150 g/kg) resulted in a good silage quality. Results of the second trials howed that lactic acid fermentation positively affected the nutritional quality of the plants; since the concentration of some antinutritional substances and crude fibre content were reduced (P > 0.05).Fermentation of the tested aquatic macrophytes is highly recommendable to use them as fish feed supplement.

Key words: alternative fish feed, antinutritional factors, heavy metals, processing methods

Introduction

In view of the increasing prices of ingredients for aquafeeds, the search for cheap and nutritionally balanced ingredients for fish feed has become an urgent need for the aquaculture sector. Particularly, the evaluation of the nutritional value and potential of locally available nutrient sources is an important aspect for the substitution of conventional fish feed ingredients.Aquatic macrophytes are among the most common plant materials in the tropical floodplain systems. They contribute to more than 60 % of the primary production in the Amazon floodplain (Leite et al 2002). In the Orinoco, they are also reported to be highly productive and to increase rapidly during high water (Vásquez 1989). In other freshwater ecosystems, they grow rapidly and remain during the whole year.

Due to their abundance, they contribute to eutrophication of water bodies (Xieet al 2004). Therefore, many researchers are looking for methods to control them effectively and others to convert them into utilizable resources (NAS 1976; Brabben 1993; Rodriguez and Preston 1996; Franklin et al 2008), both in industrial processes and at farm level (Leng 1999; Xiao et al 2009; Dordio et al 2010). In sustainable production systems, they have been frequently described as suitable alternative animal feed (Pípalová 2003; Sétliková and Adámek 2004; El-Sayed 2003; Bairagi et al 2002; Yılmaz et al 2004; Kalita et al 2007). However, their potential as fish feed should be evaluated on a local basis because of their variable composition, which is highly depending on the water quality conditions where the plants grow (Boyd 1971).

As other plant materials, aquatic macrophytes contain antinutritional substances that restrict their use as feed. To improve their nutritional value for fish and subsequently to increase their incorporation level into fish diets, they are commonly fermented.For instants, El-Sayed (2003) reported that fermentation is necessary when water hyacinth (Eichhornia crassipes) is included at levels of 20% or more into Nile tilapia diets. Likewise, Bairagi et al (2002) reported in their study that fermented Lemna leaf meal was better utilized than raw Lemna leaf meal in diets for rohu (Labeo rohita).

Although aquatic macrophytes are commonly found within the tropics, theyhave been poorly investigated as alternative or supplemented feed for the majority of native fish in Colombia. This may be due to the lack of information on their nutritional characteristics, fermentation properties and processing. Since this information is essential to increase their potential as fish feed, two separated trials were conducted to evaluate the fermentation properties (Trial I) and the effect of anaerobic fermentation on the nutritional quality and the content of antinutritional substances (Trial II) of Lemna minor, Spirodela polyrhiza, Azolla filiculoides and Eichhornia crassipes.

Materials and methods

Fermentation properties of the selected aquatic plants (Trial I)

The aquatic macrophytes Spirodela polyrhiza (Giant Duckweed), Azolla sp. (fern Azolla) and Eichornia crassipes (Water Hyacinth) used in trial I were obtained from a commercial distributor (Wakus GmbH Berlin, Germany). Lemna minor (Duckweed) was obtained from the Botanical Garden of Berlin. The plant material was washed, oven dried and stored in plastic bags for preliminary analysis in laboratory. Eichhornia crassipes was previously separated into leaves and roots and chopped into small parts.

For fermentation, freshly harvested plants were mixed with a part of the oven dried aquatic macrophytes of the same sample to complete 350 to 450 g/kg dry matter. A total of 8 mixtures (two replicates per plant species) were used for lactic acid fermentation by addition of the commercial silage inoculants and of molasses at 150 g/kg as water soluble carbohydrates (WSC) source. Afterwards, the mixtures were vacuum packed into gas tight plastic bags according to the method described by Johnson et al (2005). Silages were maintained in an incubator for 60 days at 25°C.

Bacterial culture

For lactic acid fermentation, the commercial silage inoculants BIO-SIL® based on the lactic acid bacteria (LAB) strain Lactobacillus plantarum DSM 8862 and DSM 8866 (Dr. Pieper Technologie-und Produktentwicklung GmbH, Germany) was used. The inoculant solution was prepared according to the manufacturer’s information and used on a fixed dose of 2 ml/kg of plant material for a final inoculation rate of 3 x 10⁵cfu/g.

The raw plant material was preliminarily analyzed in triplicate to determine the buffering capacity (BC) by electrometric titration (Automatic TitratorTyp AT 3 equipped with a glass electrode), nitrate content by potentiometric (ion selective electrode) and WSC content by anthrone method (Lengerken and Zimmermann 1991). The ratio WSC/BC was used to estimate the fermentability coefficients (FC).

Silages were also analyzed in triplicate. Acid concentration was determined by high-performance liquid chromatography (HPLC) according to Weiß and Kaiser (1995). Alcohols were determined chromatographically (GC). Ammonia was determined by Conway method according to Lengerken and Zimmermann (1991). The evaluation of the fermentation quality of silages was done on the basis of a chemical investigation according to the DLG (Deutsche Landwirtschafts-Gesellschaft) guidelines for silage quality proposed by Weißbach and Honig (1992).

Calculations

The fermentability coefficient (FC) was used to judge the ensiling potential and was calculated according to the parameters on the silage fermentation:

-        FC=DM (%) + 8 WSC/BC (Schmidt et al 1971).

The minimum dry matter content (DM_min) was calculated following the equation proposed by Kaiser et al (2002) as a function of nitrate content. As the DM_min content considered acceptable for ensiling in farm scale is 200 g/kg, whereas a DM content of 450 g/kgis considered as inhibitory limit value for clostridia growth in anaerobic conditions (McDonald et al 1991), samples were set to be ranged from 350 to 450 g/kgDM content.

Effect of fermentation on the nutritional quality of the selected aquatic plants (Trial II)

The aquatic macrophytes Lemna minor (Duckweed), Spirodelapolyrhiza (Giant Duckweed), Azolla filiculoides (fern Azolla) and Eichhornia crassipes (Water Hyacinth) used in trial II were harvested as wild or uncultivated material from water bodies in northern Colombia. After collection and taxonomical identification, raw plant material was treated according to the procedures described above. A total of 12 mixtures, three replications per plant species, were prepared for fermentation. A sample of each mixture was taken before the vacuum packing to be analyzed for proximate composition. After 60 days of fermentation, samples were opened and analyzed again. Proximate analysis of the unfermented and fermented aquatic plants was performed following the AOAC (2005) procedures.

Chemical analysis

Samples of unfermented plant material were sent to a private laboratory to test the mineral and heavy metal content. Determination were done by atomic absorption spectrophotometry according to the AOAC methods 985.35 and 983.27 (AOAC 1995) and to the AOAC methods 995.11 and 999.11 (AOAC 2005). For amino acid composition samples of the unfermented material were also sent to the Nutrition and Food Research Center in Freising (Germany), where the determinations were done according to the methods of the European Commission due to regulation 159/2009. Amino acids were separated by ion chromatography and photometrically determined after Ninhydrin reaction by an amino acid analyzer (Biochrom 30).

Samples of unfermented and fermented plant material were also sent toa specialized laboratory for the determination of antinutritional substances. Trypsin inhibitory activity was determined by the enzymatic-colorimetric method using benzoyl-DL-arginine-p-nitroanilide (BAPA) as a substrate (Kakade et al 1969; Smith et al 1980). Hydrolysable and condensed tannins were determined by the spectrophotometric method using the Folin-Denis reagent (Price and Butler 1977) and vanillin hydrochloric acid (V-HCl) (Earp et al 1981), respectively. Phytates (as phytic acid activity) were determined by the enzymatic-spectrophotometric method at 492 nm (Cat No. K-PHYT, Megazyme International Ireland Ltd., Wicklow, Ireland). Oxalates were determined by HPLC.

Statistical Analysis

To assess the separate and combined effect of the plant material (four species) and treatment (unfermented and fermented) on the nutritional composition (ash, protein and fibre content) of aquatic macrophytes a 4 x 2 factorial analysis of variance (ANOVA) was conducted. The means and standard error of means (SEM) for ash, protein and fibre content as a function of the two factors are presented in Table 5. The F test and Tukey's test for Post Hoc comparisons (P<0.05) were applied. All statistical analyses were performed using the SPSS (version 19) software package.

Results

Fermentation properties of selected aquatic plants (Trial I)

Table 1 shows the characteristics of the raw and lactic acid fermented aquatic macrophytes. Results indicated that dry matter content and water soluble carbohydrates (WSC) of all tested plants were very low, whereas crude fibre and ash content were relatively high. In addition, Spirodela and Lemna presented a high buffering capacity (BC). Thus, the fermentability coefficients of the raw aquatic macrophytes revealed a heavily fermentable material (FC<35).

The dry matter of the silages ranged within desirable values (395 - 446 g/kg). The pH values ranged from 3.92 to 4.26 indicating good silage quality. Ammonia-N content was low and ranged from 5 to 58 g/kg in CP, except for the stems of Eichhornia which reached a content of 164 g/kg ammonia-N in CP. Acetic acid content was also low in all samples. Silages were practically butyric and propionic acid-free. In contrast, concentrations of  lactic acid were high and ranged from 74.2 (stems of Eichhornia) to 86.8 g/kg (leaves of Eichhornia). The evaluation of silage quality by DLG points for the tested silages obtained the highest mark.

Effect of fermentation on the nutritional quality of the aquatic plants (Trial II)

Table 2 shows the chemical composition of the tested aquatic macrophytes grown in rural zones of Colombia. The crude protein content ranged between 157 and 212 g/kg DM, except for stems of Eichhornia with the lowest content (97.8 g/kg). Aquatic macrophytes have a relatively high crude fibre and ash content, and a low content of crude lipids. However, they are rich in minerals. Heavy metal concentrations of arsenic, selenium and mercury were not detectable. Cadmium and lead were detectable in low concentrations.

Table 3 shows the concentration of antinutritional substances in the raw and fermented aquatic macrophytes. Trypsin inhibitor, phytates (as phytic acid activity), soluble tannins and oxalates were detectable in all the raw aquatic plants, although the amounts did not exceed the tolerable limits for fish. Condensed tannins were not detectable in raw Lemna and Azolla, but were present in raw Spirodela and Eichhornia leaves. Trypsin inhibitor and oxalates were significantly reduced by the fermentation process, as well as phytates, except for Azolla, whose content of phytates did not change. Soluble and condensed tannins were not detectable in the fermented aquatic macrophytes.

The amino acid profile of tested local macrophytes and amino acids requirements of the tropical fish Nile tilapia (Oreochromis niloticus) and Pacu (Piaractus mesopotamicus) are presented in Table 4. The protein of the raw material resulted in a similar amino acid profile among plants and contained 5.30 to 6.28 g/100g lysine and 1.72 to 2.04 g/100 g methionine in the dietary protein. The tested aquatic macrophytes showed to be rich in aspartic acid and glutamic acid. 

The nutritional composition of the unfermented and fermented macrophytes is presented in Table 5. All tested variables showed significant differences at both factors except for ash content between treatments. The interaction between the plant species and treatments was significant and was not always in the same direction (disordinal interaction). Whereas crude fibre was significantly reduced (P < 0.05) in all fermented plants, crude protein varied among the plants species and resulted significantly higher (P < 0.05) in fermented Lemna and Spirodela and significantly lower (P < 0.05) in fermented Azolla and Eichhornia when compared to the respectively unfermented plant material. Ash content did not change.

Discussion

Fermentation properties of the selected aquatic plants (Trial I)

A minimum WSC content of 75g/kg DM (Pahlow et al 2003) and an adequate amount of lactic acid bacteria are required to establish a good fermentation. The tested aquatic macrophytes presented a low WSC content (<50 g/kg DM), and additionally, a relatively high buffering capacity. They showed very low WSC/BC ratios and consequently low fermentation coefficients (FC<35). Therefore, the addition of molasses as source of WSC is required to improve fermentation. Likewise, as natural populations of lactic acid bacteria are often low in number and hetero-fermentative on the plant material producing end-products other than lactic acid (Ennahar et al 2003), the inclusion of LAB inoculants is also desirable to improve the silage potential of the tested aquatic macrophytes.

In fact, these components were applied and the amount of fermentation end-products in the aquatic macrophytes silages was comparable to those reported for common silages as grass, corn, and alfalfa. In general, the pH values were lower than 4.5 as recommended by DLG (2006), and lactic acid concentrations were closely related to those reported by Kung (2001) for good quality alfalfa silage (70-80 g/kg lactic ac.) and grass silages (60-100 g/kg lactic ac.). The DLG points obtained in this study indicated very good silage fermentation.

Effect of fermentation on the nutritional quality of the aquatic plants (Trial II)

Characteristics of the raw aquatic macrophytes

The nutritional composition of the raw aquatic macrophytes in trial II was characterized by a high ash and crude fibre content, a limited lipid content (17-33 g/kg), an acceptable protein content (160-210 g/kg DM) considering the requirement of fish, and a well balanced amino acid composition. The content of ash and fibre for the tested plants in this study was comparable with values reported for several aquatic plants by Bairagi et al (2002), El-Sayed (2003), Kalita et al (2007) and Leterme et al (2009). Low content of lipids was also reported by Bairagi et al (2002)for Lemna polyrhiza (15 g/kg) as well as by El-Sayed(2003) for Eichhornia crassipes (10 g/kg). In contrast, Kalita et al (2007) reported a highercontent of lipids for Lemna minor (50 g/kg) from northeast India when compared to the present study. The deficit of lipids should be supplemented by additional components when the aquatic macrophytes are included into fish diets.  

The crude protein content of Eichhornia crassipes found in trial I differs from values found in literature and from the findings of trial II for which the plants were harvested in natural ecosystems. A higher nutrient content can be explained by the ability of Eichhornia to take up nutrients from the water. Mainly, this occurs when they grow in nutrient-rich water provided with large amounts of chemical nutrients as is usual for cultured aquatic plants as those used in trial I. 

Aquatic macrophytes are rich in minerals, exceeding the fish requirements but not the critical values for fish. Mineral composition of the raw aquatic macrophytes revealed a comparable content among plants, except for Azolla. Concentrations of calcium and phosphorus were similar in all the plants. Calcium and potassium were the most abundant minerals tested. Phosphorus content in Lemna and Spirodela ranged within the requirements reported for common fin fish (NRC 1993), whereas the Ca:P ratio was higher than the fish requirements.The reduction of calcium in the mineral mixture used in formulated diets must be considered.

Azolla showed the highest amount of zinc (161 mg/kg), copper (14.8mg/kg) and chromium (18.2mg/kg). Although the requirement of zinc for the majority of fish is much lower, varying from 15 to 30 mg/kg (NRC 1993), higher levels of supplemental zinc are frequently included in the practical diets to compensate the reduced zinc bioavailability caused by other dietary factors such as phytates. In some fish species (trout and carp) the tolerable limit of zinc in diets has been reported as 1900 mg/kg (Jeng and Sun 1981; NRC 1993). Likewise, the requirement of copper for fish, which varies from 3 to 5mg/kg, is much lower than in the fern Azolla. However, it does not exceed the tolerable limit forfish diets, which has been reported as 150 mg/kg (NRC 1993). Mineral concentration should be considered before the inclusion of Azolla into fish diets depending on the fish species and its particular tolerable limits.

The heavy metal concentration is particularly important as aquatic plants tend to accumulate them. In this study, the concentrations of the heavy metals arsenic, selenium and mercury were not detectable. Cadmium ranged in very low amounts from 0.48 to 1.31 mg/kg, whereas lead ranged from 3.14 to 3.98 mg/kg. Cadmium has been reported to be absorbed through the gastrointestinal tract of fish (NRC 1993) and causes liver necrosis and mortality at doses of 5 mg/kg of body weight. In contrast, Hodson et al (1978) reported that dietary lead was not absorbed by rainbow trout fed lead in different amounts. In this study the concentrations of heavy metals were not critical, but if aquatic macrophytes were used as exclusive feed source for fish, heavy metal contamination of feed, particularly cadmium retention, must be considered since it reduces fish growth, feed conversion and can be toxic.

Characteristics of the fermented aquatic macrophytes

Except for phytates content in Azolla, the antinutritional substances, trypsin inhibitor, phytates,tannins(hydrolyzed and condensed), and oxalates were significantly reduced by the lactic acid fermentation. According to Francis et al (2001) the tolerable limit of trypsin inhibitor is below 5 g/kg TI for the most cultured fish. Likewise, phytates (as phytic acid) and sodium phytates have been reported to cause depression in growth and food conversion efficiency of fish at levels of 5 g/kg and 10 g/kg in diets (Spinelli et al 1983,Hossain and Jauncey 1993). The level of trypsin inhibitor (lower than 1.37 mg/g TI), phytates (lower than 1.5 g/kg phytic ac.), tannins (not detectable) and oxalate (about zero) in fermented aquatic macrophytes did not exceed the critical value for commonly cultured fish.

Crude fibre was significantly lower in the fermented aquatic macrophytes when compared to the unfermented samples. This decrease in the fibre content could be explained by the partial acid hydrolysis of hemicelluloses resulting from the microbial utilization (Jones 1975).Crude protein content was also affected by fermentation. However, the effects of lactic acid fermentation on the protein content are conditional and strongly depend on the plant species. Thus, changes can be explained by differences in the chemical properties of the plant species, by the effect of molasses supplementation, and by the processes occurred on the plant material over the six weeks of fermentation. The decreases of crude protein in fermented Azolla and Eichhornia may be related to a slower acidification in the silage leading to an increase in proteolysis (Cussen et al 1995), whereas the increases of crude protein in fermented Lemna and Spirodelamay have been occurred through microbial synthesis (Wee 1991).

In general, lactic acid fermentation is highly recommendable before the inclusion of aquatic macrophytes into fish diets. As high fibre content of plant ingredients has a negative impact on digestibility (De Silva et al 1990), the raw aquatic macrophytes would not be recommended as exclusive nutrient sources. However, aquatic macrophytes silages may be used for the partial replacement of protein sources or as mineral source for the supplementation of basic fish feed in farming. Further research must be addressed to evaluate the optimum dietary inclusion level of the tested aquatic macrophytes in fish diets and the effect of their inclusion on fish performance. The establishment of easy practicable fermentation methods on-farm should be considered for the inclusion of the silage into fish diets.

Acknowledgements

The first author gratefully acknowledgesthe PhD scholarship award of the German Academic Exchange Services (DAAD). The authors would also like to thank Prof. Dr. Richter for her support with statistical question marks, theWAKUS GmbH and the Botanical Garden of Berlinfor providing the plant material used in the Trial I, and to Dr. Pieper Technologie-und Produktentwicklung GmbH (Germany) for providing the commercial silage inoculants BIO-SIL® used in the experiments. This work was financially supported by project COLCIENCIAS Cod. 1117-452-21305 and the University of Magdalena.

Literature cited

AOAC International 1995 Official methods of analysis of AOAC International.2 vols. 16th edition.Arlington, VA, USA.

AOAC International 2005 Official methods of analysis of AOAC International.18th edition. Gaithersburg, MD, USA.

Bairagi A, Sarkar G K, Sen SK and Ray AK 2002 Duckweed (Lemna polyrhiza) leaf meal as a source of feedstuff in formulated diets for rohu (Labeorohita Ham.) fingerlings after fermentation with a fish intestinal bacterium. Bioresource Technology 85, p.17-24.

Bicudo A J A, Sado R Y and Cyrino J E P 2009 Dietary lysine requirement of juvenile pacuPiaractusmesopotamicus(Holmberg, 1887). Aquaculture, v. 297, n. 1-4, p. 151-156.

Boyd C E 1971 Leaf protein from aquatic plants.In: FAO, Fish.Tech.Pap. T187.

Brabben T 1993 Research needs for aquatic plant management in developing countries. J. Aquat. Plant Manage. 31, p. 214-217.

Cussen RF, Merry RJ, Willians AP, Tweed JKS 1995 The effect of additives on the silage of forage of differing perennial ryegrass and white clover contents. Grass and forage sciences 50, p. 249-258.

De Silva S S, Shim K F, Ong A K 1990 An evaluation of the method used in digestibility estimations of a dietary ingredient and comparisons on external and internal markers and time of faeces collection in digestibility studies in the fish Oreochromisaureus (Steindachner). Reprod. Nutr. Dev. 30, p. 215–226.

DLG Ausschuss für Futterkonservierung 2006 Grobfutterbewertung. Teil B: DLG-Schlüssel zur Beurteilung der Gärqualität von Grünfuttersilagen auf der Basis der chemischen Untersuchung. DLG e.V. (Hrsg.): DLG-Information 2/2006, 5 S. from: http://www.dlg.org/fileadmin/downloads/fachinfos/futtermittel/grobfutterbewertung_B.pdf

Dordio A, Palace-Carvalho A J, Martins-Teixeira D, Barrocas-Dias C, Paula-Pinto A 2010 Removal of pharmaceuticals in microcosm constructed wetlands using Typha spp. and LECA.Bioresource Technology 101, p.886–892.

Earp C F, Akingbala J O, Ring S H and Rooney L W 1981 Evaluation of several methods to determine tannins in sorghums with varying kernel characteristics. Cereal Chemistry, v. 58, n. 3, p. 234-238.

El-Sayed A-F M 2003Effects of fermentation methods on the nutritive value of water hyacinth for Nile tilapia Oreochromis niloticus (L.) fingerlings. Aquaculture,v.218, p. 471–478.

Ennahar S, Cai Y and Fujita Y 2003 Phylogenetic Diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Applied and environmental microbiology, v. 69, n. 1, p. 444–451.

Francis G, Makkar H P S and Becker K 2001 Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, v. 199, p. 197-227.

Franklin P, Dunbar M and Whitehead P 2008 Flow controls on lowland river macrophytes: A review: Science of the total environment, v 400, n.1-3, 369-378.

Hodson P V, Blunt B R and Spry D J 1978 Chronic toxicity of water-borne and dietary lead to rainbow trout (Salmo gairdneri) in Lake Ontario water. Water Research,v. 12, n. 10, p. 869–878.

Hossain M A and Jauncey K 1993 The effect of varying dietary phytic acid, calcium and magnesium levels on the nutrition of common carp, Cyprinuscarpio. In: Kaushik, S.J., Luquent, P. (Eds.), Fish Nutrition in Practice. Proceedings of International Conference, Biarritz, France, June 24–27, 1991, p. 705–715.

Jeng S S and Sun L T 1981 Effects of dietary zinc levels on zinc concentration in tissues of Common carp. The Journal of Nutrition, v. 111, p. 134-140.

Johnson H E, Merry D R, Davies D R, Kell D B, Theodorou M K and Griffith G W 2005 Vacuum packing: a model system for laboratory-scale silage fermentations. Journal of Applied Microbiology, v. 98, p. 106-113.

Jones I D 1975 Effect of processing by fermentation of nutrients. In: Bairagi A., SarkarGhosh K., Sen S.K., Ray A.K., 2002. Duckweed (Lemnapolyrhiza) leaf meal as a source of feedstuff in formulated diets for rohu (Labeorohita Ham.) fingerlings after fermentation with a fish intestinal bacterium. Bioresource Technology,v. 85, p. 17-24.

Kaiser E, Weiß K and Polip I 2002 A new concept for the estimation of the ensiling potential of forages. In: Gechie L.M., Thomas C. (eds): Proc. 13^th Int. Silage Conf. Auchincruive, Scotland, p. 344-358.

Kakade M L, Simons N and Liener I E 1969An evaluation of natural vs. synthetic substrates for measuring the antitryptic activity of soybean samples. Cereal chemistry, v. 46, p. 518-526.

Kalita P, Mukhopadhyay P K and Mukherjee A K 2007 Evaluation of the nutritional quality of four unexplored aquatic weeds from northeast India for the formulation of cost-effective fish feeds. Food Chemistry, v. 103, p. 204–209.

Kung L Jr. 2001 Silage fermentation and additives. In: Proceedings of Alltech’s 17th Annual Symposium. (K.A. Jacques and T.P. Lyons, eds). Nottingham University Press, Nottingham, UK, p. 145-159, from:http://en.engormix.com/MA-feed-machinery/formulation/articles/silage-fermentation-additives-t369/800-p0.htm

Leite R G, Araújo-Lima C, Victoria R L and Martinelli L A 2002 Stable isotope analysis of energy sources for larvae of eight fish species from the Amazon floodplain. Ecology of freshwater fish, v. 11, p. 56-63.

Leng R A 1999 Duckweed: A tiny aquatic plant with enormous potential for agriculture and environment. FAO Animal production and health paper.

Lengerken J and Zimmermann K 1991 Handbuch Futtermittelprüfung. Deutscher Landwirtschaftsverlag, Berlin.

Leterme P, Londoño A M, Muñoz JE, Súarez J, Bedoya CA, Souffrant WB and Buldgen A 2009 Nutritional value of aquatic ferns (Azollafiliculoides Lam. and Salviniamolesta Mitchell) in pigs. Animal Feed Science and Technology, v. 149, p. 135-148.

McDonald P, Henderson A R and Heron S J E 1991 The biochemistry of silage. 2nd edition, Chalcombe publications, Marlow, UK.

National Academy of Sciences (NAS) 1976 Making aquatic weeds useful: Some perspectives for developing countries.Washington, D.C., USA.

National Research Council (NRC) 1993 Nutrient requirements of fish. National Academy Press, Washington, D.C., USA, p. 124.

Pahlow G, Munk R E and Driehuis F 2003 Microbiology of ensiling.Silage Science and Technology.Edited by D. R. Buxton, R. E. Muck and J. H. Harrison.Madison, USA, 2003, p. 31-94.

Pípalová I 2003 Grass carp (Ctenopharyngodonidella) grazing on duckweed (Spirodela polyrhiza). Aquaculture International,v. 11, p. 325–336.

Price M L and Butler L G 1977 Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain. Journal of agricultural and food chemistry,v. 25, n. 6, p. 1268–1273.

Rodriguez L and Preston T R 1996 Comparative parameters of digestion and N-metabolism in Mong Cai/Large white cross piglets having free access to sugar cane juice and duckweed. Livestock research for rural development, v. 8, p. 72-81.

Santiago C B and Lovell R T 1988 Amino acid requirements of growth of Nile tilapia.J. Nutr., v. 118, p. 1540-1546.

Schmidt L, Weißbach F, Wernecke K D and Hein E 1971 Erarbeitung von Parametern für die Vorhersage und Steuerung des Gärungsverlaufes bei der Grünfuttersilierung zur Sicherung einer hohen Silagequalität. Forschungsbericht, Rostock.

Sétliková I andAdámek Z 2004 Feeding selectivity and growth of Nile tilapia (Oreochromis niloticus L.) fed on temperate-zone aquatic macrophytes. Czech J. Anim. Sci., v. 49, n. 6, p. 271–278.

Smith C, Megen W V, Twaalfhoven L and Hitchcock C 1980 The determination of trypsin inhibitor levels in foodstuffs. Journal of the Science of Food and Agriculture, v. 31, n. 4, p. 341–350.

Spinelli J, Houle C R and Wekell J C 1983 The effect of phytates on the growth of rainbow trout (Salmogairdneri) fed purified diets containing varying quantities of calcium and magnesium. Aquaculture, v. 30,p. 71–83.

Vasquez E 1989 The Orinoco river: a review of hydrobiological research. Regulated Rivers Research and Management, v. 3, p. 381-392.

Wee K L 1991 Use of non-conventional feedstuff of plant origin as fish feeds is it practical and economically feasible. In: Bairagi A, SarkarGhosh K, Sen S K and Ray AK 2002 Duckweed (Lemna polyrhiza) leaf meal as a source of feedstuff in formulated diets for rohu (Labeorohita Ham.) fingerlings after fermentation with a fish intestinal bacterium. Bioresource Technology, v.85, p.17-24.

Weiß K and Kaiser E 1995 Milchsäurebestimmung in Silageextrakten mit Hilfe der HPLC. Wirtschaftseig. Futter. 1995, S.41, p. 69-80.

Weißbach F and Honig H 1992 Ein neuer Schlüssel zur Beurteilung der Gärqualität von Silagen auf der Basis der chemischen Analyse. Proc. 104.VDLUFA-Kongreß, Göttingen; p.489-494.

Xiao L, Yang L, Zhang Y, Gu Y, Jiang L and Qin B 2009 Solid state fermentation of aquatic macrophytes for crude protein extraction.Ecological Engineering, v. 35, p. 1668-1676.

Xie Y, Yu D and Ren B 2004 Effects of nitrogen and phosphorus availability on the decomposition of aquatic plants. Aquat. Bot., v. 80, p. 29–37.

Yılmaz E, Akyurt I and Günal G 2004 Use of duckweed, Lemna minor, as a protein feedstuff in practical diets for common carp, Cyprinuscarpio, Fry. Turkish Journal of Fisheries and Aquatic Sciences, v. 4, p. 105-109.

Received 14 March 2011; Accepted 17 October 2011; Published 4 November 2011

Go to top