Nutritional Potential and Apparent Digestibility of Organic Matter, Protein, and Energy of Two Duckweeds (Lemna Minor and Lemna Eaquinoctialis) In Juvenile Tilapia Oreochromis Niloticus (Brazil Strain)

The aquaculture practice represents an important alternative for increasing protein sources, and for reducing the food insecurity in Sub-Saharan African countries. Studies have reported the critical issue of fishery and aquaculture associated with nutrition improvement, food security, and economic growth in developing and emergent countries [1]. Other studies analysed this situation by showing that there are few knowledge on influences of climate change and demographic transition on aquaculture products. Indeed, aquaculture plays an increasing role in aquatic food security [2]. The World Food Organization reported that the consumption of fish has increased at 3.0% yearly, representing about twice the proportion of annual world population growth (1.6%), and the per capita consumption rate estimated at 20.2 kg [3]. The need of animal protein covered by fishing is increasing, with a fish production estimated at 178 million tons worldwide, where up to 88 million tons come from aquaculture [3]. Aquaculture is strongly dependent on food representing 60% of aquaculture production costs [4]. For the composition of farmed fish feed, the fishmeal and oils are main important ingredients because of their high contents on proteins and essential fatty acids, which are necessary for the growth of fishes [5]. These compounds are costly and limited access in developing countries. The use of fish from fisheries for the production of fishmeal and fish oil, for farmed fish feed in aquaculture reduced the availability of natural fish stocks that are already overexploited [6]. 

There is a need to investigate new sources of improved farmed fish feeds in aquaculture practices in Sub-Sahara Africa, particularly in Côte d’Ivoire. Moreover, some alternatives sources of feed have been experienced by many authors to increase in context-specific for ensuring food security and the sustainable management of natural resources. For example, recently a study showed that microalgal biomass has the potential to support the growth of fish and shrimp aquaculture [7]. Authors revealed that fish and microalgae have similar nutritional profiles and their use as highly digestible feed reduced production costs, feed waste generation, and the risk of eutrophication. Other investigations recommended the reduction of the feed production costs in aquaculture through the replacement of fishmeal and oil by additional protein sources [8]. Many plants have the potential to replace fishmeal in aquaculture [9]. Usually, food ingredients for aquaculture are cereals, oil cakes, oil seeds, and aquatic plants [4,10,11]. Duckweed is the aquatic plant used as food supplements, with the potential to produce a biomass for fish farming and animal husbandry [12]. Moreover, their protein, vitamin, micro and macronutrient content makes them suitable for fish feed [13]. Specifically, Tilapia fed by Lemna valdiviana and Heterobranchus longifilis by Lemna pauciscostata have shown high growth performance rates [14,15]. 

In Côte d'Ivoire, located in West Africa, the national fishery production is estimated at 116 028 tons/year. This quantity is still low for covering the fish demand for the population that is estimated 618 182 tons/year [16]. Overall, 549 233 tons are imported to fix the demand of fish requirements. In this context, many efforts are undertaken by the government in Côte d’Ivoire, through piloted projects and scientific research to increase fish production locally [16]. This challenge faced some difficulties, because there are few investigations of food supplements, particularly plant food ingredients in aquaculture for reducing the fish feed production cost. It is therefore, necessary to provide scientific evidence on aquaculture potential addressing the critical issue of nutritive characteristics, physico-chemical quality of fishes, and associated risk for improving decision making in Côte d’Ivoire. This applied research assessed the nutritional profile and digestibility potential of two duckweeds (L. minor and L. eaquinoctialis) in Tilapia juvenile (O. niloticus, Brazilian strain), in order to improve a feed-based ingredient for aquaculture practice in Côte d’Ivoire.

Study design 

The overall methodology of this study used experimental designs comprising duckweed leaves production, and fish cultivated in six experimental ponds. The apparent digestibility coefficient of leaves by Tilapia O. niloticus, and physico-chemical characteristics of the aquatic environment were subsequently assessed. 

Duckweeds production in experimental ponds 

The culture of duckweeds (L. minor and L. eaquinoctialis) was carried out at the laboratory of the Centre de Recherches Océanologiques, in two experimental ponds (13.78 m³ each) following the protocol developed by [17]. Indeed, each pond comprised a polyethylene liner filled with water (5000 L). On the first day of the experiment, 5 kg of pig manure from the piggery were added to two ponds. Then, 100 g of L. minor and L. eaquinoctialis, harvested from the natural environment, were used to seed the ponds. After one week of the cultivation process, a biomass of duckweeds had formed on the surface of the ponds. The sample of this biomass was harvested for chemical composition analyses. 

Experimental design 

The digestibility of duckweeds was assessed through the experimental design using six aquaria (45 L of water each). Practically, these aquaria integrated a closed-circuit system tank that were connected to an electric pump (5 L/min, 550 Watts). Every day, 30% of water in aquaria was renewed. Fishes (35.73±0.08 g) were then randomly distributed in aquaria with 10 fishes per aquarium. Fishes were adapted to the experimental conditions for one week by feeding with a commercial diet (35% of protein). Wastes in aquaria were cleaned by siphoning, and three batches were used for feed testing (L. minor and L. eaquinoctialis). Physico-chemical parameters analyses concerned in situ variables, such as temperature (T°), pH, and dissolved oxygen of the water of aquaria, daily assessed at 8:30. In each aquarium, the average values of temperature, pH and dissolved oxygen were 27.2°C; 7.3 and 4.7 mg/L, respectively. Every week, the aquarium water was sampled for laboratory analyses of phosphorus and ammoniacal nitrogen. The values of these parameters were ranged between 2.29 and 2.39 mg/L, and 0.41 and 0.43 mg/L, respectively. 

Feeding and faeces collection 

Duckweed was harvested with a dip net and rinsed with tap water. Fishes were fed with fresh duckweed manually and apparent satiety twice a day at 9:30 AM and 2:30 PM. Uneaten leaves were recovered 45 minutes after feeding. Fish faeces collection started three days after the beginning of the trial, by siphoning at one-hour intervals before the second feeding phase, where faeces from each triplicate were stored at -20°C until analysis. The digestibility test was performed over 15 days. 

Analytical methods 

Analytical methods were used for the physico-chemical characteristics of duckweeds and fish faeces. Indeed, the composition of duckweeds, particularly L. minor, L. eaquinoctialis and fish faeces were assessed in the laboratory to estimate dry matter after drying at 105°C for 24 hours. The content of total organic matter, hydrolysable organic matter, resistant organic matter to hydrolysis and minerals were determined according to the method described by [18], modified by [19]. The analysis of proteins, lipids and fibres were done by a standard method [20]. Indeed, Proteins were determined by the Kjeldahl method, lipids were with hot hexane soxhlet approach and fibres by acid and alkaline hydrolysis. The ash content was determined by incineration of samples of duckweeds and faeces in a muffle furnace at 550°C for 24 hours [21]. The non-nitrogenous extractives content were determined by the method used by [22] and the energy by [23]. The essential amino acid profile was determined by the method of the High-Performance Liquid Chromatography (HPLC,) (Waters Alliance, Model 2695, USA). 

Assessment of apparent digestibility coefficient 

The Apparent Digestibility Coefficient (ADC) of fresh duckweeds leaves was assessed for four compounds (organic matter, proteins, lipids, energy) following the method of [24] quoted by [25], using minerals (Ash) as reference material. The apparent digestibility coefficient was determined as follows:

Ni (%)=[(N_Ff/Min_Ff –N_F / Min_f)/(N_Ff/Min_Ff)]×100

With:

Ni: Nutrient content in duckweeds

N_Ff : Percentage (%) of nutrient content in duckweeds

N_f: Nutrient content in fish faeces (%)

Min_Ff: Mineral content in duckweeds (%)

Min_f: Mineral content in faeces (%)

Statistical analysis 

Statistical analyses were performed using STATISTICA 7.1 software (Informer Technologies Inc, France). Practically, descriptive statistics were implemented for assessing physico-chemical characteristics of duckweeds. The inference for analyzing the significant difference between L. minor and L. eaquinoctialis the apparent digestibility coefficient were done using ANOVA statistics One-way Analysis of Variance. Additional multiple comparisons of means were conducted with Tukey test at 5%.

Chemical composition of duckweeds L. minor and L. eaquinoctialis 

(Table 1) shows chemical composition of duckweeds L. Minor (LM) and L. Eaquinoctialis (LE) used as feed in the experimental ponds. Total Organic Matter (TOM) contents were ranged from 974.73 to 978.49 mg g^-1, Hydrolysable Organic Matter (HOM) from 954.84 to 967.74 mg g^-1, Hydrolysable Resistant Organic Matter (HROM) from 10.75 to 19.89 mg g^-1 and minerals (Min) from 21.51 to 25.27 mg g^-1. Furthermore, the results showed that the proportion of hydrolysable organic matter (967.74 mg g^-1) in L. minor are higher (p < 0.05) than those observed in L. eaquinoctialis (HOM: 954.84 mg g^-1). But, hydrolysable resistant organic matter (19.89 mg g^-1) and mineral (25.27 mg g^-1) contents in L. eaquinoctialis are higher (p < 0.05) than those in L. minor (HROM: 10.75 mg g^-1; Min: 21.51 mg g^-1). Lipid (3.15%), ash (2.42%), and energy (19.12 kj g^-1) determined in L. minor are statistically higher (p < 0.05) than those obtained in L. eaquinoctialis (1.49% lipid, 1.13% ash and 18.41 kj g^-1 energy). Protein contents estimated at 38.80% in L. minor were also high, with essential amino acid concentration of leucine were 5.29 g/16gN, 4.22 g/16gN for arginine, valine (3.66 g/16gN), phenylalanine (3.65 g/16gN), tryptophan (3.64 g/16gN) and 3.18g/16gN for lysine. The lowest values are observed for methionine (0.99 g/16gN) and histidine (1.39 g/16gN) (Table 2). The protein/energy ratio of L. minor is estimated at 20.29 and 16.14 for L. eaquinoctilias. 

Table 1: Approximate composition of L. minor and L. eaquinoctialis collected from the experimental ponds.

In each line the values with different letters are significantly different (p < 0.05). 

DM: Dry matter;

TOM: Total Organic Matter;

HOM: Hydrolysable Organic Matter;

HROM: Hydrolysable Resistant Organic matter;

Min: Mineral (ash); NNE: Non-nitrogenous extractives. 

Table 2: Essential amino acids composition in proteins of cultivated L. minor and L. eaquinoctialis

On each line, values with letters indicate a statistically significant difference (p < 0.05). 

Anti-nutritional contents in L. minor and L. eaquinoctialis 

(Table 3) shows the composition of oxalate, tannin, saponin, and phytate in L. minor and L. eaquinoctialis. The concentrations of the compounds varied for L. minor and L. eaquinoctialis. Indeed, the concentration of oxalate was estimated at 2.06 mg/100g and 0.13 mg/100g for saponin, and particularly higher in L. minor. High concentrations of tannins (1.40 mg/100g) and phytates (0.81 mg/100g) were observed in L. eaquinoctialis. Among the investigated anti-nutritional contents, oxalate had the highest concentration, and saponin with the lowest levels in both duckweeds species. 

Table 3: Anti-nutritional substances in L. minor and L. eaquinoctialis. 

On each column, values with different letters indicate a statistically significant difference (p < 0.05). 

Heavy metal contents in L. minor and L. eaquinoctialis 

(Table 4) shows the concentration of metals in duckweeds collected from the experimental ponds. The concentration of lead was estimated at 1.215 mg kg^-1 and 0.042 mg kg^-1 for cadmium in L. eaquinoctialis. The lead was estimated at 0.009 mg kg^-1 and 0.000 mg kg^-1 for cadmium in L. minor. The concentration of mercury was 0.001 mg kg^-1 for L. eaquinoctialis and 0.002 mg kg^-1 for L. minor. The results indicated that a statistically significant difference (p < 0.05) was observed between heavy metals contents of L. minor and L. eaquinoctialis. 

Table 4: Heavy metal contents of Lemna minor and Lemna eaquinoctialis from experimental ponds. 

On each column, values with different letters indicate a statistically significant difference (p < 0.05). 

Assessment of apparent digestibility coefficients of duckweeds 

Table 5 shows the Apparent Digestibility Coefficients (ADC) of duckweeds in O. niloticus. The apparent digestibility coefficient of L. minor was estimated at 39.08% for Total organic matter, Hydrolysable organic matter (45.93%), protein (56.30%) and energy (44.38%). Furthermore, L. eaquinoctialis was estimated at 31.74% for Total organic matter, Hydrolysable organic matter (41.68%), protein (46.20%) and energy (37.69%). The results showed a significant difference (p < 0.05) between the apparent digestibility coefficients of L. minor and L. eaquinoctialis in O. niloticus. 

Table 5: Apparent Digestibility Coefficient (ADC), organic matter, proteins, and gross energy of duckweeds. 

In each line the values with different letters are significantly different (p < 0.05).

This study assessed the nutritional potential and the apparent digestibility of L. minor and L. eaquinoctialis on juvenile O. niloticus. It was observed that L. minor and L. eaquinoctialis had high nutritive potentials as fish feeds for aquaculture in Côte d’Ivoire. 

This study revealed that investigated duckweeds had a significant biomass content of organic matter, with important quantity particularly of hydrolyzable organic matter (967.74 mg g^-1 for L. minor and 954.84 mg g^-1 for L. eaquinoctialis). This result is close to the study reported in periphyton, a preferred natural food source of phytophagous fishes, such as tilapia [26,27]. Additionally, the assessment of the analytical composition showed high concentration of lipids, proteins, and amino acids in L. minor leaves. Total lipid contents remained higher in L. minor, with high values close to those recorded (3 to 7% DM) in duckweeds grown in nutrient rich medium [28,29]. Revealed that a nutrient poor medium induced low lipid content in duckweeds. The results of this study show that L. minor grown under the same conditions as L. eaquinoctialis, would be less demanding of nutrient resources. Protein contents obtained in L. minor in the present study are similar to the finding reported by other author in similar contexts [30,13]. Protein contents and Protein/Energy ratio evaluated are close to values reported by [31] who showed that dietary protein contents (35 - 40% with P/E ratio of 20 mg kJ^-1) allow a good growth of tilapia O. niloticus. Among anti-nutritional substances (oxalates, tannins, phytates, saponins) in the investigated duckweeds, the highest value of oxalate was observed in both duckweeds species. These results corroborated the observations made by [32] in the duckweed of Lemna species. Overall, the concentrations of anti-nutritional substances in duckweeds remained low than those reported in aquatic plants used as fish feeds in aquaculture, where concentrations varied from 0.6% - 3.5% for calcium oxalate, tannin (0.25% - 0.93%) and phytate (0.004% - 0.005%) [33]. The investigated duckweeds in the experimental ponds, could not be considered as risk for fish health, particularly concerning heavy metals such as lead, cadmium, and mercury. These chemical concentrations in this study were in line with European community standards for heavy metals in the raw material of animal feeds, with thresholds fixed at 10 mg kg^-1 for lead, cadmium 1 to 2 mg kg^-1 and mercury 0.1 mg kg^-1 [34]. 

Furthermore, the apparent digestibility coefficient of total organic matter, hydrolyzable matter of L. minor and L. eaquinoctialis show middle digestion of duckweeds organic matter in O. niloticus. These results are consistent with those obtained in O. niloticus reared in extensive fish ponds with a diet based primarily on natural nutrient resources [27]. The digestibility performance of the organic matter appeared to be consubstantial with plants, which in the natural state exhibit low nutrient availability associated with low digestibility [9]. However, the digestibility of the protein fraction and energy remained higher in fish fed with L. minor compared to L. eaquinoctialis. Overall, the apparent digestibility values observed for the duckweed-fed fish in this study remained low compared to those obtained when duckweeds are incorporated into the compound diet. Studies have already shown the relevance of protein apparent digestibility coefficient of 77% in O. niloticus fed with 20% by fresh L. minor [35]. 

The lower values of apparent digestibility coefficients in this study could be explained by the presence of anti-nutrients that could interfere with nutrient utilization [36], and consequently could reduce their digestibility by O. niloticus. Some substances such as tannins have the property of binding or precipitating proteins and various organic compounds, including amino acids and alkaloids [37]. Tannins are considered as inhibitors of the enzymatic activity of some enzymes, mainly lipase, proteolytic enzymes, alpha-galactosidase, cellulase, pectinase and amylase [38]. Therefore, the presence in duckweeds of anti-nutritional substances, suggest the application of adapted treatments for their efficient use in fish diets [39].

This study showed that L. minor and L. eaquinoctialis have important hydrolyzable organic matter. Among the two tested fresh duckweeds species, L. minor showed the best protein and energy utilization coefficient. However, adequate treatment of fresh duckweeds would improve the ingestion of these leaves by the fish as a source of protein.

1. Béné C, Arthur R, Norbury H, Allison EH, Beveridge M, et al. (2016) Contribution of fisheries and aquaculture to food security and poverty reduction: Assessing the current evidence. World Development Elsevier 79: 177-196.
2. Garlock T, Asche F, Anderson J, Love DC, Osmundsen TC, et al. (2022) Aquaculture: The missing contributor in the food security agenda. Global Food Security 32: 100620.
3. FAO (2022) La situation mondiale des pêches et de l’aquaculture, FAO.
4. Nates SF (2016) Introduction, In S.F. Nates (Ed) Aquafeed Formulation, Academic Press, San Diego.
5. Yeo MG, Blé CM, Otchoumou AK, Dabonné S, Alla YL, et al. (2017) Digestibility and growth performance in fingerlings of tilapia Oreochromis niloticus fed with diet containing high-carbohydrate ingredients. International Journal of Fisheries and Aquatic Studies 5: 171-175.
6. Médale F, Kaushik S (2008) Evolution des recherches en nutrition piscicole à l’INRA: Substitution des produits d’origine marine dans l’alimentation des poissons d’élevage. Productions Animales 21: 87-94.
7. Tacon AG, Metian M (2008) Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquaculture 285: 146-158.
8. Ahmad A, Hassan S, Banat F (2022) An overview of microalgae biomass as a sustainable aquaculture feed ingredient: Food security and circular economy. Bioengineered Taylor and Francis 13: 9521-9547.
9. Oliva-Teles A, Enes P, Peres H (2015) Replacing fishmeal and fish oil in industrial aquafeeds for carnivorous fish. Feed and feeding practices in aquaculture 203-233.
10. Daniel N (2018) A review on replacing fish meal in aqua feeds using plant protein sources. International Journal of Fisheries and Aquatic Studies 6: 164-179.
11. Otchoumou AK, Blé CM, Etchian OA, Alla YL, Niamké SL, et al. (2012) Effects of increasing dietary protein levels on growth, feed utilization and body composition of Heterobranchus longifilis (Valenciennes, 1840) fingerlings. African Journal of Biotechnology 11: 524-529.
12. Jha S, Pokharel B (2015) Duckweed: The hidden treasure. Nepalese Journal of Biosciences 5: 1-20.
13. Chakrabarti R, Clark WD, Sharma JG, Goswami RK, Shrivastav AK, et al. (2018) Article| Mass Production of Lemna minor and Its Amino Acid and Fatty Acid Profiles | University of Stirling. Frontiers in Chemistry 6: 479.
14. Mohedano R, Rodríguez J, Fracalossi D (2005) Lemna valdiviana, uma planta que além de tratar efluentes alimenta os peixes cultivados. Panorama da Aquicultura 15: 33-40.
15. Effiong BN, Sanni A, Fakunle JO (2009) Effet de remplacement partiel de la farine de poissons avec des lentilles d’eau (Lemna pauciscostata) sur la performance de croissance de fingerlings de Heterobranchus. Rapport et Avis 1: 76-8.
16. Yayo N’cho AJ, Aka KS, Tano K (2020) Faible niveau de production halieutique nationale face à la demande croissante de la population en Côte d’Ivoire. Fiche Technique et Document de Vulgarisation 1: 24-29.
17. Tavares FA, João Bosco RR, Fracalossi DM, Juan E, Roubach R (2008) Dried duckweed and commercial feed promote adequate growth performance of tilapia fingerlings. Biotemas 21: 91-97.           
18. Buddington RK (1980) Hydrolysis-Resistant Organic Matter as a Reference for Measurement of Fish Digestive Efficiency. Transactions of the American Fisheries Society 109: 653-656.
19. Bowen SH, Lutz EV, Ahlgren M (1995) Dietary protein and energy as determinants of food quality: Trophic strategies compared. Ecology 76: 899-907.
20. AOAC (1990) The Official methods of analysis of the Association of Official Analytical Chemists (15th ed) 1230.
21. AOAC (1995) Official methods of analysis (16th edn) Association of Official Analytical Chemists 250.
22. Kerdchuen N (1992) L’alimentation artificielle d’un silure africain, Heterobranchus longifilis (Teleostei: Clariidae): Incidence du mode d’alimentation et première estimation des besoins nutritionnels.
23. Luquet P, Moreau Y (1989) Energy-protein management by some warmwater finfishes. Advances in Tropical Aquaculture, Workshop at Tahiti, French Polynesia 751-755.
24. Conover RJ (1966) Assimilation of Organic matter by zooplankton 1. Limnology and Oceanography 11: 338-345.
25. Getachew T (1987) A study on an herbivorous fish, Oreochromis niloticus L, diet and its quality in two Ethiopian Rift Valley lakes, Awasa and Zwai. Journal of fish Biology 30: 439-449.
26. Ble MC, Arfi R, Yeboua AF, DIOPOh KJ (2006) Food quality of the trophic sources provided by an acadja (Ebrié lagoon, Ivory Coast, West Africa). Life & Environment 255-264.
27. Ble MC, Arfi R, Yeboua AF, Diopoh KJ (2007) Qualité nutritive de l’alimentation naturelle du tilapia Oreochromis niloticus en élevage extensif dans des étangs de barrage (Côte d’Ivoire). Bulletin Français de la Pêche et de la  Pisciculture 385: 1-16.
28. Hasan MR, Chakrabarti R (2009) Use of algae and aquatic macrophytes as feed in small-scale aquaculture: A review. Food and Agriculture Organization of the United Nations (FAO).
29. Appenroth KJ, Sree KS, Böhm V, Hammann S, Vetter W, et al. (2017) Nutritional value of duckweeds (Lemnaceae) as human food. Food Chemistry 217: 266-273.
30. Sharma JG, Kumar A, Saini D, Targay NL, Khangembam BK, et al. (2016) In vitro digestibility study of some plant protein sources as aquafeed for carps Labeo rohita and Cyprinus carpio using pH-Stat method. Indian Journal of Experimental Biology 54: 606-611.
31. Jauncey K, Ross B (1982) A guide to tilapia feeds and feeding. Institute of Aquaculture
32. Goopy JP, Murray PJ (2003) A review on the role of duckweed in nutrient reclamation and as a source of animal feed. Asian-australasian journal of animal sciences 16: 297-305.
33. Kalita P, Mukhopadhyay PK, Mukherjee AK (2007) Evaluation of the nutritional quality of four unexplored aquatic weeds from northeast India for the formulation of cost-effective fish feeds. Food Chemistry 103: 204-209.
34. CE (2002) Communautés Européennes numéro 221/2002 de la commission du 6 février 2002. Portant fixation de teneurs maximales pour certains contaminants dans les denrées alimentaires. Journal Officiel des Communautés Européennes 37/4, 37/5 et 37/6.
35. El-Shafai SA, El-Gohary FA, Verreth JA, Schrama JW, Gijzen HJ (2004) Apparent digestibility coefficient of duckweed (Lemna minor), fresh and dry for Nile tilapia (Oreochromis niloticus L.). Aquaculture Research 35: 574-586.
36. Akande KE, Doma UD, Agu HO, Adamu HM (2010) Major antinutrients found in plant protein. In Their effect on nutrition. Pakistan Journal of Nutrition 9: 827-832.
37. Yilkal T (2015) Important antinutrionnel substances and inherent toxicants of feeds. Food Science and Quality Management 36: 40-47.
38. Selle PH, Cowieson AJ, Cowieson NP, Ravindran V (2012) Protein-phytate interactions in pig and poultry nutrition: A reappraisal Nutrition research reviews 25: 1-17.
39. Yeo MG, Blé MC, Otchoumou AK, Dabonne S,  Alla YL, et al. (2017) Digestibility and growth performance in fingerlings of tilapia Oreochromis niloticus fed with diet containing high-carbohydrate ingredients. International Journal of Fisheries and Aquatic Studies 5: 171-175.