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INTRODUCTION
The development of intensive aquaculture requires optimizing the use of nutrients by the cultured species, for this the selection of raw materials is necessary, taking into account the availability of plant- based foods with potential use in diets for omnivorous fish such as tilapia. Therefore, the culture actions from the stages of larva and small fish receive the most attention in aquatic farms (Das et al. 2018).
The search for alternative foods for agricultural production emphasizing the use of available natural resources as a main line is an important aspect; one of the greatest challenges has been to find sources of easily acquired proteins (Tacon and Metian 2015).
In this sense there are local products such as aquatic plants (unconventional foods), which are not universally used in animal feeding, but well used can be an important element in sustainable production systems, by virtue of their low cost, recognizing that some of them can turn into environmental contaminants (Abdel-Tawwab 2008).
Several authors have considered the aquatic macrophytes as a plague due to their rapid growth, which sometimes invade lakes and cause several problems. However, if its proliferation power is properly managed, its nutrient absorption capacity and the bioaccumulation of other water compounds make it a useful tool in the wastewater treatment. In the world and particularly in Asia, farmers produce and harvest aquatic plants for different purposes, including green manure and animal food source (Islam and Nishibori 2017).
Therefore, the objective of this study was to evaluate the effect on growth and survival indicators of inclusion levels of water fern meal (Azolla filiculoides) in diets for red tilapia hybrid small fish (Oreochromis mossambicus x O. niloticus).
MATERIALS AND METHODS
Location and experimental ecology. The experiment was carried out at the Estación Acuícola ACUIPASO, Bartolomé Maso, Granma province, Cuba. In this stage, the following climatic variables were recorded; average temperatures of 24.6 to 28.2°C; relative humidity between 42 and 60 %, average rainfall between 269.40 and 294.42 mm, and light hours from 9.16 to 11.04.
Meal production. The plants were collected from a reservoir of the Estación de Alevinaje. For the Azolla filiculoides production, two concrete pools were built with a dimension of 10 m wide by 20 m long and 1.5 m deep each. A total of 50 g/m2 of seed from the fresh plant were sown and later to fertilize, 240 kg of pig excreta were deposited in different parts of the pools at the beginning of the experimental stage (Pérez et al. 2014). Later, it was washed three times with distilled water and put to dry in the sun, on polyethylene mat with a total area of 32 m2; four turns were made during the day until 4:00 p.m., for three days; then the dehydrated plant was collected and stored in a cool place (Fasakin et al. 2001). A 250 μm hammer mill with a sieve was used to prepare the meal. Once obtained, it was stored in polyethylene bags at room temperature.
Chemical composition. Foods were measured in quadruplicate such as Dry matter (DM), Crude protein (CP), Ashes, Crude Fiber (CF), Ether extract (EE) according to AOAC (2000). The digestible energy (DE) was estimated theoretically from the conversion factor of 3.80 Kcal/g DM of plant-based food (De Graaf and Janssen 1996).
Determination of amino acids. The total amino acids were quantified by acid hydrolysis with 6 N HCl and the amino acid separation was performed on a Beckman 6300 High Performance Analyzer. The analysis was done in quadruplicate.
Inclusion levels of Azolla filiculoides meal. Formulation, elaboration of the diets and preparation of the experimental diets: For the establishment of inclusion levels of Azolla filiculoides meal (table 1), the results obtained by Priyo et al. (2011) were taken as a reference, who used meals from Lemna sp. and Azolla mexicana in the polyculture of tilapia (Oreochromis sp.) small fish. Based on this experience, productive results and the literature reviewed, the water fern meal was used at inclusion levels of 10, 20, and 30 %.
Table 1 Experimental diets according to replacement levels by Azolla meal (dry base)
| Azolla filiculoides, % | ||||
|---|---|---|---|---|
| Ingredients, % | 0 | 10 | 20 | 30 |
| Azolla meal | 0 | 10 | 20 | 30 |
| Fish meal | 25.0 | 18.0 | 16.0 | 14.0 |
| Soybean meal | 33.5 | 42.5 | 43.2 | 43.4 |
| Wheat meal | 27.6 | 15.6 | 6.9 | 0.3 |
| Starch | 5.0 | 5.0 | 5.0 | 3.4 |
| Fish oil | 0.2 | 0.2 | 0.2 | 0.2 |
| Sunflower oil | 2.7 | 2.7 | 2.7 | 2.7 |
| P. Minerals1 | 2.0 | 2.0 | 2.0 | 2.0 |
| P. Vitamins2 | 2.0 | 2.0 | 2.0 | 2.0 |
| Alginate | 2.0 | 2.0 | 2.0 | 2.0 |
| Total | 100 | 100 | 100 | 100 |
1mg/kg of the diet: (Roche Chemistry Inc.). Magnesium sulfate 5.1; Sodium chloride 2.4; Potassium chloride 2; Ferrous sulfate 1; Zinc sulfate 0.2; Cupric sulfate 0.0314; Manganous sulfate 0.1015; Cobalt sulfate 0.0191; Calcium iodate 0.0118; Chrome chloride 0.051.
2mg/kg of the diet: (Roche Chemistry Inc.). Thiamin 60; Rivoflavin 25; Niacin 40; Vitamin B6 50; Pantothenic acid 75; Biotin 1; Folate 10; Vitamin B12 0.2; Choline 600; Myoinositol 400; Vitamin C 200; Vitamin A 5000 UI; Vitamin E 100; Vitamin D 0.1; Vitamin K 5.
The bromatological composition of the diets used in the experiments is shown in table 2, where the Azolla filiculoides meal is included at levels of 10, 20, 30 % for red tilapia small fish. The methodology proposed by (Méndez-Martínez et al. 2018a) was taken into account for its formulation and elaboration.
Water quality. During the bioassay, the physical-chemical indicators of the water were recorded: temperature (ºC), dissolved oxygen concentration (mg/L) and pH. The flow of this liquid, coming from the supply well, was maintained at 60 L minute (Méndez-Martínez et al. 2018a)
Preparation of experimental diets. All the diets (table 2) were formulated using Excel Solver from Windows 2010, in which fish meal was partially replaced by Azolla filiculoides meal(10, 20 and 30 %). All the ingredients were sieved through a 250 μm mesh, and each diet was prepared by mixing all the macro-ingredients in an industrial blender until a uniform mixture was obtained. The micro-ingredients were mixed by hand in a plastic container before adding them to the macro-ingredients. Soya lecithin and fish oil were mixed until a homogeneous mixture was obtained and then water was added. The 3 mm granules were extracted with a meat grinder and dried for 8 hours at 45 °C in an airflow oven. Subsequently, the dried granules were packed in plastic bags and kept refrigerated at -4 ° C until use. The elaborated diets showed the required compaction, due to the correct process of milling and mixing of each ingredient, in addition the characteristics of the agglutinant were taken into account according to Méndez-Martínez et al. (2018a).
Table 2 Composition and contribution of the diets tested according to replacement levels by Azolla filiculoides meal
| Azolla filiculoides, % | Requirements | ||||
|---|---|---|---|---|---|
| Nutrients | 0 | 10 | 20 | 30 | Small fish |
| DM (%) | 93.40 | 93.60 | 93.70 | 93.60 | - |
| CP (%) | 35.00 | 35.00 | 35.00 | 35.00 | 35.00 |
| CF (%) | 2.20 | 3.10 | 4.90 | 5.70 | 8 |
| Threo (%) | 0.71 | 0.72 | 0.76 | 0.79 | 1.70 |
| Val (%) | 0.92 | 0.94 | 1.00 | 1.06 | 1.20 |
| Met (%) | 0.45 | 0.40 | 0.39 | 0.39 | 0.75 |
| Iso (%) | 0.99 | 1.02 | 1.06 | 1.09 | 1.39 |
| Leu (%) | 1.23 | 1.34 | 1.44 | 1.54 | 1.46 |
| Lys (%) | 1.40 | 1.30 | 1.29 | 1.28 | 2.20 |
| Hist (%) | 0.49 | 0.48 | 0.48 | 0.49 | 0.75 |
| Arg (%) | 0.95 | 1.04 | 1.10 | 1.16 | 1.81 |
| Tyip (%) | 0.93 | 0.93 | 0.95 | 0.98 | 0.43 |
| DE, MJ/g food | 0.0126 | 0.0117 | 0.0113 | 0.011 | 0.0126 |
DM: dry matter; CP: crude protein; EE: ether extract, CF: crude fiber; A: ash, FNE: Free Nitrogren Extract; Threo: threonine, Val: Valine, Met: methionine, Iso: Isoleucine, Leu: leusin, Lys: lysine, Hist: histidine, Arg: arginine, Trip: tryptophan. DE: Digest- ible energy.
Reception of red tilapia small fish. The hybrid red tilapia small fish (Oreochromis mossambicus x O. niloticus) were acclimatized in a reception tank prepared with water at zero parts per thousand salinity, aerated at 7 m/L and without chlorine. The most important physical-chemical indicators (temperature and pH) were taken, and then the tilapia bags were placed inside the tank with water and the water was tempered at 26 °C until the contents were completely empty, keeping the small fish in acclimation process for 7 days.
Bioassay with small fish. The culture of the organisms was developed for a period of 60 days. A total of 120 small fish of red tilapia hybrid (Oreochromis mossambicus x O. niloticus) were used with an age of 26 days and initial average weight of 0.7 g ± 0.5 g, weighed on a 0.01 g Sartorius precision scale, and initial average length of 2.1 to 2.7 cm. The small fish were placed in the experimental tanks and 4 groups were formed: the control and 3 diets where the Azolla filiculoides meal was included at 10, 20 and 30 %. For each of the treatments, 3 tanks (repetitions) with 10 small fish per each were used, 30 small fish per treatment, for 12 tanks in total. The fish were fed the diet at 8 % of their live weight on a fresh basis and the daily amount offered in each tank was divided into 3 frequencies a day at the following times: 7:30 a.m., 11.30 a.m., and 3:30 p.m.
Controlled productive indicators. The animals were individually weighed on a digital scale (± 0.01 g, PE 3600 Mettler-Toledo) and the total length was determined with a digital vernier gauge (± 0.001 mm, GT-MA15). All calculations were obtained from the means of the three replications. The following formulas were applied to obtain productive and nutritional indicators.
Where:
Wx: is the final body weight (g), Wi: is the initial body weight (g) and t: is the duration of the experiment (days) (Arnauld et al. 2017).
Experimental design and statistical analysis. A completely randomized design with four treatments and three repetitions was used. A simple classification variance analysis was applied to the obtained results, considering diets as the only variation factor. The difference between the means was quantified by the Duncan(1955) test. For the normal distribution of the data the Kolmogorov-Smirnov (Massey 1951) test was used and for the variances the Bartlett (1937) test.
RESULTS AND DISCUSSION
The physical-chemical indicators of the water (oxygen concentration, pH and temperature) were in the optimum average value (3-10 mg/L, 6.5-9, 24- 32 °C) in the 47 days that the experiment lasted (table 3).
Table 3 Physical-chemical indicators of water during the red tilapia small fish culture
| Indicators | Mean | SD |
|---|---|---|
| Oxygen, mg/L | 5.34 | 0.67 |
| Temperature, °C | 24.33 | 0.46 |
| pH | 8.02 | 0.08 |
The dissolved oxygen is an environmental factor that controls metabolic activity since oxidative capacity depends on it, the number of molecules that can be catabolized and the amount of energy obtained to perform metabolic work. The value of the previous indicator obtained in this study is in the range of that was reported by Llanes et al. (2011), 5-7 mg L-1 and explained that the species (Oreochromis mossambicus x O. niloticus) has the ability to take oxygen on the surface when the concentration in the medium is low (less than +3 mg L-1), although this would lead to a decrease in food intake (Buenaño-Buenaño et al. 2018 and Méndez-Martínez et al. 2018a).
During the experimental period, the temperature values obtained, as average of 27.12 °C, were in the normal ranges (24-32 °C). Among the physical factors, one of great importance in the aquatic species culture is the water temperature since it is closely related to the oxygen concentration and vice versa, due to the directly proportional relation between both factors. On the other hand, these variations influence on the fish nutrition since, by decreasing the temperature values below the established limits for each species, the oxygen dissolved in the water also decreases and these animals reduce the intake and adjust their metabolism to changes in the environment, although increasing the temperature within tolerable limits leads to an increase in protein requirements (Cruz-Velásquez et al. 2014) aspects to take into account to manage this species during the nursery stage.
This aquatic macrophyte (Azolla filiculoides) has a high water content, which has an impact on its use, hence in this research it has been determined to use dehydrated (meal) to reduce the volumes to be used in the diet, and to obtain better use of these, and can be used in minor species of farm, fish and crustaceans. Due to its high biomass production, it is considered promising for its use in the breeding of aquatic species (Gangadhar et al. 2015, Pérez et al. 2014 and Radhakrishnan et al. 2014).
This macrophyte has high water content but the meal obtained in this research showed 91.44 % DM (table 4), an adequate value for the meal of this type of plant since it helps its conservation. These results coincide with Carranco et al. (2002) and Yu et al. (2016) that when evaluating seven aquatic species found values between 84-92 % DM.
Table 4 Chemical and amino acid composition of Azolla filiculoides meal
| Indicator | Mean, % | SD |
|---|---|---|
| Dry matter | 91.44 | 0.04 |
| Crude protein | 20.74 | 0.06 |
| Ether extract | 2.95 | 0.01 |
| Crude fiber | 19.34 | 0.03 |
| Ash | 29.8 | 0.03 |
| FNE | 18.6 | 0.06 |
| Amino acids based on CP | ||
| Threonine | 2.30 | 0.05 |
| Valine | 3.14 | 0.04 |
| Methionine | 1.40 | 0.07 |
| Isoleucine | 2.40 | 0.03 |
| Leusin | 3.80 | 0.104 |
| Lysine | 2.65 | 0.04 |
| Histidine | 1.78 | 0.03 |
| Arginine | 2.30 | 0.2 |
| Triptophan | 0.97 | 0.05 |
The protein levels were of 20.74 %, results that are below those reported by Carranco et al. (2002) and Méndez-Martínez et al. (2018a) with 31.26 % in Azolla mexicana. The potential value of aquatic plants as food for herbivores has been emphasized more in comparison with terrestrial plants. The nitrogen content of trees, shrubs and grasses, many intakes as forage, is similar to or lower than aquatic macrophytes and algae. Gangadhar et al. (2015) and Brouwer et al. (2016) reported tenors between 20 and 30 %, the chemical composition of Azolla sp. usually varies according to the place where the plant grows, the change of season and the nutrients content of the water and the one found in this plant was mainly due to the favorable ecological conditions that exist in the water reservoirs.
This approach is based on the plant's ability to rapidly absorb nutrients from the environment due to the degradation of the organic matter carried out by bacteria adhering to their roots.
Regarding the crude fiber content, 19.34 % was obtained, which is considered of great interest due to the impact of this nutrient on the voluntary intake and digestibility of animals, especially monogastric. This value is higher than those reported by Gangadhar et al. (2015) and Gökçınar and Bekcan (2015) (13.1 - 16.5 %), so it must be taken into account when preparing the diet, so that it remains within the permissible range for these organisms (Méndez- Martínez et al. 2018a b).
The Ethere Extract reached 2.95 % higher when compared to studies where percentages of 0.78 were found (Méndez-Martínez et al.2018b). It is important to highlight that although amino acids are the main biomolecules for fish, this indicator provides essential fatty acids for organic functionality, as well as material that cannot be saponified characterized by phytosterols and liposoluble vitamins.
The ash content was 29.8 %, higher than those reported by Carranco et al. (2002) and Ngugi et al. (2017) who reported values between 8-20 % and argued that the float plants have more of this component compared to the submerged and these, in turn, than the emergent and marginal vegetation. This may be due to contact with water and environmental factors such as dust and residual wastes from the treated water in which they are.
Regarding the amino acid composition Azolla meal is rich in threonine, valine, isoleucine, leucine and lysine, it is essential the presence of ten essential amino acids in the diet of fish: arginine, lysine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan and valine, since they intervene in the growth and development, as well as in the formation of the tertiary structure of protein, constituting the essential amino acids up to 50 % of the protein, results that coincide with Wu et al. (2016), who reported that it is important to mention that the values of Azolla were higher in lysine and methionine when compared with those reported for soybean meal and meat and bone meal (Das et al. 2018).
Omnivorous fishes such as tilapia have a certain degree of metabolic efficiency of foods from vegetable origin, due to the structural adaptations they have in their digestive system. However, at a global level it is common for fish foods to include fishmeal. Fishmeal is found in almost all commercial diets, because it is very digestible and rich in essential amino acids, mainly lysine, but still efforts are made to find other foods that can be included in fish diets and thus reduce the cost of them (Ochieng et al. 2014 and Kollah et al. 2015).
The results of the final live weight, ADG and absolute growth rate (table 5) did not show significant differences between the control and 10 % of Azolla, which in turn differed from the rest of the treatments. This indicator (average daily gain) is considered by Abdel-Tawwab (2008) in Tilapia zilli, as one of the main criteria for evaluating productivity in this species and in this sense, Cruz-Velásquez et al. (2014) showed similar results to those obtained in this research. When evaluating the use of aquatic plants (Lemna minor, Spirodela polyrhiza and Azolla filiculoides) fermented in small fish of Cachama blanca and Tilapia del Nilo, obtained gains of 4.16 -5.84g/day. On the other hand Gökçınar and Bekcan (2015) when replacing fishmeal with Azolla (10, 20 and 30 %) found higher results in final weight, ADG, final length concluding that this plant is a source of protein of vegetable origin that can be used for fish culture.
Table 5 Productive performance of the small fishes that intake Azolla filiculoides meal.
| Indicators | % Azolla filiculoides | SE ± | P | |||
|---|---|---|---|---|---|---|
| 0 | 10 | 20 | 30 | |||
| Initial weight (g) | 0.771 | 0.763 | 0.755 | 0.770 | 0.001 | 0.075 |
| Final weight (g) | 6.742a | 6.556a | 5.781b | 5.915b | 0.026 | 0.007 |
| Weight gain (g/day) | 5.971a | 5.793a | 5.026b | 5.145b | 0.015 | 0.033 |
| Absolute growth rate | 3.61a | 3.58a | 3.39b | 3.40b | 0.469 | 0.006 |
| Feed conversion (g/g) | 1.840a | 1.850a | 1.890b | 1.880b | 0.0023 | 0.012 |
| Food efficiency | 0.551a | 0.550a | 0.523b | 0.521b | 0.002 | 0.041 |
| Initial lenght (cm) | 2.600 | 2.401 | 2.525 | 2.602 | 0.078 | 0.083 |
| Final lenght (cm) | 7.242a | 7.266a | 6.451b | 6.455b | 0.569 | 0.004 |
| Survival (%) | 100a | 100a | 98b | 95c | 1.267 | 0.010 |
ab Different letters in the same row differ to P <0.05 (Duncan 1955)
Results similar to the one obtained this research expressed Gangadhar et al. (2015) when using inclusion levels of 10, 20, 30 and 40 % of Azolla sp. in rations for Labeo fimbriatus and concluded that the good productive performance of the fish was due to the correct formulation of the diet and good zootechnical management.
The best results for the conversion and food efficiency were for the control treatments and 10 %, without differences between them, where the conversion is below 1.90 g/g and food efficiency varied between 0.52-0.55, so that the conversion index obtained in this study can be attributed to the management and acceptance and nutritional contribution of some aquatic macrophytes such as Azolla sp, which covered the nutritional requirements of the small fish.
In this sense, Ngugi et al. (2017), showed results similar to those obtained in this research when evaluating Amaranthus hybridus meal as a partial substitute of fishmeal for Nile tilapia, obtaining feed conversion values of 1.53 and 1.87g/g and average gains of 3.78-5.55 g , stating that this vegetable product can replace up to 80 % fishmeal in the diet of O. niloticus, without compromising the growth, use of nutrients and their digestibility.
CONCLUSIONS
The Azolla filiculoides have adequate chemical and amino acid composition so that it could be used in the feeding of red tilapia small fish, with 10 % of inclusion in the diet, results in accordance with the control diet were obtained in the productive indications without affecting the water quality.
