Artemia in Freshwater Prawn diets

Abstract

A 105-day feeding study was conducted to evaluate the effect of partial (0%, 20%, 40%, 60% and 80%) and complete (100%) substitution of dietary fish meal (FM) with frozen Artemia on growth performance and feed utilization of Giant freshwater prawn (Macrobrachium rosenbergii) post larvae (PL) reared in fiberglass tanks under controlled conditions. A total of 1080 M.rosenbergii PL, average weight of 6.10 ± 0.4 mg, were equally distributed over 18 fiberglass tanks, (1 m3 each, and triplicate per treatment). Six experimental isocaloric (14 MJ/kg/digestible energy) and isonitrogenous (35% crude protein) diets were formulated.

Control diet (D1, 100% FM) was formulated with FM as the main protein source (0% Artemia). Diets D2-D6 were substituted with five dietary protein levels of frozen Artemia (20%, 40%, 60%, 80% and 100%), respectively. Results showed that growth performance of M. rosenbergii PL fed different Artemia based diets was either equal or superior to prawn fed control FM diet. In addition, increasing dietary frozen Artemia replacement level from 20% up to 100% led to a significant (P ≤ 0.05) increase in survival, growth and feed utilization, which indicate that the essential nutrient requirements needed for M. rosenbergii PL was covering with improving feed satiation of PLs. It could be concluded that frozen Artemia biomass instead of fishmeal in M. rosenbergii PL diets can improve survival rate, growth performance, feed utilization, contributing to reducing the use of FM in practical diets and the nursery phase could be shorter and more profitable.

Key words: Dietary protein, fish meal, Artemia, replacement, M. rosenbergii, growth.

1. Introduction

Freshwater prawns belong to the family Palaemonidae which includes the brackish and freshwater grass shrimp and the larger river shrimp. Freshwater prawn, Machrobrachium rosenbergii, is currently popular with small scale aquaculture all over the world. It has a potential development for rural aquaculture, considerable employment and income could be generated, thereby bringing prosperity to rural poor [1]. Global expansion of freshwater prawn farming was very rapid since 1995 [2], which led to increase demand for PLs from hatcheries.

Farming of freshwater prawn is environmentally sustainable, since it is practiced at lower grow-out density [2]. In Egypt a major constraint in the large scale aquaculture of this species is the scarcity of seed at required quantities and in all locations. A majority of seed (post larvae, PL) used in grow out farming of M. rosenbergii comes from hatcheries [3]. Existing hatcheries in the country are not producing up to their installed capacity due to various constraints. In small scale hatchery system, back-yard system and small tanks nursery system normally produce up to fivesixth days of post larvae stages (PL5-6). These stages cannot be stocked directly in the grow-out ponds. Therefore nursing of post larvae from the small hatchery is still necessary. Nursing of PL can be done in many ways such as in concrete tanks, earthen ponds or in net cages. After the prawn PL metamorphosed to the PL stages of PL5 or PL6 they are harvested and transferred to the nursery tanks to avoid over crowding and to vacate the larval rearing tanks for the next operation.

Freshwater prawns are omnivorous scavengers that feed on a variety of bottom organisms and organic materials (detritus) by smell, taste and feel, rather than eyesight. The food habits of prawns vary during different life stages. Artificial diets are now utilized in the production of all life history stages of crustacean animals. Formulated, commercially available feeds are preferred for use in aquaculture operations due to their lower cost, ease of storage, and reduced incidence of bacterial contamination compared to live or frozen foods [4, 5].

Prawn farmers prefer to stock PL older than 10 days to ensure a high survival rate in their grow-out ponds.  However, feed is the single largest running cost item for M. rosenbergii culture, as it constitutes 30%-70% of the operational costs [6]. Hence, development of formulated feeds to attain higher survival, better growth and more efficient feed conversion ratios is needed. The growth of the prawn and shrimp aquaculture industry has resulted in a high demand diets, and feed manufacturers have responded by investing effort into developing formulated diets. Therefore, any management that improves feeding efficiency of M. rosenbergii PL is important in reducing feed costs. 

Diet is a fundamental aspect in larval culture of decapods crustaceans [7, 8]. Protein and lipids were the most critical components for maximal survival and growth of early stages of crustacean [9]. Generally, live food supplies all the necessary nutrients for development and can contribute with exogenous digestive enzymes that aid in digestion [10, 11]. 

Early stages of prawns PL, like larval penaeid shrimp, are ideally reared on live Artemia and are cannibalistic when the diet provided is unpalatable or insufficient. It was reported that as an alternate to live Artemia, a frozen Artemia can result in reasonable growth, survivorship [12] and supports growth rates approximately 60% of that of live Artemia [13]. Although prawns fed the frozen Artemia in the diet, showed a better growth performance and feed utilization, the cost of its purchase or production is prohibitive for large-scale hatchery use compared to dried Artemia [14]. The food for culture of M. rosenbergii PL consists basically of newly hatched Artemia nauplii [15] supplemented with inert food [16]. Feed consumption by PL is dependent uponnumerous factors related to culture conditions specific for site; at the same time, scientific knowledge is not enough to base an adequate feeding schedule. In general, hatcheries either maintain a constant Artemia supply (about 5 nauplii m-l day-l) or increase the density over culture period [17-19].

Most Egyptian feed manufacturers are using expensive imported FM as a protein source for aqua-feeds [20]; on the other hand, the higher cost of feed mainly responds to the cost of FM [21]. Therefore, assessment of cheaper or more readily available alternative protein sources such as fisheries by-products, processing or other sources that may reduce the use of FM in feeds is necessary. Among the alternative animal protein sources, Artemia biomass (adult Artemia) may be considered to be a suitable ingredient for replacing dietary FM for fish and crustacean because of its high nutritional value [14, 22-26]. Frozen adult Artemia included in a mixed diet improved the reproductive performance of Penaeus vannamei [27, 28].

The advantage of using Artemia biomass as alternative protein sources in the nursery phase of freshwater prawn diets lies not necessarily in their nutrient composition, but in their local availability and low prices. Unfortunately, live adult Artemia is excessively expensive to purchase, and labor intensive to produce. For these reasons, frozen Artemia was used in the present study to evaluate the effect of it´s totally or partially replacing FM as a protein source on growth performance and feed utilization of freshwater prawn post larvae, M. rosenbergii, reared in tanks under controlled conditions. 

Experimental grow-out production was carried out at Animal and Fish Production Department, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt for 105 days. Freshwater prawn PL with an average initial body weight of 6.10 ± 0.4 mg (PL6) was obtained from Hatchery of Mariut Fish Farm Company, Alexandria Governorate, Egypt. Prior to trial, M. rosenbergii PLs were acclimated to experimental condition for two weeks. During this period, PLs were fed daily ad libitum a fine minced tilapia flesh, three times a day (09:00, 12:00 and 16:00 h).

Six experimental treatments were assigned in triplicate. Experiments were conducted in fiberglass tanks at stocking density of 60 PL/tank, each holding 1 m³. Each culture tank was provided with four 15-cm long 8 mm diameter black polyvinyl chloride (PVC) pipe to minimize the cannibalism during the molting as suggested by Mariappan and Balasundaram [29]. All fiberglass tanks were cleaned daily from faeces and supplied with de-chlorinated tap water. The turnover rate of water was 0.7 m²/tank/day and PL was held under natural light (12:12, light:dark schedule). The tanks were aerated by electric blower. Each tank was stocked with 60 PL/m²; these stocking densities were based on the tank bottom area (1 m²) according to FAO [30]. Mortality and limb loss were recorded at 08:00, once a day. 

Six experimental isocaloric (14 MJ kg^-1 digestible energy) and isonitrogenous (35% crude protein) diets were formulated (Table 1) to investigated the effects of partial (0, 20%, 40%, 60% and 80%) and complete (100%) replacement of dietary fish meal (FM) with frozen Artemia biomass on growth performance and feed utilization of freshwater prawn, M. rosenbergii PL reared in fiberglass tanks. The control diet (D1, 100% FM) was formulated with FM as the main protein source (0% Artemia). Diets 2-6 each dietary FM was substituted with five different dietary levels of frozen Artemia biomass (20%, 40%, 60%, 80% and 100%). Dry ingredients were ground by screen diameter (1.0 mm) using a homogenous mixture grinder. The experimental pellet diets (2 mm) were processed by blending the dry ingredients through a laboratory hand pellet machine (locally made). The pelleting temperature did not exceed 60 °C and all diets were air dried for 4 h (moisture content of about 10%). All diets was packed in cellophane bags and cooled at -4 °C prior to use. During the 105 days experimental period, prawns larvae were fed daily ad libitum, three times a day at (08:00, 12:00 and 17:00), then the daily feeding ration was computed and re-adjusted according to mass live weight every 15 days. 

Gross energy content of diets were calculated according to caloric values of Brett [31] using the values of 23.6, 39.5 and 17.2 kJ/g for crude protein, crude fat and total carbohydrate, respectively. Dietary digestible energy (DE) was estimated using values of 20.9, 37.6 and 8.4 kJ/g for crude protein, crude fat and carbohydrate, respectively according to Das et al. [32].

To estimate the growth performance indices during the experimental period, initial and final as well as intermediate samples weights of individual prawns were measured using digital electronic balance (0.001 g accuracy). The first sampling was conducted after PL stocking and at 15 days interval thereafter. Survival rate was calculated according to the following formula; (SR%) Survival (S) = (final number of prawns/initial number of prawns) × 100. Moreover, mean final body weight (Wt) of each experimental treatment was determined by dividing total prawn weight in each tank by number of prawns. On the other hand, weight gain (WG), specific growth rate (SGR%), feed conversion ratio (FCR), protein efficiency ratio (PER), protein productive value (PPV%), energy retention (ER%) were calculated according to Ballestrazzi et al. [33] using the following equations: 

Survival (S) = (final number of prawns /initial number of prawns) × 100.
WG = Final body weight (g) - Initial body weight (g).
SGR = (ln FBW - ln IBW)/t × 100; where: FBW is final body weight (g); IBW is initial body weight (g);
ln = natural logarithmic; t = time in days.
PER = weight gain (g)/protein intake (g).
PPV = [protein gain (g)/protein intake (g)] × 100.
ER = [energy gain (kJ)/energy intake (kJ)] × 100.
ECR = Cost of diet ($ kg-1) × Feed conversion ratio (FCR)
The apparent as-fed FCR was calculated per aquarium as:

FCR = Total weight of feed consumed/Wet biomass gain.

Water quality parameters, including temperature, dissolved oxygen, pH and ammonia, were monitored to ensure that water quality remained well within the limits recommended for the PL prawn. Water temperature was recorded daily at 13:00 using a mercury thermometer suspended at 30 cm depth. Dissolved oxygen (DO) was measured at 8:00 using YSI model 56 oxygen meter (Yellow Springs Instrument, Yellow Springs, OH, USA) and pH by using a pH meter (Orion pH meter, Abilene, TX, USA). Ammonia and alkalinity were measured at weekly intervals according to Standard Methods for the Examination of Water and Wastewater [34].

At the beginning of the trial, random pooled samples of three healthy individual groups (150 PL each) of M. rosenbergii PL were selected, weighed and immediately frozen at -20 °C for assessment of the initial proximate body composition. At the termination of the feeding trial, 15 prawns were randomly sample from each experimental treatment (5 prawn each replicate) to assess final proximate body composition. The proximate composition of prawns and diet samples were determined according to Association of Official Analytical Chemists [35]. Moisture was determined after oven drying (105 °C) for 24 h (MEMMERT Drying Oven, GE-174, Memmert GmbH, Germany). Ash was measured by incineration at 550 °C for 12 h (Thermo Scientific Heraeus M 110 Muffle Furnace, Thermo Fisher Scientific Inc., Waltham, MA 02454, Germany). Crude protein was determined by micro-Kjeldhal method, N% × 6.25 (using Kjeltech autoanalyzer, Model 1030, Tecator, Höganäs, Sweden) and crude fat by soxhlet extraction with diethyl ether (40-60 °C) (Soxtec System HT6, Tecator, Höganäs, Sweden).

Data were statistically analyzed by ANOVA using MSTAT-C version 4 software [36]. Duncan´s multiple range test was used to compare differences between treatment means when significant F values were observed [37], at P  0.05 level. Responses to dietary frozen Artemia inclusion were examined by linear regression. All percentage data were arc-sin transformed prior to analysis [38]. However, data are presented untransformed to facilitate comparisons. 

All conditions of the experimental evaluation in the present study were apparently satisfactory, and fell under the optimal standards defined for nutritional evaluations in freshwater prawn, M. rosenbergii PL. All the water quality parameters were within the acceptable range for M. rosenbergii. Water temperature ranged from 27.4 to 28.5 ± 0.5 °C, dissolved oxygen from 5.7 to 6.4 ± 0.9 mg/L, pH from 6.9 to 7.6 ± 0.2 and ammonia from 0.21 to 0.24 ± 0.1 mg/L.

Results of survival and growth performance indices including final body weight (FBW, Fig. 1), weight gain (WG), specific growth rate (SGR %/day) of M. rosenbergii PLs are presented in Table 2. Freshwater prawn PLs fed 100% Artemia diet (D6) realized, significantly (P ≤ 0.05), the highest values of survival (95.0%), final body weight (1.76 g), weight gain (1.70 g) and specific growth rate (3.10 % / day). Meanwhile, M. rosenbergii PLs fed 100% fish meal diet (control diet, D1) significantly (P ≤ 0.05) achieved the lowest values of the same parameters (85.0%, 1.46 g, 1.39 g, 2.92%/day, respectively). 

On the other hand, results in Table 3 illustrated feed utilization including feed intake (FI), feed conversion ratio (FCR); protein efficiency ratio (PER), protein productive value (PPV %) and energy retention (ER%) of M. rosenbergii PLs. It was observed that post larvae fed 100% Artemia diet (D6) realized significantly (P ≤ 0.05) the highest values of feed intake (9.18 g /105 day /PL) and energy retention (21.76%), while PLs fed 100% fish meal diet (control diet, D1) were significantly (P ≤ 0.05) lower (7.13 and 19.22, respectively) and no significant (P ≤ 0.05) differences was observed for FCR, PER and PPV% between all treatments. In addition, the lowest value of ER% was recorded for M. rosenbergii PL fed the diet D2 [20% Artemia (18.88%)].

Table 4 illustrates the regression analysis between Artemia inclusion in PLs diets and growth performance with feed utilization parameters. Positive relationships were observed between Artemia inclusion levels and FBW; WG; SGR%; FCR; PPV%; ER%, while relationship with PER was negative. 

Table 5 reveals the proximate analysis of whole body composition for different experimental M. rosenbergii PL. It was observed that M. rosenbergii PL fed 60% Artemia diet (D4) recorded the highest value of whole body moisture content (77.15%) significantly (P ≤ 0.05); meanwhile the lowest value (73.95%) was observed for the prawn fed diet 40% Artemia (D3). Prawn PL fed the diet containing 80% Artemia biomass recorded the highest significant value (P ≤ 0.05) for crude protein (15.16%), ether extract (5.33%) and gross energy (626.54 kJ 100g-1), meanwhile those PLs fed the control diet (D1) showed the lowest value of CP and higher value of ash significantly (P ≤ 0.05). 

Culture of the giant freshwater prawn, M. rosenbergii, in temperate climates (such as Egyptian climate conditions) is characterized by a four-phase production system [3]. The hatchery and two nursery phases occur indoors, during the winter months of the year, while production of market-sized prawns is completed in outdoor ponds during the warm season [39]. Most investigations of M. rosenbergii commercial potential pond culture in temperate areas have indicated that nursed juvenile prawns, rather than new PL, must be stocked into ponds to achieve maximum profitability because the grow-out season is relatively short [40]. On the other hand, while prawns can obtain substantial nutritional benefit from natural foods at relatively low biomass densities [41], higher levels of biomass are likely to be more dependent on prepared diets [42]. In addition, freshwater prawns are more aggressive than penaeids and thus are not well suited to intensive culture [30]. 

In Egypt, the cost of producing nursed juveniles has emerged as a major factor influencing the profitability of the nursery operators and prawn farmers [39]. However, further in depth formation for relevant practical diets to freshwater prawns are to be developing to sustain higher growth, survival and feed efficiency of prawn PL. In the light of this, the present results elucidate the highest survival percentage, growth indices and feed utilization for prawn M. rosenbergii PL fed Artemia-based diets compared to control FM diet.

Aqua-feeds cost is the highest recurring cost in aquaculture, ranging from 30% to 70% of variable costs, depending on the intensity of the operation [6]. Animal origin ingredients such as fish meal (FM), poultry by-product meal and other rendered animal meals are considered among the most suitable protein sources for shrimp and prawn feeds. Most of studies carried out on shrimp during the two-three first months of independent life under controlled conditions have shown mean survival rates around 50%, with growth values between 90-200 mg, depending mainly on the species and feeding conditions [43-48]. 

From Artemia nutritional value point of view, the  reason of using freezed Artemia procedure is that the nutritional value of newly hatched Artemia with an undigested yolk sac are an excellent source of nutrition and without frequent feedings, the nutritional value of uneaten Artemia in the water column decreases over time because the nutrients contained in the yolk sac are continually being removed to satisfy growth and metabolic needs. 

Reference to this concept, Nyström and Savolainen et al. [49, 50] attributed better survival and growth of juvenile signal crayfish (Pacifastacus leniusculus) to the availability of non quantified live feed. In addition, supplemented a dry diet with known amounts of live Artemia nauplii and Daphnia, also obtained good results [51]. In these three studies, authors reported the highest survival rates obtained so far under intensive conditions (around 80% after 100 days), which are in the same range compared to our present results. Moreover, Sasaki and Capuzzo [52] reported that the initial freezing process of Artemia and/or conditions during shipping and storage could lead to degradation of essential phospholipids. On the other hand, the superior values of survival percentage, growth indices and feed utilization in the present study may be attributed to the non exposure of experimental frozen Artemia to such degradation factors and then, used frozen Artemia retained their properties well.

In the present study, results indicated that the growth performance of M. rosenbergii PLs fed Artemia-based diets was inferior to prawn fed control FM diet. Moreover, the present results can be suggested that the SGR% of M. rosenbergii PLs fed the Artemia-based diets (2.93%-3.10% day-1) was comparable to the prawn fed control FM diet (2.92% day-1). Moreover, prawn fed Artemia-based diets demonstrated a similar or superior range of survival (85%-95%) than prawn fed control FM-based diet (85%). 

On the other hand, regression analysis indicated that survival and growth rate of prawn PLs increased significantly (P ≤ 0.05) with increasing dietary Artemia protein inclusion level from 0% up to 100%, and the highest survival and growth performances was recorded for prawns fed D6 diet (100% Artemia). From nutritional point of view, the improve growth induces and feed utilization of prawn PL fed Artemia-based diets than control FM-based diet may attributed to better nutrients balance in Artemia-based diets and palatability, which may result in better feed satiation of the prawns and hence high survival (lower cannibalism) and good growth performance. With respect to the previous results of the present study, in agreement with Abelin et al. [53] who reported that PL30-45 of P. monodon and PL15 of P. vannamei fed a diet containing a freeze-dried Artemia meal had a significantly better growth as compared with the FM-based diet. Furthermore, Naegel and Rodriguez-Astudillo [54] demonstrated that feeding L. vannamei shrimp PL with dried Artemia biomass resulted in a significantly higher survival and larger size compared with four commercial feeds and three crustacean meals. Recently, Anh et al. [14] demonstrates the potential of Artemia biomass as a diet ingredient in aquaculture.

Considering the different costs of FM and Artemia, used in the present study, some considerations can be made, where collected Artemia biomass was very easy and inexpensive method. Hence, it is difficult to estimate its cost compared to the price of imported FM (8.5-13.5 E.P. equal 1.47-2.33 $/kg). This study shows that up to 100% replacement of FM with frozen Artemia biomass improves the economical conversion rate (ECR) values (Table 6). Prawn PLs fed the diet containing 100% Artemia biomass were the cheapest and are recommended for culturing prawn M. rosenbergii PL. 

In conclusion, the results of present study demonstrated that the formulated diets containing frozen Artemia biomass ensured that the essential nutrient requirements for M. rosenbergii PL were met with the experimental prawn´s nutritional requirements and resulted in excellent growth performance, feed utilization and high survival.

Therefore, use of Artemia biomass as a protein source of early stages of freshwater prawn, M. rosenbergii culture may improve the cost-effectiveness, contribute to reduce the use of FM in practical diets and also, the nursery phase can be shorter and more profitable. 

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