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Available online at http://www.urpjournals.com International Journal of Research in Marine Sciences Universal Research Publications. All rights reserved Original Article Determination of Total Ammonia Excretion in Embryo, Zoea and Post-larval Stages of the Giant clawed prawn, Macrobrachium rosenbergii (De Man, 1879) Suresh Babu CH 1 *, Shailender M 1, Krishna P V 1, Kishor B 2 1. Acharya Nagarjuna University, Nagarjuna Nagar, Andhra Pradesh, India 2. Andhra University, Vishakhapatnam, Andhra Pradesh, India Corresponding Author: Suresh Babu. CH Acharya Nagarjuna University, Nagarjuna Nagar, Andhra Pradesh, India. Email: chsbabu008@yahoo.co.in Received 18 June 2013; accepted 04 July 2013 Abstract Present study was conducted to determine the dry mass and the total ammonia-n (TAN) excretion in embryos, Zoea (I XI) and post-larvae (PL) stages of Macrobrachium rosenbergii at 1, 5, and 10 d after metamorphosis (PL1, PL5, and PL10). Tested animals were collected according to their developmental stages, and placed into incubation chambers ( 50 ml) for 2 hours to calculate the TAN excretion of the animal. After this period, analyses were carried out for individual TAN determination. Individual TAN excretion generally increased throughout the larval development and varied from 0.05 ± 0.021 μg TAN/individual/h in embryo to 0.945 ± 0.232 μg TAN/individual/h in PL10. There was no significant difference between embryo and zoea (P >0.05), whereas in post larvae (PL1, PL5, and PL10) differed (P <0.05) from each other. Higher increments in individual ammonia- N excretion were observed between Zoea V to Zoea X and PL1 PL10. The lowest value was found in embryo (0.05 ± 0.021 μg TAN/mg DM/h) and the maximum values in Zoea-V and PL10 (0.22 ± 0.34 and 0.64 ± 0.63 μg TAN/mg DM/h, respectively). Results indicate that metabolic rate is proportional to the body mass in M. rosenbergii, during early life stages. Variations in ammonia excretion during this phase may be associated mainly with body size. Based on the results of the present study may be useful in developing and optimizing rearing techniques of M. rosenbergii, such as rearing tank size, stocking density and culturing methods. 2013 Universal Research Publications. All rights reserved Key Words: Macrobrachium rosenbergii, embryos, different larval stages, dry mass, total ammonia excretion Introduction: Macrobrachium rosenbergii (de Man) is a giant freshwater prawn commonly called Scampi is an important commercial species for its highest protein content, low cholesterol and for its delicious meat. In India, giant freshwater prawn is distributed throughout the revarine regions in India and mainly in the Southern region where average temperature does not change much between night and day times and seasons. Though the prawn hatchery technology has advanced over the decade, the hatchery production is more often hampered by severe mortalities caused mostly by bacteria. Increasing ammonia concentration in larvae and rearing tanks water has been reported to reduce the survival rate of larvae and post larvae. The Scampi, M. rosenbergii, has a large geographic distribution in South-East Asia specially India, Thailand, Malaysia, Australia [1-4]. Some populations live in brackish water areas very far from the sea and complete their lifecycle in freshwater. On the other hand, the populations that occur close to the coast are dependent on brackish water for larval development [5]. The larvae hatch as a typical free-swimming palaemonid zoea and pass through eleven zoeal stages [6-7]. They are planktonic; backward swimming actively, tail first, upside-down within the water column and exhibit positive phototoxis. After metamorphosis, postlarvae (PL) are miniature of adult prawns, swim with the dorsal side uppermost and assume a more benthic lifestyle. Both larvae and PL are mainly omnivorous [8]. M. rosenbergii presents high potential for aquaculture [6]. Some characteristics that may contribute to the rearing feasibility are fast larval development (16-20 d) in clear water, green water and reused water systems (30 ± 1 0 C and 10 12 salinity) and high survival rates and productivity in hatchery, nursery, and grow-out phases [9]. Furthermore, meat has a firmer texture and a more marked taste compared with Macrobrachium rosenbergii, hence being better accepted by consumers [10]. Information on metabolism and the processes related to the use of energy throughout the ontogenetic development is necessary to improve culture practices. However, studies related with 33 International Journal of Research in Marine Sciences 2013; 2(2): 33-38

metabolic rates in M.rosenbergii are limited. In most of the aquatic crustaceans, particularly decapods larvae, ammonia is the major waste product [11]. Ammonia is excreted mainly by gills and may occur in ionized (NH 4 +) and unionized (NH 3 ) forms in the water. Unionized ammonia increases with increasing ph and temperature, but decreases as salinity rises [12]. However, ph is by far the major factor influencing NH 3 /NH 4 + ratios. Unionized ammonia has a higher toxicity because it can more easily diffuse across lipid membranes. It may reach toxic levels to rearing animals, if tank system and management are not properly designed. High ammonia concentration inhibits larval excretion [11] and respiration [13]. Therefore, quantifying ammonia excretion during ontogenetic development allows the establishment of suitable densities for prawn rearing and transportation in clear and recirculated water culture systems. Thus, the objective of the present study was to find the ammonia excretion rates in life cycle stages of hatchery reared M. rosenbergii. Materials and Methods This present experiment was conducted at the Kakati Aqua Tech Ltd, flood bank of River Krishna, Undavalli village, Vijayawada, Andhra Pradesh, India. This hatchery was well equipped with all facilities to complete life cycle of prawn larvae. M. rosenbergii brooders contain orange and brown/black eggs were obtained from culture ponds located in Bhimavaram area located in Andhra Pradesh, India. They are stocked in brooders rearing tanks and acclimatized to the hatchery conditions for incubation of egg. Brooders are fed twice in a day. Rice bran pellets, clam meat cow meat were supplied alternate days to the brooders as food. Embryo, larvae, and PL of M. rosenbergii were obtained from broodstock which are maintained in the experimental brooder holding tanks. Females carrying eggs in advanced stages of embryonic development (some hours before hatching) were used for the present experiment. This phase of egg incubation is characterized by slow movements of females and the presence of visible eyes inside the transparent eggs. Animals were disinfected in formaldehyde solution at 15 mg/l for 30 min, and then kept in a hatching tank at 100 individuals/m 2 and maintained under 6-8 salinity and 30 ±1 o C temperature. It was provided with aeration, heating system, and artificial substrates for hiding. Some females had eggs extracted for dry mass and embryos excretion determination. After hatching, larvae were stocked at 100 Zoea -I/L in 100 L cylindrical tanks. Water was kept at 30 o C, ph 7.8 8.2 ppm and 12 salinity until metamorphosis to post larvae. Larvae were fed on newly hatched Artemia nauplii (55 60% crude protein) from Zoea-II to Zoea-V (3 7 nauplii/ml/d). After this stage, they were fed on a 45% crude protein based egg custard inert diet (25 μg/ml/d) [2, 14] plus Artemia nauplii (8 10 nauplii/ml/d), supplied twice daily until metamorphosis in to post larvae [15]. PL were transferred to 1000 L tanks filled with freshwater at 10 PL s/l and fed on commercial pelleted diet (Higashi Maru No. 3 feed) twice a day (3 5 g/tank/d, 35% crude protein). In this study, PL1 is referred as the stage immediately after metamorphosis while PL5 and PL10 were PL at 5 and 10 d after metamorphosis, reared in freshwater at 30 ±1 0 C. Individual dry mass (DM) was determined by groups of 10 (embryo and Zoea-I Zoea-XII) or 10 (PL) individuals. They were gently rinsed with distilled water, dried with filter paper, placed in preweighted cartridges, and dried for 48 h at 70 0 C. Thus, dry samples were weighed on analytical balance at the nearest 1 μg. Ten replicates were obtained for each developmental stage. Table 1. Age, dry mass (DM), number of individuals, and biomass: volume ratio in incubation chambers used to determine ammonia-n excretion rates during early life stages of M. rosenbergii Larval Stage Age (days) Dry mass (μg) Animals /Crate Biomass :Volume Embryo 0 72.5 ± 0.4 40 58.0 Zoea-I 1 61.2 ± 0.5 20 24.5 Zoea-II 2 65.4 ± 0.2 15 19.6 Zoea-III 3 92.5 ± 1.6 10 18.5 Zoea-IV 5 121.6 ± 1.9 8 19.5 Zoea-V 6 315.7 ± 1.2 8 50.5 Zoea-VI 8 379.8 ± 1.8 8 60.8 Zoea-VII 11 468.2 ± 2.4 8 74.9 Zoea-VIII 13 578.0 ± 3.1 8 92.5 Zoea-IX 14 625.2 ± 6.7 6 75.0 Zoea-X 16 782.4 ± 10.2 6 93.9 Zoea-XI 17 857.3 ± 17.4 4 68.6 Post Larvae-1 19 957.2 ± 26.9 2 38.3 Post Larvae-5 24 1358.7 ± 59.8 2 54.3 Post Larvae-10 29 2297.4 ± 118.6 2 91.9 For the determination of total ammonia- N rectangular plastic crates (approximately 50 L volume) sealed with silicon tablets [16-17] were used as incubation chambers to measure the ammonia-n excretion of test animals. Crates were individually identified and the exact volume was gravimetrically determined. An orifice of 1.5 mm in the center of the cover enabled the elimination of air bubbles from inside the chamber during closure. Chambers were hermetically closed inside 1-L beakers filled with water (12 salinity) to avoid formation of bubbles inside the 34 International Journal of Research in Marine Sciences 2013; 2(2): 33-38

containers. After being closed, inside water was isolated from air by a plastic tablet 1.7-cm diameter silicon seal so that the tension between orifice water and the silicon seal could block inside water and air gas exchange. Animals were fed in excess just before excretion measurements. Embryos were kept in incubation chambers filled with 8 salinity brackish water for 2 h. Larvae and PL in the same development stage [18] were sorted and stocked into the chambers for 2 h. Larvae were incubated in 12 salinity brackish water, whereas PL were in freshwater (0 ). The number of individuals used for each developmental stage was determined according to individual dry mater (Table 1). The biomass and volume ratio was calculated by dividing the total dry mass of individuals by the chamber volume and varied from 18.5 to 93.9 μg/ml. Brackish water at 8 and 12 salinity and freshwater were used as control chambers without animals for embryos, larvae, and PL, respectively. Samples and controls were kept in water bath at 30 ± 1 o C for 2 h. Variation in total ammonia-n (TAN = unionized plus ionized ammonia as nitrogen) contents was calculated by the difference between values obtained in sample (with animals) and control (no animals) units. For TAN analysis [19] separate water samples was used. TAN excretion was expressed as individual (μg TAN/individual/h) and for specific dry mater (μg TAN/mg DM/h) rates. Differences among means were tested by oneway ANOVA (F-test), followed by Duncan s multi comparison test. Data of the ammonia excretion (in μg TAN/individual/h) and DM (in μg) were logarithmically transformed and then subjected to linear regression analysis (SPSS Software). Slopes were compared to 1 using a t -test [20]. Differences were considered significant at P <0.05. Figure 1. Individual dry mass (DM) and age (A) of early developmental stages of M. rosenbergii Figure 2. Individual total ammonia-n (TAN) excretion rates during the early life stages of M.rosenbergii Results: In the present study revealed that the individual dry matter was increased throughout the larval development (Table-1; Fig-1). The highest increases in subsequent stages were verified between the Zoea IV Zoea V (194%) and Zoea VIII Zoea IX (157%), while the lowest value was found between Embryo and Zoea I (4.2%). Individual ammonia-n excretion did not differ between embryo Zoea IV and Zoea V Zoea IX (P > 0.05), but differed between PL1 PL3 and PL7 PL10 (P <0.05) (Fig. 2A). The SD minimum observed value was 0.05 ± 0.021 μg TAN/individual/h in embryo, and the maximum was 0.98 ± 0.63 μg TAN/individual/h in PL14 (Fig- 2). Significant (P <0.05) increases in individual ammonia-n excretion were observed between Zoea IV Zoea V, PL1 PL10. The SD mean excretion from Zoea I Zoea IV and Zoea V Zoea IX were 0.11 ± 0.03 and 5.04 ± 0.08 μg TAN/individual/d, respectively. In PL1, PL5, and PL10, they were 0.42 ± 0.11, 0.57 ± 0.17, and 0.98 ± 0.09 μg TAN/PL/d. Massspecific excretion rates presented two groups, embryo Zoea II (P >0.05) and Zoea III PL10 (P >0.05). Stages ZIII, ZV, and PL1 presented high SD. Discussion The mass of M. rosenbergii increases during development stages from Zoea-I onward. This pattern was previously observed in M. rosenbergii [2, 21-24], and for other caridean larvae, such as Sesarma rectum [25], Crangon crangon, and Crangon allmanni [26]. A particularly high increase in M. rosenbergii body mass occurred from Zoea IV to Zoea V (194%), suggesting that important morphological and physiological modifications occur in Zoea V. On the other hand, [02] did not observe significant growth in M. rosenbergii larvae from Zoea I to Zoea III or higher growth rate from Zoea IV to Zoea V in larvae maintained in individual 100-mL plastic bowls, fed only Artemia nauplii. Probably, culture conditions have an important effect on larvae growth. In the present work, individual ammonia excretion generally increased during larval development and results showed five phases: embryo Zoea V, Zoea VI Zoea XI, PL1, PL5, and PL10. In the first phase, animals present low mass and use internal energy reserves from yolk, but from Zoea II to Zoea IV, also ingest exogenous food, [27-30]. In the second phase (Zoea VI Zoea XI), larvae increased noticeably in size, actively feed on inert diet plus Artemia nauplii [31] and the percentage of nitrogen in biomass is significantly higher [30, 32-33]. During this phase, the yolk supply is depleted and exogenous feeding becomes essential. It is likely that the increase in body mass, coupled with a switch to active feeding on exogenous food, is responsible for the increase in ingested food and consequently the increase in the individual ammonia excretion from the first to the second phase. In the last phases (PL5 PL10), there is an increase in individual ammonia excretion rates due to a great increase in animal biomass and consequently in the ingested food [34-35]. Therefore, it seems that the use of exogenous food is the main factor to increase excretion rates. However, many other processes related with the use of energy and protein metabolism may affect ammonia excretion during development [36], which probably caused the very high variability exhibited by data. The high standard deviation observed in Zoea V may be due to metabolic adjustments related to the change from endogenous to exogenous feed, whereas in Zoea VII the high variability may be associated with the beginning of feeding on inert food. In PL1, high variability may be related to the change in life strategies after metamorphosis. Larvae shifted from a planktonic to a benthic habitat and were transferred from the 10 salinity water to freshwater. In 35 International Journal of Research in Marine Sciences 2013; 2(2): 33-38

spite of decreasing salinities shown to increasing ammonia excretion in M. rosenbergii [37] and Marsupenaeus japonicas [38], this trend did not occur in the present study. However, since the diets supplied to the experimental animals had decreasing amounts of protein as the prawn attained PL stages and salinities decreased to zero (PL feed 35% protein diet and was maintained in freshwater), a compensatory effect may have occurred. Bertalanffy s classification [39] showed three metabolic types describes the relationship between metabolic rate and body size. In the first, the metabolic rate is proportional to the surface of the animal or the 2/3 power of the body mass. Salinity, temperature, feed diet, and feed schedule have varied in these different studies. Although metabolic differences may be partially attributed to variation in culture conditions, it seems that metabolism pattern is quite variable in decapod crustacean. Information on metabolic rates, such as ammonia excretion, is very important to design aquaculture systems and protocols operations. Our data suggest that a set of 1000 larvae produce a load of about 1 and 5 mg TAN/d during Zoea I Zoea V and Zoea VI Zoea XI phases, respectively. Thus, present values may be used as a base in M. rosenbergii culture for determination of stocking densities in rearing tanks or in transport bags, the frequency of water exchange in flow-through systems, and for estimating the dimensions of biofilters in recirculating systems. Biofilters used in freshwater prawn hatcheries have varied from 4 to 20% the size of the rearing tank [40]. This size was based on the daily maximum expected total ammonia load of M. rosenbergii and the bacterial carrying capacity of the filter [41-45]. 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