World Journal of Zoology 5 (2): 110-114, 2010 ISSN 1817-3098 IDOSI Publications, 2010

High Density Rotifer Culture as Live Food for Larval Rearing in Carp Hatcheries
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Kazi Ahmed Kabir, 1Refat Laila Baby, 2Imtiaj Hasan, 1 M. Niamul Naser and 1MD. Shahadat Ali

Department of Zoology, University of Dhaka, Bangladesh Department of Biochemistry and Molecular Biology, Rajshahi University, Bangladesh

Abstract: Rotifer is the most dominant zooplankton in all the freshwater aquatic ecosystems and considered as ideal food for fish larvae. High density culture of Brachionus calyciflorus, one of the most common rotifer species, was conducted in laboratory condition. Starting from the step of isolation and inoculation to production and preservation of resting eggs were successfully carried out by simplification and adaptation of existing high density culture method to local ingredients and environment. Fresh algae- Phacus, algae paste, Bakers yeast and mushroom powder was provided as feed in the B. calyciflorus culture vessel of 100 L glass tanks 4 times daily. Nutritional Enrichment of the target species was carried out by egg yolk and cod liver oil. Present study reveals that embryonic development of B. calyciflorus requires 0.5 days, female attains reproductive maturity after1-1.2 days, Interval period between two spawning was 3.5-3.8 hours and the doubling time was 2.26 days. The average final density during this culture was obtained 685 35 individual.mlG1 while the maximum was 721 individual.mlG1. Resting eggs were enumerated 1800/mg. The study also found that as the density of rotifer increases the ratio of egg production decreases but also the rate of growth per day continues to increase. Key words: Brachionus calyciflorus % High density culture % Resting egg production and preservation % Density and egg production ratio INTRODUCTION In nature, zooplankton is one of the primary foods of fish larvae. Two of the dominant zooplankton groups in freshwater of Bangladesh are Rotifera and Copepoda. These two groups are the preferred prey for shrimp and fish and are commonly used in aquaculture farms. The intensive larval rearing of carps depends on a large supply of zooplankton. The successful use of rotifers in the commercial hatchery operations of the red sea bream (Pagrus major) encouraged investigations in the development of mass culture techniques of rotifers [1]. Twenty five years after the first use of rotifer in larviculture feeding several culture techniques for the intensive production of rotifers are being applied worldwide. The availability of large quantities of this live food source has contributed to successful hatchery production. Success of rotifers as a culture organism are manifold, including their, planktonic nature, tolerance to a wide range of environmental conditions, high reproduction rate of 0.7-1.4 offspring.femaleG1.dayG1 [2]. Moreover, their small size and slow swimming velocity make them a suitable prey for fish larvae that have just resorbed their yolk sac but cannot yet ingest the larger food. However, the greatest potential for rotifer culture resides in the possibility of rearing these animals at very high densities. Densities of 2000 individuals.mlG1 have been reported by [3]. Even at high densities, the animals reproduce rapidly and can thus contribute to the build up of large quantities of live food in a very short period of time. Last, but not the least, the filter-feeding nature of rotifers facilitates the inclusion into their body tissues of specific nutrients essential for the larval predators. MATERIALS AND METHODS Physical and Chemical Conditions: The physical and chemical conditions are very important factor for rotifer culture. The free ammonia with a density of 3-5 mg lG1 inhibits reproduction of rotifers. Standard water quality parameters were maintained during the culture process (Table 1).

Corresponding Author: Kazi Ahmed Kabir, Department of Zoology, University of Dhaka, Bangladesh

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World J. Zool., 5 (2): 110-114, 2010

Table 1: Water quality standards for rotifer culture (modified from [4]) Parameters DO pH Free Ammonia (NH3) Temperature Optimum level 5-7 mglG1 6-8 <1mglG1 25-30C

A synthetic medium consisting of 96 mg NaHCO3, 60 mg CaSO4.2H2O, 60 mg MgSO4 and 4 mg KCl in 1 L of deionized water was prepared to boost up the density and growth of rotifers. Isolation and Inoculation: Wild zooplanktons were collected from Curzon Hall pond by plankton net of 64 m mesh size. The concentrated plankton was then shifted to a 1 liter conical flask, poured with filtered pond water. Basudine, an organo-phosphoric acid ester applied at a rate of 1.5 mg.lG1. This concentration was arrived at through series of toxicity experiments to determine the safe concentration for the rotifer [5]. At this concentration, crustacean including copepods and cladocerans, aquatic insects including mosquito larvae will fail to survive thereby allowing the rotifers to multiply in the absence of predators. The inoculum water containing them is completely renewed with 7.5 mg.lG1 Furazolidone (a disinfectant), 10 mglG1 Oxytetracycline, 30 mglG1 Sarafloxacin, or 30 mglG1 Lincospectin to get the mono-specific population of Brachionus calyciflorus [6]. Stock Culture: The inoculum was first disinfected. The disinfection consisted of killing the free-swimming rotifers but not the eggs with a cocktail of antibiotics (e.g. erythromycin 10 mg.lG1, chloramphenicol 10 mg.lG1, sodium oxolinate 10 mg.lG1, penicillin 100 mg.lG1, streptomycin 20 mg.lG1). The eggs were then separated from the dead bodies on a 50 m sieve and incubated for hatching and the offspring used for starting the stock cultures. Starter Culture: The stock culture was up scaled to starter culture .The rotifers were carried out in static systems consisting of erlenmeyers of 500 ml placed 2 cm from fluorescent light tubes (5000 lux). The temperature readings in the erlenmeyers were 26-28C. The rotifers were stocked at a density of 50 individuals.mlG1 and fed 400 ml freshly-harvested algae (Phacus 1.6.105 cells.mlG1); approximately 50 ml of algae being added every day to

supply enough food. After 3-4 days they were rinsed on a submerged filter. The concentrated rotifers were then distributed in several 15 L. bottles filled with 2 L. water at a density of 50 individuals.mlG1 and a mild tube aeration provided. Fresh algae (Phacus 1.6 105 cells.mlG1) were supplied daily. Every other day the cultures were cleaned (double-screen filtration) and restocked at densities of 200 rotifers.mlG1. After one week the 15 L bottles were completely filled and the cultures were ready to be used for inoculation of mass cultures. Mass Culture: Mass culture of rotifer was made in a 100 L glass tank with provision of water exchange and harvesting. Initially 50 individuals. mlG1 were stocked. Feeding schedule of rotifers was set according to their feeding habit and maintained strictly to get a healthy and fast growing culture (Table 2). The rotifers were enriched with oil emulsions before harvesting them to feed the fish larvae to get the maximum nutritional output. The oil emulsion was very simple and easy to prepare. The ingredients were egg yolk and cod liver oil. They were blended properly to mix and prepare an oil emulsion (Table 3). Counting the Rotifer Number: Sedgwick-Rafter (S-R) Counting Cell was used to count the number of rotifers by using the following strip counting method:
N= C 1000mm3 L D W S

Here, N C D W S = = = = = Number of organism / ml Number of organisms counted Depth of a strip (S-R cell depth) mm Width of a strip (Whipple grid image width) mm Number of strips counted.

Then the result was multiplied by dilution factor that was 5. Thus the actual numbers of plankton/ml were obtained. Harvesting: From the 3rd day of mass culture 50% of rotifer was harvested regularly. The harvesting was done by a rotifer collecting screen with a mesh size of 50 m. The tank bottom was cleaned at one day interval to avoid any organic matter or debris that might cause depletion of water quality.

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Table 2: Feeding schedule for mass culture of rotifer (modified from [7] Tank (100 L) ---------------------------------------------------------------------------------------------------------------------------------------------------------------Fresh Algae Day 0 Day 1 Day 2 Day 3 Early morning 12 L 05 L 08 L 08 L Late morning 12 L 05 L 08 L 08 L Mid afternoon 12 L 05 L 08 L 08 L Late afternoon 25 L 10 L 15 L 15 L Tank (100 L) ---------------------------------------------------------------------------------------------------------------------------------------------------------------lgae Paste Day 0 Day 1 Day 2 Day 3 Early morning Nil 200 ml 250 ml 250 ml Late morning Nil 200 ml 250 ml 250 ml Mid afternoon Nil 200 ml 250 ml 250 ml Late afternoon Nil 400 ml 500 ml 500 ml Tank (100 L) ---------------------------------------------------------------------------------------------------------------------------------------------------------------Bakers Yeast Day 0 Day 1 Day 2 Day 3 Early morning Nil 1.5 mg 2.0 mg 2.5 mg Late morning Nil 1.5 mg 2.0 mg 2.5 mg Mid afternoon Nil 1.5 mg 2.0 mg 2.5 mg Late afternoon Nil 3.0mg 4.0 mg 5.0 mg Tank (100 L) ---------------------------------------------------------------------------------------------------------------------------------------------------------------Mushroom Powder Day 0 Day 1 Day 2 Day 3 Early morning Nil 1.0 mg 1.5 mg 1.5 mg Late morning Nil 1.0 mg 1.5 mg 1.5 mg Mid afternoon Nil 1.0 mg 1.5 mg 1.5 mg Late afternoon Nil 2.0 mg 3.0 mg 3.0 mg Note: Rotifer were received their last feeding no earlier than 4 pm. Table 3:Composition of oil emulsion used for rotifer enrichment [8] Constituents Quantity Egg yolk 01 piece.100 LG1 Cod liver oil 15 ml. 100 LG1 Table 4:Reproductive activity of Brachionus calyciflorus. Category of reproductive activity Result 0.5 1-1.2 3.5-3.8 7 1 182

Time for embryonic development (days) Time for young female to spawn for the first time (days) Interval between two spawning ( hours) Length of life (days) Average number of eggs spawned by a female during her life

Production and Preservation of Resting Eggs: Resting egg production scheme was done in concrete tank. The method followed to give shock in the favorable living environment was set with the working procedure of resting egg production provided by [9]. This was achieved by limiting food supply and instant change in temperature. The resting eggs were deposited at the bottom due to its weight. The water of the tank was replaced by brine solution and the eggs float on the surface. The floating eggs were collected from the surface by the collecting screen of 25 m mesh size. The collected eggs were dried in incubator at 35C temperature. The dried eggs were preserved in a previously autoclaved and sterilized sealed vial. RESULTS Reproductive Activity and Production of Rotifer: High density rotifer culture under the supply of mixed diets was enough successful. The maximum density attained 721 individuals.mlG1. At the temperature of 112

27-28C Brachionus calyciflorus showed well reproductive activity, attained reproductive maturity within 36-40 hours and produced enough egg to make their doubling time 50-55 hours. The reproductive activities of Brachionus calyciflorus at the temperature of 27-28C were observed and documented (Table 4). The growth performance of Brachionus calyciflorus was also satisfactory. Increase in density within the culture vessel was monitored regularly. The health condition of the culture species was also good. The movement rate of B. calyciflorus in static water was recorded as 1m/sec with a resting phase for 2-3 seconds in an average interval of 5-7 seconds. Though growth at beginning of culture was moderate the population became double in a day at its log growth phase, but the average doubling time was 2.26 days (Table 5). In the first day of stocking culture density reached 200 individuals. mlG1

World J. Zool., 5 (2): 110-114, 2010

Table 5:Growth performance of Brachionus calyciflorus. Age of production Day 1 Day 2 Day 3 Day 4 Number of individual / ml 200 282 10 490 27 685 35 Average growth per day 0.307 Doubling Time(days) 2.26

Table 6: Effect of density on rotifer egg production. Density of Rotifer (Brachionus calyciflorus/ml) 200 400 600 721 Rotifer Egg Production Ratio 27 20 18 16

Fig. 1: Growth Performance of Brachionus calyciflorus

Fig. 2: Effect of density on rotifer egg production 113

World J. Zool., 5 (2): 110-114, 2010

from the initial stocking density 50 individuals. mlG1. Density continued to increase as the culture age progressed and finally it attained average 68535 individuals.mlG1. The maximum density observed was 721 individuals.mlG1 (Fig. 1). We observed that increase in density of rotifer influences the egg production of the females (Table 6). As the culture age progresses the density (individuals.mlG1) rises up and the egg production slows down. Even then the rate of growth per day continues to increase (Fig. 2). Rotifer Resting Egg Production: Resting eggs were collected after 7 days of stopping feed supply to the culture vessel. Resting eggs were dried for preservation and enumerated 1800 / mg in average. The eggs were further introduced into a new culture medium and 76% hatching was recorded at 27C temperature within 40 hours. DISCUSSIONS Live food production for carp larval rearing is an integrated process, which includes culture of both algae and rotifers. Satisfactory growth can only be achieved if adequate food is supplied to the larval culture vessel. In addition the food should have met the nutritional requirement of the fish larvae. Hence it is required to be nutritionally enriched. The density in rotifer culture obtained in present study was maximum 721 individuals.mlG1. But 1300 individuals.mlG1 for Brachionus rubens was recorded by [10]. In addition [11] got a density of 2000 per ml for Brachionus plicatilis. Though both results are much higher than the present output, it has to be considered that the growth rate of marine rotifers is always higher than that of freshwaters. Rotifers reared intensively on microalgae support more dense culture. But in present research the effort was taken to decrease the pressure on algae culture, a sophisticated technology and to adopt alternative diets like bakers yeast, mushroom powder, Spirulina dust etc to make the process easy, less risky and popular among the hatchery operators of Bangladesh. In this sense the result was acceptable. REFERENCES 1. Fukusho, K., 1989. Biology and Mass Production of The Rotifer, Brachionus plicatilis. Int. J. Aq. Fish. Technol., 1: 232-240.

Dhert, P., et al. 1995. Production and evaluation of resting eggs of Brachionus plicatilis originating from the P.R. of China. In: Lavens, P.E. Jaspers and I. Roelants, (Eds.), Larvi 95 Fish and Shellfish Larviculture Symposium. European Aquaculture Society, Special Publication, Gent, Belgium, 24: 315-319. 3. Harita, H., 1979. Rotifer Culture in Japan. In: Styczynska-Jurewicz, E.T. Backiel; E. Jaspers and G. Persoone, (Eds), Cultivation of Fish Fry and Its Live Food. European Mariculture Society, Special Publication, 4: 361-375. 4. Arimoro, F.O. and P.C. Ofojekwu, 2004. Some aspects of the culture, population dynamics and reproductive rates of the freshwater rotifer, B. calyciflorus fed selected diets. J. Aquatic. Sci., 19(2): 95-98. 5. Agbon, A.O., P.C. Ofojekwu, I.C. Ezenwaka and W.O. Alebeleye, 2002. Acute toxicity of diazinon on rotifers, Cyclops, mosquito larvae and fish. J. Appl. Sci. Environ. Managt., 6(1): 18-21. 6. Arimoro, F.O., 2006. Culture of the freshwater rotifer, Brachionus calyciflorus and its application in fish larviculture technology, Afr. J. Biotechnol., 5 (7):536-541 7. Donaldson, J., 1991. Commercial production of rotifer at Coast Oyster Company. In: W. Fulks, K.L. Main, (eds). Rotifer and microalgae culture systems. The Oceanic Institute, Honolulu, pp: 229-236. 8. Watanabe, T., M. Ohta, C. Kitajina and S. Fujita, 1982. Improvement of dietary value of brine shrimp Artemia salina for fish larvae by feeding them T3 highly unsaturated fatty acids. Bulletin of the Japanese Society of Scientific Fisheries. Nippon Suisan / Gakkaishi, 48(12):1775-1782. 9. Hagiwara, A., M.D. Balompapueng, and K. Hirayama, 1995. Mass production and preservation of marine rotifer resting eggs. Page 314. In: Lavens, P.E. Jaspers and I. Roelants, (Eds.), Larvi 95 Fish and Shellfish Larviculture Symposium. European Aquaculture Society, Special Publication, Gent, Belgium, pp: 24-314. 10. Vijverberg, J., 1989, Culture techniques for studies on growth, development and reproduction of rotifers and cladocerans under laboratory and in situ conditions: a review . Freshwat. Biol., 21: 317-373 11. Reed, H., 2000. High density rotifer culture technique for small hatcheries. J. Appl. Sci. Environ. Managt., 4(2): 11-18.

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