Usefulness of Spirulina sp. Meal as Feed Additive for Giant. Freshwater Prawn, Macrobrachium rosenbergii. Heisuke NAKAGAWA1) and Gabriel

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1 Usefulness of Spirulina sp. Meal as Feed Additive for Giant Freshwater Prawn, Macrobrachium rosenbergii Heisuke NAKAGAWA1) and Gabriel GOMEZ-DIAZ2) 1)Faculty of Applied Biological Science, Hiroshima University, Higashi-hiroshima, 739, Japan 2)Oversea Fish ery Cooperation Foundation, 17-22, Akasaka-2, Minato-ku, Tokyo 107, Japan Abstract Juvenile giant freshwater prawns Macrobrachium rosenbergii with individual weights between g were reared with purified diets supplemented with various levels of Spirulina meal (0, 5, 10, 15, and 20%) for 60 days in duplicate indoor aquaria. The Spirulina meal significantly improved growth, survival, and feed utilization irrespective of supplementation level. Improved growth and feed utilization by feeding Spirulina was due to enhancement of protein assimilation. Biochemical composition of abdominal muscle was not influenced by feeding Spirulina. On the other hand, midgut gland lipids were highly dependent on Spirulina level. Triglycerides and free fatty acids were elevated by Spirulina, and were highest with feed supplementation at 5% and 15%, respectively. Supplementation of more than 15% Spirulina depressed lipid level of midgut glands. Accordingly, it is implied that inclusion of Spirulina in the diet is important for controlling the growth performance of prawns. The freshwater prawn is an omnivorous species, and the natural diet is to include a certain quantity of vegetable matter besides detritus, aquatic insects, and earthworms. Lingl1) reported that the gut contents of the freshwater prawn were composed mainly of detritus and organic matter. Costa and Wanninayake2) reported that about 20% of the diet of wild prawns is composed of vegetable matter. Cohen et al.3) concluded that algae facilitate the growth of larvae, but negligible quantities of algae were ingested directly. In fish, algae as feed additive are known to have important physiological effects4-7). Among the examined algae, Spirulina showed superior effects in red sea bream Pagrus major. The present study was designed to assess the possible role of Spirulina meal as a feed additive in giant freshwater prawn in terms of growth, feed utilization, and body composition. Materials and Methods Rearing and Feeding Conditions Initial weight of prawns used in the experimental ranged from 0.35 to 0.66g. Rearing conditions were as described in previous papers8,9). Twenty prawns were assigned to a 50 l aquarium (water volume 45 1), equipped with a sand filter recirculating system and plastic-covered chicken wire as the habitat to minimize cannibalism. Duplicate aquaria were set up for each test group. Water temperature was kept constant Received : April 21, 1995 Key words: Giant freshwater prawn, Spirulina, Feed additive, Midgut gland lipids

2 H. Nakagawa and G. G. Diaz (1995) at 28 Ž throughout the experiment. Photoperiod was kept under natural conditions without artificial control. Only intermolt stage C prawns were selected for biochemical determinations. Molt stage was identified according to Pebbles10) Diets The purified diets were prepared with a fixed protein/starch ratio of 1:1 as shown in Table 1, according to the methods described previously8,9). Ingredients, proteins and starch were partly replaced by Spirulina meal obtained from a commercial source in proportions of 0, 5, 10, 15, and 20% (groups 1-5). The sausage-type diet was stored at -20 Ž until feeding. Fresh shortnecked clam, Ruditapes philippinarum, meat was fed to group 6 as a control diet. Prawns were fed daily with about 10% of the body weight and residual food was weighed. The duration of the experiment was 60 days. Biological and Biochemical Analyses FE (%)=(wet weight gain/food wet weight eaten) ~100 PER=weight gain/dry protein weight eaten BP=final total weight-initial total weight MGR (%)=(weight of midgut gland/body weight) ~ 100 The abdominal muscle and midgut glands obtained from about 10 prawns were from each group, which were pooled and submitted to analyses to determine proximate composition and lipid analyses. Moisture and ash were measured by standard methods. Crude protein was determined by the Kjeldahl method. Lipid was extracted by the method of Bligh and Dyer11). Analysis of lipid class composition was carried out on an Iatroscan TH-10 (Iatron Laboratories). Lipid class was identified by TLC on Kieselgel 60 plates using n-hexane/diethyl ether/acetic acid (80: 20:1, v/v/v) as developing solvent. Partial glycerides were identified by the method described previously8). Gross energy was estimated from the energy value of each dietary ingredient, measured by bomb calorimetry (Shimadzu, CA-3). Feed efficiency (FE), protein efficiency ratio (PER), biomass production (BP), and midgut gland ratio (MGR) were calculated as follows: Statistical Analyses All data were subjected to analysis of variance and Table 1. Dietary composition for giant freshwater prawn *1C ahpo4 E 2 (H20), 5; pollack liver oil, 5; cholesterol, 1; mineral mixture No. 2 (ICN Biochemicals), 2; vitamin mixture (Halver premix), 2; sodium carboxymethyl cellulose, 2.5. *2 C ontains trimethylamine hydrochloride as an attractant. *3 G ross energy measured by bomb calorimetry.

3 Spirulina as Feed Additive in Prawn Table 2. Effects of feeding Spirulina on growth performance and biometric parameters of giant freshwater prawn Values show average of two aqualia. *M ean }SD. Initial body weight, g. Values in the same column followed by different letters are significantly different (p<0.05). Duncan's Multiple Range Test to determine significant differences. Probabilities of 0.05 or less were considered statistically significant. Results Table 2 shows the mean values of growth, survival, and feed utilization in two aqualia during 60 days rearing. The survival of prawns fed clam meat was inferior to that of those fed the various purified diet. Feeding Spirulina at a level of more than 10% improved survival. Growth performance and feed utilization were markedly improved by feeding Spirulina irrespective of dietary level. Feed efficiency did not seem to be affected to a great extent by the Spirulina level in the diet, although the Spirulina-fed groups showed somewhat higher values than group 1. The same trend was apparent in protein efficiency ratio. Spirulina did not influence midgut gland ratio. Table 3 shows the proximate composition of the abdominal muscle. Protein content was similar in all the groups regardless of dietary Spirulina level, although lipid content was slightly depressed by the Spirulina. Fig. 1 shows lipid level and lipid class composition of abdominal muscle and midgut gland. Muscle lipid class composition was not sensitive to Spirulina level. Phospholipids and cholesterol were the main lipid class and did not change markedly with addition of Spirulina at various levels. Negligible amounts of triglycerides were found in the abdominal muscle, and Table 3. Effects of feeding Spirulina on muscle proximate composition of giant freshwater prawn Fig, 1. Effects of feeding Spirulina on lipid content and lipid class composition (g/100g tissue) of abdominal muscle and midgut gland of juvenile giant freshwater prawn. For explanation of dietary groups, see Table 1. TG: triglycerides, FA: fatty acids, PG: partial glycerides, Cho: cholesterol, PL: phospholipids.

4 H. Nakagawa and G. G. Diaz (1995) phospholipids showed no specific relationship with the dietary Spirulina level. The lipid contents of the midgut glands varied with addition of Spirulina in the diet. The midgut glands of clam-fed prawns had extremely low triglyceride levels. The lipid content of group 1 was two thirds that in group 3 (Spirulina 10%), and that of clam meat-fed prawns was only one third (4.3 %). Triglycerides, levels of which were negligible in the abdominal muscle, were the main component in the midgut gland. Free fatty acids increased with dietary Spirulina level and showed a maximum at a supplement level of 15%. In contrast, triglyceride level showed an opposite trend with a maximum in the group fed the 5 % Spirulina diet. Discussion The effects of vegetable matter as feed additives on growth and survival have been studied in several decapods12-16). The addition of vegetable matter in Penaeus aztecus diet elevated feed efficiency12), and growth and health were improved by feeding fresh leaves in the giant freshwater prawn14). These results were obtained regardless of the plant species substituted (Ailanthus alitissima and Malva parviflora). In the present experiment, dietary Spirulina elevated growth performance and feed utilization, which effects were irrespective of Spirulina level. Muscle composition was unaffected by the supplementation level. The midgut gland is an organ where lipid metabolism is actively taking place17) and is, therefore, important to assess as an indicator of trophic condition. Beneficial effects can not always be expected with any kind of vegetable source. Excessive dietary Spirulina resulted in negative effects on growth and feed utilization in striped jack, Pseudocaranx dentex18) and red tail prawn, Penaeus penicillatus*. Accordingly, the present results suggested the influence of negative substances of Spirulina on growth performance. Inclusion of vegetable matter in the diet improved the efficiency of Homarus americanus15), but when fed in excess it reduced growth of M. rosenbergii19). The presence of mimosine in Leucaena liecocephala leaves fed to the black tiger prawn Penaeus monodon had physiologically detrimental effects. Phytates present in vegetable tissue would reduce growth, feed efficiency, and protein digestibility of fish20,21) Accordingly, special care should be taken to determine the presence of detrimental substances in plant materials for practical use as feed additives. The advantageous effects observed in feeding trials of vegetable matter have been presumed to be derived from certain chemical substances, such as carotenoids, feeding stimulants, vitamin source, antioxidant, and protectors from vitamin degradation14,22,23) Dietary fiber is consumed to a certain extent by the giant freshwater prawn24). Thus, the contribution of dietary fiber and synergistic effects of vitamins can not be disregarded. The results on growth performance in prawns given Spirulina as a feed additive would be due to high protein assimilation in the presence of Spirulina. Growth and survival of the clam meat fed prawn were significantly lower than the composed diet fed prawn. The results support the hypothesis that dietary algae may be important to the regulation of physiological condition in the ominivorous prawn. Dietary algal substances are important for controlling the normal physiological condition in fish5,7). The beneficial effect of Spirulina as a feed additive was also confirmed here in the prawn. With regard to the practical use of algae in prawn feed, further studies are required to clarify the mechanism of the efficacy of dietary algae. Acknowledgements We wish to thank Dr. S. Kasahara for providing valuable suggestions and Riken Vitamin Co., Ltd. for kind supplying the vitamin mixture and pollack liver oil. References 1) Ling, S. W. (1969) : The general biology and development of Macrobrachium rosenbergii (de Man). FAO Fish Rep., No. 57, 3, * Liao, W.-L. and M. A. Bolanos (1993) : Effects of Spirulina-supplemented diets on the red tail prawn, Penaeus penicillatus. VI th International Symposium on Fish Nutrition and Feeding. Hobert, No. 56.

5 Spirulina as Feed Additive in Prawn 2) Costa, H. H. and T. B. Wanninayake (1986) : Food, feeding and fecundity of the giant freshwater prawn Macrobrachium rosenberigii from natural habitats in Sli Lanka. In J. L. Maclean, L. B. Dizon and L. V. Hosillos (eds.), The First Asian Fisheries Forum. Asian Fisheries Society, Manila, Philippines, pp ) Cohen, D., A. Finkel, and M. Sussman (1976) : On the role of algae in larviculture of Macrobrachium rosenbergii. Aquaculture, 8, ) Montgomery, W. L. and S. D. Gerking (1980) Marine macroalgae as foods for fishes: an evaluation of potential food quality. Env. Biol. Fish., 5, ) Nakagawa, H. (1985) : Usefulness of Chlorellaextract for improvement of the physiological condition of cultured ayu, Plecoglossus altivelis (Pisces). Tð thys, 11, ) Indergaard, M. and J. Minsaas (1991) : Animal and human nutrition, In gseaweed Resources in Europe, Use and Potential, M. D. Guirez and G. Blinden (eds.), John Wiley & Sons Ltd., pp , ) Mustafa, Md. G. and H. Nakagawa (1995) : A review: dietary benefits for algae as an additive in fish feed. Israeli J. Aquaculture, (in press). 8) Gomez-Diaz, G., H. Nakagawa, and S. Kasahara (1988) : Effect of dietary protein/starch ratio and energy level on growth of the giant freshwater prawn Macrobrachium rosenbergii. Nippon Suisan Gakkaishi, 54, ) Gomez-Diaz, G. and H. Nakagawa (1990) : Effects of dietary carbohydrates on growth and body components of the giant freshwater prawn, Macrobrachium rosenbergii. Aquat. Living Resour., 3, ) Pebbles, J. B. (1977) : A rapid technique for molt staging in live Macrobrachium rosenbergii. Aquaculture, 12, ) Bligh, E. G. and W. J. Dyer (1959) : A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, ) Venkataramiah, A., G. L. Lakshmi, and G. Gunter (1975) : Effect of protein level and vegetable matter on growth and food conversion efficiency of brine shrimp. Aquaculture, 6, ) AQUACOP (1976): Incorporation des proteines vegetales Bans un alimant compose pour crevettes Macrobrachium rosenbergii. Aquaculture, 8, ) Harpaz, S. and E. A. Schmalbach (1986): Improved growth and health of Malaysian prawn, Macrobrachium rosenbergii, by addition of fresh leaves to the artificial diet. Aquaculture, 55, ) Syslo, M, and J. T. Huges (1981): Vegetal matter in lobster (Homarus americanus) diet (Decapoda, Astacidea). Crustaceana, 41, ) Vogt, G., E. T. Quinitto, and F. P. Pascual, (1986): Leucaena leucocephala leaves in formulated feed for Penaeus monodon : a concrete example of the application of histology in nutrition research. Aquaculture, 59, ) D' Abramo, L. R., N. A. Baum, C. E. Bordner, D. E. Conklin, and E. S. Chang (1985): Diet-dependent cholesterol transport in the American lobster. J. Expt. Mar. Biol. Ecol., 87, ) Watanabe, T., W.-L. Liao, T. Takeuchi, and H. Yamamoto (1990): Effect of dietary Spirulina supplementation on growth performance and flesh lipids of cultured striped jack. J. Tokyo Univ. Fish., 77, (in Japanese, with English summary). 19) Stern, H. L., D. A. Armstrong, A. W. Knight, and D. J. Chippendale (1976): Survival and growth of juveniles of the giant Malaysian prawn, Macrobrachium rosenbergii, fed natural plant diets. Proc. World Maricult. Soc., 7, ) Spinelli, J., D. R. Houle, and J. C. Wekell (1983) The effect of phytates on the growth of rainbow trout (Salmo gairdneri) fed purified diets containing varying quantities of calcium and magnesium. Aquaculture, 30, ) NRC-NAS (1983) : Nutrition requirements of warmwater fish. National Research Council- National Academy of Sciences, Washington D. C., pp ) Fujimoto, K. and T. Kaneda (1980) : Screening test for antioxygenic compounds from marine algae and fractionation from Eisenia bicyclis and Undaria pinnatifida. Bull. Jpn. Soc. Sci. Fish., 46, ) Garces, C. L. and J. M. Heinen (1993) : Evaluation

6 of beef liver, fruits, and vegetables as dietary supplements for indoor nursery culture of freshwater prawn Macrobrachium rosenbergii. J. World Aquacult. Soc., 24, ) Fair, P. H., A. R. Fortner, and M. R. Millikin (1980) : Effects of dietary fiber on growth, assimilation, and cellulase activity of the prawn, (Macrobrachium rosenbergii). Proc. World Maricult. Soc., 11,