Aquaculture, 110 (1993) 141-150 Elsevier Science Publishers B.V.

, Amsterdam AQUA 50044

141

Dietary effects of different feeds on the bioch&mical composition of the rotifer (Brachionus plicatilis Miiller )
M. CariC, J. Sanko-Njire and B. Skaramuca
Biological Institute, Dubrovnik, Croatia (Accepted 5 June 1992)

ABSTRACT CariC, M., Sanko-Njire, J. and Skaramuca, B., 1993. Dietary effects of different feeds on the biochemical composition of the rotifer (Brachionus plicutilis Miiller ) . Aquaculture, 110: 14 1- 150. The rotifer (Brachionus plicutilis Miiller) was cultured on phytoplankton monocultures (Dunaliella tertiolecta, Tetraseimis suecica, Nannochloropsis sp., Phaeodactylum tricornutum), natural nanoplankton and bakers yeast. Biochemical composition of rotifers harvested at exponential, stationary and death phases was determined. The moisture content declined in all samples in their stationary phase of growth. Ash levels were highest at the death phase. Lipids and carbohydrates were observed to decline between the exponential and the death phases. Protein levels increased in the stationary phase. Growth rate, doubling time, lipid, carbohydrate and protein content of rotifers fed Phaeodactylum tricornutum and Nannochloropsis sp. indicate that these monocultures are the most suitable diets for the rotifer. Rotifers fed on these algae are expected to satisfy the nutritional demand of fish larvae.

INTRODUCTION

The rearing of the majority of marine fish larvae requires the use of live food. The rotifer, Bruchionus plicutilis, and the crustacean, Artemiu salina, are the two organisms most extensively used as food. Although Bruchionus plicutilis Miller does not fall in the category of natural live food for fish rearing, it has been widely used due to its ideal size, quick reproductive rate and ability to feed on different unicellular algae and bakers yeast. The nutritional quality of live food is very important for the survival of fish larvae. Therefore, along with the production of sufficient quantities of rotifers, it is also necessary to ensure proper nutritional quality to satisfy the needs of the larvae. Considerable differences have been noted in the survival rate of larvae fed on rotifers reared on diverse phytoplankton cultures (Howell, 1977;
Correspondence to: M. CariC, Biological Institute, P.O. Box 39, 50000 Dubrovnik, Croatia.

0044-8486/93/$06.00

0 1993 Elsevier Science Publishers

B.V. All rights reserved.

142

M. CARIC: ET AL.

Scott and Baynes, 1979; Dendrinos and Thorpe, 1987). The chemical composition of rotifers is similar to that of the algae upon which they feed (BenAmotz et al., 1987). The aim of this study was to investigate the nutritional effects of phytoplankton monocultures (Dunaliellu tertiolecta, Tetraselmis suecica, Nunnochloropsis sp., Phaeodactylum tricornutum), natural sea nanoplankton and the bakers yeast (Saccharomyces cerevisiae) on the biochemical composition of the rotifer. In order to determine water, ash, total lipid, protein and carbohydrate contents, rotifer samples were taken at the exponential, stationary and death phases.
MATERIALS AND METHODS

Growthconditions Yeast: Bakers yeast was suspended in distilled water and 1 ,ug (about 1.3 x 1O4ceils) was provided to the rotifer each day (Fujita, 1979). Algue: The unicellular algae (used as food for rotifers) Dunaliellu tertiolecta, Tetraselmissuecica, Nannochloropsissp., Phaeodactylum tricornutum and natural sea nanoplankton, were from stocks held in the Biological Laboratory at Dubrovnik for the last 5 years, cultivated on pasteurized (30 PU) natural sea-water enriched with nutrient medium (Guillard and Ryther, 1962 ) . They were cultured in 50-l plastic bags at 22 C with aeration and a 12 h light (480 lux) : 12 h dark regime. Salinity was maintained at 37.7 x 10p3 and pH 8.0. Culture density was determined by microscopic counts in a Btirker-Turk chamber. Zooplankton: Rotifer Brachionus plicutilis (Miiller) from a stock kept in culture at the Biological Laboratory in Dubrovnik for the last 4 years was used. The average length and width of the lorica (in pm) were 170.8 x 119.8. The average dry weight was 2 15 ng/ind. As many as 7- 16 rotifers were added to the algal cultures in the late exponential growth phase. Daily estimates of rotifer numbers in the cultures were determined. Usually 1 x 1OSrotifers were sampled for chemical analysis at exponential (I ) , stationary (II ) and death (III) phases by separation on 53-pm-aperture nylon mesh and rinsing with distilled water to remove extraneous salts. The sample was then left on an absorbent surface for 20 min to remove as much extraneous water as possible. Growth rate was calculated according to the formula: K= 1/t lnN,/N,, where N, = initial numbers of rotifers and N,= maximum number of rotifers after time t in days. Doubling time was calculated according to the formula: D= In 2/K. Analyticalmethods Moisture was determined by drying to constant weight at 60 C and ash content by ashing at 800C (Lovergrove, 1966). For biochemical analysis,

DIETARY EFFECTS ON BIOCHEMICAL COMPOSITION OF ROTIFER

143

samples were homogenized in a tight-fitting Dounce homogenizer, dried at 60 C and ground to a fine powder in order to ensure efficient extraction of various macromolecules and reduce errors in measurement that may occur due to non-homogenized samples. For analysis the pulverized material was stored desiccated at - 25 C (Giese, 1967). Lipids were extracted using a chloroform-methanol ( 1: 2 ) mixture (Bligh and Dyer, 1959 ) and total lipids were estimated by the sulphophosphovanillin method (Barnes and Blackstock, 1973). TCA was added to the lipid-free pellets to precipitate protein (Holland and Hannant, 1973) and the supernatant was analyzed for total carbohydrates using a phenol-sulfuric acid method reported by Kochert ( 1978 ), which is based upon procedures developed by Dubois et al. ( 1956). The precipitated protein was assayed as described by Bradford ( 1976 ) after hydrolysis in 1 NNaOH for 30 min at 100C. Each experimental treatment was analysed by triplicate samples. Data were subjected to analysis of variance (ANOVA) and SNK multiple range test.
RESULTS

The species of phytoplankton showed different growth dynamics. T. suecica needed 10 days, D. tertiolecta 8 days, Nannochloropsis sp. and P. tricornutum 5 days and nanoplankton 3 days to reach the late exponential growth phase. The concentration values of phytoplankton cells in the late exponential growth phase, in addition to pertinent data on growth of rotifers fed on the different regimes, are presented in Table 1. The fastest growth rates for rotiTABLE 1 Growth rate and doubling time of Bruchionusplicutilis fed on different phytoplankton natural sea nanoplankton and bakers yeast Type of food Algal cell density (cells/ml) Number of rotifers Initial (ind./ml) Maximum (ind./ml) Time to reach max. number (days) 7 8 7 8 11 9 monocultures.

Growth rate (K)

Doubling time (days)

Phaeodactylum tricornutum Dunaliella tertiolecta Nannochloropsis sp. Tetraselmis suecica Nanoplankton Bakers yeast

1.5x lo6 4.5x lo6 6.2x lo6 1.3x 10s 7.7x lo6 0.9x lo5

15.5 16.2 9.6 7.2 8.8 7.0

539.2 151.8 284.2 133.4 228.0 37.4

0.51 0.28 0.49 0.36 0.30 0.19

1.36 2.47 1.41 1.93 2.31 3.65

144 TABLE 2 The chemical composition Growth cycle of rotifers fed on different food at three growth phases

M. CARIt ET AL.

Rotifers fed on Nanoplankton Bakers yeast ,85.9b .86.8 s93.6 P. T. tricornutum suecica .88.3aab .88.4 .89.Fb .5.8a,b .7.3 ,13.0 .16.0 bl1.1 .8.5 .12.7b .11.7 ,10.5, .3.5 .2.4 .2.0 .34.0s .35.1s ,25.5b .42.0a*b b51.8 .38.8. ,4 1.OBBb .34.3b .39.4s s48.0 .35.3 .42.0 ,17.2 ,15.7s bl 1.1 .15.8 J3.2 ,9.5, .5.0s .4.6b b2.1 ,44.7a .47.6 .42.2 ,88.2a,b .88.2 .88.3 Nannochloropsis sp. D. tertiolecta .*b86.0b .83.0 b90.0b

Moisture (O/a wet wt)

I II III 1 II III I II III 1 II III I II III

.89.6 ,875 ,89.0,b ,6.9 08.7 ,10.6 .19.48 bl 1.5* b 10.3= .4.8a,b s2.9 ,2.8 .50.0 .52.0 ,42.3

Ash (%drywt)

Lipid (O/o dry wt)

Carbohydrate (%drywt)

Protein (%drywt)

I - exponential, II - stationary, III - death phases of growth. Means in the same line followed by different superscripts are significantly different (PC 0.05). Means in the same column for each growth cycle preceded by different subscripts are significantly different (P~0.05). Initial rotifer inoculum (day 0) contained 86.1% moisture, 8.8% ash, 11.7% lipid, 3.3% carbohydrate and 39.8% protein.

fers were achieved with Nannochloropsissp. and P. tricornutum and the slowest with natural nanoplankton. The chemical composition of rotifers fed on the different regimes at exponential, stationary and death phases is presented in Table 2. In most samples, water content reached its lowest value at the stationary phase, with the Nannochloropsis sp.-fed rotifers having the lowest value (P-C 0.05 ) of all treatments. This phase significantly differs from other growth phases. In all feeding regimes, highest ash levels were recorded in the death phase, probably due to a decline in organic matter. This phase significantly differed from other phases. Highest ash content was found in nanoplankton- and P. tricornutum-fed rotifers, which was probably consistent with the siliceous nature of diatom cell walls. At the stationary phase, no significant differences were observed in the carbohydrate content of rotifers reared on bakers yeast, P. tricornutum, Nannochloropsissp. and D. tertiolecta.Significant differences were found between

DIETARY EFFECTS ON BIOCHEMICAL COMPOSITION OF ROTIFER

145

146

M. CARIC ET AL.

growth phases for all feeding regimes except T. suecica. In the death phase, however, the carbohydrate content of rotifers fed on bakers yeast was significantly higher than all others (P~0.05). Protein levels increased during the stationary phase and reached the highest values in nanoplankton- and P. tricornutum-fed rotifers. Noteably lower values (P-C 0.05 ) were found for bakers yeast- and Nannochloropsissp.-fed rotifers in this phase, which statistically differed from the other two phases only in P. tricornutum-and T. suecica-fed rotifers. Lipids were observed to decline from the exponential to the death phases, with the exception of the rotifer samples fed on bakers yeast. The highest lipid value was found in the exponential phase of the nanoplankton-fed rotifers. High content of lipids (PC 0.05 ) in this phase of growth was observed in the rotifers fed on P. tricornutum, Nannochloropsissp. and D. tertiolecta. However, similar differences were not observed in the stationary growth phase, except for the rotifers fed on Nannochloropsissp., where significant (PC 0.05 ) differences in the content of lipid occurred in the stationary phase as well, and thus without statistical difference from the exponential phase. The statistical data of rotifer chemical composition are presented in Table 3. Two-way variance analysis indicated that both factors, the growth cycle and diet, significantly affected the chemical composition of the rotifers. Significant interaction between diet and growth cycle was found in all parameters except protein. One-way ANOVA indicated that diet significantly affected each nutrient category in all the phases of the growth cycle, whereas only ash levels were significantly affected by the growth cycle in all types of food. The same was observed for carbohydrate and lipid content in all types of food except T. suecica. On the contrary, it was only rarely that moisture and protein levels were significantly affected by the growth cycle.
DISCUSSION

The growth rate and chemical composition of rotifers fed on different species of algae and bakers yeast were observed. According to the changes in the biochemical composition of the rotifers during the growth cycle, they were harvested in the appropriate growth stage. Moisture content was observed to decline in all samples at the stationary phase of growth and increase at the death phase. Significantly lower water level in the growth cycle was found in Nannochloropsissp.-fed rotifers. Ash content increased in all the treatments from the exponential to the death phases, probably due to a decline in organic matter. The increased content of water and ash at the death phase is in agreement with a generally well established fact that a relative decrease of organic components such as carbohydrates, lipids and proteins is always accompanied by a relative increase of ash and water (Katavic et al., 1985).

DIETARY EFFECTS ON BIOCHEMICAL COMPOSITION OF ROTIFER

147

Carbohydrates were observed to decline in most regimes from the exponential to the death phases. Dietary carbohydrate is extensively used as a source of energy. While simple carbohydrates provide the starting point for many biological syntheses, fish can be grown on diets completely devoid of carbohydrate (Cowey and Sargent, 1972). The natural diet of many carnivorous fish contains relatively small quantities of this material. A ration poor in carbohydrate entails the use of either lipid or protein to provide necessary calories. For that reason carbohydrates are included in formulated feeds for fish primarily as a low cost source of energy to spare dietary protein for growth rather than energy. Relative protein-sparing action of carbohydrates is also highly variable among fish species (Millikin, 1982 ) . In all feeding treatments, highest protein levels were observed at the stationary phase of growth which may be attributed to rapid protein synthesis in growing rotifers. Protein requirement of fishes ranges from 30 to 50%, depending upon fish species, age, feeding habit and dietary essential amino acid requirement (Milhkin, 1982 ). The amino acid profiles of rotifers do not seem to deviate significantly from those of other zooplanktonic organisms, either cultured or of wild origin, which are recognised as being beneficial for growth of larvae (Caste11 et al., 1986). Furthermore, Dendrinos and Thorpe ( 1987) reported that no apparent significant differences in amino acid composition were found between Brachionus plicatilisfed on algae and those fed on yeast. According to our data, the concentration of protein at the exponential and stationary phase of growth varies from 34.0 to 52.0% depending upon feeding treatment. It is to be expected that this protein concentration should meet the protein requirements of fish larvae. The percentage of lipids decreased during the growth cycle. Lipids are important elements of cell structure and a major energy source in most zooplankton organisms (Dawirs, 1987) and marine fish larvae. Lipid in general and specifically n-3 HUFA have been observed to have an essential role in fish larval diets (Watanabe et al., 1978; Scott and Middleton, 1979). These fatty acids most likely form a part of the cellular membranes of the larvae and therefore are crucial in determining the rates of enzymatic processes taking place at these sites (Lubzens et al., 1989). The rotifer can synthesize de novo some n-3 HUFA (Lubzens et al., 1985), but not in quantities sufficient to satisfy the demand of fish larvae. Therefore, rotifers must be given these fatty acids through feeding. The fatty acid composition of rotifer phospholipids is largely dependent on that of their food, indicating that the ingested lipids are incorporated into the rotifer lipids. In order to supply large amounts of HUFA to marine fish larvae, rotifers must be fed on HUFA-rich food (Watanabe et al., 1983; Lubzens et al., 1985; Walford and Lam, 1987). At the exponential phase of growth, the highest lipid content was found in rotifers fed on D. tertiolecta,P. tricornutum, Nannochloropsis sp., and nanoplankton, varing from 15.8% to 19.4%. At the stationary phase of growth the Nannochloropsis-fed

148

M. CARIt ET AL.

rotifers had significantly higher lipid content. Since marine fish require a diet containing 13- 16% lipid (Caste11 et al., 1986 ), the rotifers meet this requirement. Nunnochloropsissp. contains a high amount of 20: 5n-3 (27.8%) (Watanabe et al., 1978) and P. tricornutum contains 14.7% (Ben-Amotz et al., 1987) of this fatty acid, which is an EFA for marine fish. In contrast, D. tertiolectudid not contain this fatty acid or any other polyunsaturated fatty acid of a chain length greater than 18 carbons (Pillsbury, 198 5 ) . According to these results and the high total lipid levels in rotifers fed on P. tricornutum and Nannochloropsissp., it appears that the rotifers fed on these algae would have n-3 HUFA levels high enough to meet the EFA requirements of larval fishes. Furthermore, Nannochloropsis sp. and P. tricornutum are algae that are not difficult to culture and reach the late exponential growth phase in 5 days, which, when compared with other algae cultured, (T. suecica and D. tertiolecta) in a relatively short time. Fernandez-Reiriz et al. ( 1989) have reported the growth curve and variations in the biochemical prolile of seven phytoplankton species, including P. tricornutum and T. suecica. Their data on the time needed to reach late exponential growth phase of these algae is in accordance with our results. Hirayama et al. ( 1979) investigated the nutritional effects of eight species of marine phytoplankton on population growth of the rotifer, Brachionus plicatilis. Two species, Synechococcus elongatus and Chlorella sp. were evaluated as excellent food plankton and are much smaller in cell size than the other species. During this investigation, Nannochloropsissp. and P. tricornuturn gave the best growth rate, although these algae were considerably different in cell size. It has been reported (Minkoff, 1987) that rotifer eggs and rotifer lorica are not digested by fish larvae. The contribution of female rotifers to the overall rotifer population during the growth cycle decreased from 30% to 5%. In the late exponential growth phase, the female contribution ranged from 7 to 9%. Considering the results achieved in this study, namely, maximum rotifer numbers, time to reach maximum numbers, growth rate, doubling time and the chemical composition of the rotifers fed P. tricornutum and Nannochloropsis sp., these phytoplankton species in polyculture are proposed to be a suitable diet for fish larvae. Furthermore, the rotifers should be maintained at the late exponential phase which is the most suitable for feeding purposes.
ACKNOWLEDGEMENTS

We thank Dr B.R. Howell, Fisheries Laboratory, Conwy, Great Britain, for his suggestions on the manuscript. We would like to thank two anonymous reviewers for their helpful comments. Thanks are also extended to Mrs. Nela Matkovic for translation.

DIETARY EFFECTS ON BIOCHEMICAL COMPOSITION OF ROTIFER

149

REFERENCES Barnes, H. and Blackstock, J., 1973. Estimation of lipids in marine animals and tissues: detailed investigation of the sulphophosphovanillin method for total lipids. J. Exp. Mar. Biol. Ecol., 12: 103-l 18. Ben-Amotz, A., Fishler, R. and Schneller, A., 1987. Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids. Mar. Biol., 95: 3 l-36. Bligh, E.G. and Dyer, W.F., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 9 1 l-9 17. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Castell, J.D., Conklin, D.E., Craigie, J.S., Lall, S.P. and Norman-Boudreau, K., 1986. Aquaculture nutrition. In: M. Bilio, H. Rosenthal and C.J. Sindermann (Editors), Realism in Aquaculture; Achievements, Constraints, Perspectives. European Aquaculture Society, Bredene, Belgium, pp. 251-308. Cowey, C.B. and Sargent, J.R., 1972. Fish nutrition. Adv. Mar. Biol., 10: 383-492. Dawirs, R.R., 1987. Influence of limited starvation periods on growth and elemental composition (C, N, H) of Carcinws maenas (Decapoda: Portunidae) larvae reared in the laboratory. Mar. Biol., 93: 543-549. Dendrinos, P. and Thorpe, J.P., 1987. Experiments on the artificial regulation of the amino acid and fatty acid contents of food organisms to meet the assessed nutritional requirements of larval, post-larval and juvenile Dover sole (Solea solea L. ). Aquaculture, 6 1: 12 l- 154. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Fernandez-Reiriz, M.J., Perez-Camacho, A., Ferreiro, M.J., Blanco, J., Planas, M., Campos, M.J. and Labarta, U., 1989. Biomass production and variation in the biochemical profile (total protein, carbohydrates, RNA, lipids and fatty acids) of seven species of marine microalgae. Aquaculture, 83: 17-37. Fujita, S., 1979. Culture of red sea bream, Pagrus major, and its food. In: E. Styczynska-Jurewicz, T. Backiel, E. Jaspers and G. Persoone (Editors), Cultivation of Fish Fry and its Live Food. European Mariculture Society Special Publication No. 4, Bredene, Belgium, pp. 183197. Giese, A.C., 1967. Some methods for study of the biochemical constitution of marine invertebrates. Oceanogr. Mar. Biol. Annu. Rev., 5: 159-l 86. Guillard, P.R.L. and Ryther, J.H., 1962. Studies on marine planktonic diatoms I. Cyclotella nana Hustedt and Defonula confervacea (Cleve.) Gran. Can. J. Microbial., 8: 229-239. Hirayama, K., Takagi, K. and Kimura, H., 1979. Nutritional effect of eight species of marine phytoplankton on population growth of the rotifer, Brachionus plicatilis. Bull. Jpn. Sot. Sci. Fish., 45: 11-16. Holland, D.L. and Hannant, P.J., 1973. Addendum to a micro-analytical scheme for the biochemical analysis of marine invertebrate larvae. J. Mar. Biol. Assoc. UK, 53: 833-838. Howell, B.R., 1977. Aspects of the development of cultivation techniques for flatfish. Ph.D. Thesis, M.A.F.F., Fisheries Laboratory, Lowestoft, UK, 105 pp. Katavic, I., Tudor, M., Komljenovic, J. and Ruiic, N., 1985. Changes in the biochemical composition of Artemia salina (L. ) in relation to different feeding conditions. Acta Adriat., 26( 2): 123-134. Kochert, G., 1978. Carbohydrate determination by the phenol-sulfuric acid method. In: J.A. Hellebust and J.S. Craigie (Editors), Handbook of Phycological Methods: Physiological and Biochemical Methods. Cambridge University Press, London, pp. 95-97. Lovergrove, T., 1966. The determination of the dry weight of plankton and the effect of various

150

M. CARIt ET AL.

factors on the values obtained. In: H. Barnes (Editor), Some Contemporary Studies in Marine Science. George Allen and Unwin, London, pp. 429-467. Lubzens, E., Marko, A. and Tietz, A., 1985. De novo synthesis of fatty acids in the rotifer Brachionusplicatilis. Aquaculture, 47: 27-37. Lubzens, E., Tandler. A. and Minkoff, G., 1989. Rotifers as food in aquaculture. Hydrobiologia, 186/ 187: 387-400. Millikin, M.R., 1982. Qualitative and quantitative nutrient requirements of fishes: a review. Fish. Bull., 80(4): 655-686. Minkoff, G., 1987. The effect of secondarily enriched rotifers on growth and survival of marine fish larvae. Ph.D. thesis, University of Stirling, UK. Pillsbury, K.S., 1985. The relative food value and biochemical composition of five phytoplankton diets for queen conch, Strombus gigas (Linne) larvae. J. Exp. Mar. Biol. Ecol., 90: 22 l231. Scott, A.P. and Baynes, S.M., 1979. The effect of unicellular algae on survival and growth of turbot larvae (Scophthalmus maximus L). In: J.E. Halver and K. Tiews (Editors), Finfish Nutrition and Fishfeed Technology. Proc. World Symp., 20-23 June 1978, Hamburg. Vol. I. Heenemann, Berlin, pp. 423-433. Scott, A.P. and Middleton, C., 1979. Unicellular algae as a food for turbot (Scophthalmus maximus L. ) larvae - the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture, 14: 227-240. Walford, J. and Lam, T.J., 1987. Effect of feeding with microcapsules on the content of essential fatty acids in live foods for the larvae of marine fishes. Aquaculture, 6 1: 2 19-229. Watanabe, T., Kitajima, C., Arkawa, T. and Fujita, S., 1978. Nutritional quality of rotifer Brachionusplicatilis as a living feed from the viewpoint of essential fatty acids for fish. Bull. Jpn. Sot. Sci. Fish., 44: 1109- 1114. Watanabe, T., Tamiya, T., Oka, A., Hirata, M., Kitajima, C. and Fujita, S., 1983. Improvement of dietary value of live foods for fish larvae by feeding them on w3 highly unsaturated fatty acids and fat soluble vitamins. Bull. Jpn. Sot. Sci. Fish., 49: 47 l-479.