THE USE OF MICROPARTICLES IN AQUACULTURE

F.J. GATESOUPE

INTRODUCTION

The first successes in Marine Aquaculture are linked with the more or less local opportunity to collect spat, fry or post larvae shrimp. The development of this activity is connented with the design of the hatcheries which must be capable of ensuring the production of juveniles, while not relying on the natural environment. All the species employed, begin to feed soon after hatching, although the locomotive, sensorial and digestive faculties of the larvae are just beginning to form. Thus, it is obligatory that the hatcheries furnish a food which is as similar as possible to the plankton consumed in a natural environment. It is often possible to collect plankton, but this is too hazardess, thus hatcheries generally cultivate live prey from isolated strains. These annex cultures are expensive, and from 1970 onwards, numerous tests were carried out so as to replace them by a complete artificial diet, presented in the form of microparticles. Confident with the results obtained in the rearing of the rainbow trout, where the fry accept without any difficulty, the pellets from the first feeding onwards, the precursors believed that the microparticle was but a smaller sized pellet than that used in fattening. Unfortunatly, when trout fry start feeding they are no longer larvae and have already a functional stomach: It was necessary to try out more elaborated formulas immediatly, which would hinder the particles from dropping to the bottom and from disintograting too quickly, while at the same time ensuring that the above could be adapted to the locomotive behaviour of the larvae, and that the addition of a colour solution and appetizers would stimulate their sensorial faculties. But the fundamental question remaining unanswered is that of the choice of nutritive componants adapted to the larvae.

The advantage of employing microparticles in aquaculture is not only limited to larvae: They can be employed to feed the live prey which will afterwards be fed to the larvae. Their use for filterer molluscs after their metamorphosis has taken place, can also permit the acquirement of a better knowledge of the diet of these species, whose feeding is ensured by a natural primary production.

Besides compound microparticles, there also exists natural ones: eggs or bacteria, for example, In addition, it is possible to modify the composition of live prey through their culture conditions or Feeding. FONTAINE and REVERA (1980) have even termed as “microcapsulation”the forcible feeding of rotifers with nutritive particles. On the other hand, the organisms employed as live prey are also an important source of raw material used in the composition of microparticles. indeed, between the plankton collected and the compound microparticle of purified raw materials, there exists a great variety of intermediary solutions with live or inert prey whose composition is more or less monitored (Diagrams 1). Thus, it is all the feeding techniques employed that will be of interest to us, by making a list of the food at disposal, and the results obtained in the larval rearing. We do not need to go into detail since nearly all hatcheries develop their own techniques, but instead to emphasize the tendancies and establish the different choices possible.

1. THE “NATURAL” MICROPARTICLES

Whether live or dried, numerous organisms make up the “natural” microparticles which can be directly employed to feed live prey or larvae.

1.1. Unicellular organisms

The great advantage of these organisms lies in the quality and quantity of proteins that they contain: Generally, they represent 50 to 80 % of the dry matter, having an amino-acid balance much the same as that of muscular proteins. They are also rich in nucleic acids and in certain vitamins (LITCHFIELD, 1983). In addition to the industrial products, it is possible to cultivate specified isolated marine strains (Diagram 2).

1.1.1. Bacteria

The industrial production of bacteria is advancing rapidly and is the most promising source for natural microparticles. Some of these products have already been tried out in aquaculture, as is the case of Methylophilus methylotrophus which is used successfully on oysters in fattening (NELL and WISELY, 1983). The marine bacteria are, a priori, the most interesting for aquaculture but if production techniques do exist (YAMAMOTO at al., 1978), there, to our knowledge, exists no commercialization. In addition, even marine bacteria do not seem to contain the essential Fatty acids: eicosapentaenoic and docosahexaenoic (ORO et al., 1967; ANDREE et al., 1979).

1.1.2. Yeasts

There exists a great variety of Saccaromyces cerevisiae, of commercial source, either in atomized form or live form (baker's yeast or brewers yeast, etc…) one of these which is especially adapted for aquaculture has been developed in japan (“w-yeast” - IMADA et al., 1979): Its culture medium containing squid oil gives it a high tenor in essential Fatty acids. Candida utilis yeast has been introduced into the feeding of juvenile oysters (URBAN and LANGRON, 1984). Finally, there exists culture techniques of marine yeasts (KAWANO and KAMEL., 1980)but as in the case of bacteria they contain very few or no essential fatty acids (HIGASHIHARA et al., 1983 a).

1.1.3. Algae

Mass culture of algae permits commercialization. This especially concerns fresh water chlorella, in Taïwan (CHEN, 1977) and spiruline in Mexico (which are indeed pluricellular algae - CIFERRI, 1983). These algae are available in the atomized form, principally intended for human consumption, and so very expensive. However, there exist mass productions of diatoma and marine chlorella which are meant especially for molluscs (GOLDMAN, 1979; CLAUS et al., 1980).

The controlled pure culture of algae is performed in most hatcheries (De PAUW, 1981; LE BORGNE, 1986). This, to a certain extent, permits to modulate the food quality, by diversifying the culture conditions: for example, the quantity of nitrate has an effect on the protein rates (MOSTERT and GROBBELAAR, 1981); The digestibility of proteins increases with the age of the culture (KANDATSU and KAWAGUCHI, 1979); the salinity increases the tenor in essential fatty acids, countrary to the temperature (SETO et al., 1984).

1.2. Animal live prey

If we leave out Artemia cysts which are a natural resource, the animal live prey must be cultivated from isolated strains: Then, there is no question of an alternative with other industrial products such as compound micro-particles.

1.2.1. Copepada

Marine calanoids belong to the plankton which is normally consumed in a natural environment: Thus, they are prey which are very well adapated for larval rearing, moreover as, they have an important essential fatty acid tenor (WATANABE et al., 1983). Unfortunatly, around 20 days is required before adults can be obtained and rearings such as these run very expensive on hatcheries ((STOTTRUP et al., 1986). The detrivorous and benthic harpacticoïds copepoda have the same disadvantage but they seem to feed more easily on artificial food (for example, if Tigriopus japonicusand Acartia clausi are compared - KITAJIMA, 1973).

1.2.2. Brachionus plicatilis

The performances of the parthenogenetic reproduction of this rotifer adapted to a marine environment,along with its resistance to enviornmental changes and its facility to eat, permits it to be reared in all hatcheries (POURRIOT, 1986). The most reknown and reliable technique is the culture with baker'yeast starting from an inoculum cultivated with the algae (HIRATA, 1979). The marine yeast culutres and to lesser degree bacteria, can replace the algae culture for rotifers (HIGASHIHARA et al., 1983 b). In the same way, a compound diet consisting of atomized unicellular organisms and additives (oil, vitamins, raw starch) could be effective (GATESOUPE and LUQUET, 1981). Compound feeding permits not only to improve the ensurance of these nutritional needs of the rotifer - for example, by adding vitamin B 12 and fish oil to the baker's yeast (HIRAYAMA and FUNAMOTO, 1983) but also its food quality. For the latter, only the effect of a fish oil supplementation is sure (WATANABE et al., 1983). There exists other ways of supplying a strong tenor in essential fatty acids to rotifers: we have already stated the special yeast of IMADA et al ., 1979; Enrichments through baths for time lengths varying between 30 minutes and 24 hours is also carried out just before the rotifers are distributed to the larvae: For thus, the rotifers are placed into a concentrated suspension of a algae, emulsion (WATANABE et al., 1983) or compound food (GATESOUPE and LUQUET, 1981).

Microcapsules have even been tested (TESHIMA et al., 1981). A priori, it seems surprising to search for a capsuled particle for a rotifer which is capable of feeding on soluble substances (SCOTT, 1983) and bacteria: however, the flora associated with rotifers is difficult to control (COVES et al., 1986) and microcapsules could help limiting the bacterial proliferations caused by uneaten food. We have, also, employed microcapsules for the supply of antibiotics to turbot larvae by Forcibly Feeding the rotifers, thus avoiding the need of employing bath treatment techniques (RUBIO RINCON, 1986).

1.2.3. Artemia

Artemiacysts permit obtaining nauplii which can be given diretctly to the larvae, so long as the Artemia strain is of “marine type” (WATANABE et al., 1983), in other words, that the nauplii contain an important quantity of essential fatty acids. If not so, it is necessary to proceed with a diet rich in fish oil while taking into account that nauplii dont feed until several hours have passed, after hatching (URBANI, 1959). There is a lot of data on this subject and the techniques don'tdiffer greatly from those employed for rotifers (Diagram 3); we shall refer to the more recent documents (SORGELOOS and al., 1986; LEGER et al., 1986).

1.3. Fish eggs

There exists other natural microparticles: Let us take for example fish eggs which constitute and interesting food for sea-bass Micropterus salmoides (BRANDENBURG et al., 1979) or shrimp Macrobrachium rosenbergii (MANZI and MADDOX, 1980). Unfortunately, the availability of these eggs is often not compatible with the regular supply from a hatchery.

2. COMPOUND MICROPARTICLES

We must distinguish here, two types of consumers which dictate different constraints for the utilization of microparticles: the filterers and the predators (Diagram 4).

The filterers injest large volumes of water and find a considerable part of their food in the dissolved substances (STEPHENS, 1982) and bacteria. The microparticles can then either be filtered directly or disintegrated and increase the concentration of bacteria and dissolved substances, provided however that the filterers resist the environmental alterations that this entails. With shrimp or mollusc larvae, it is advisable to use stable microparticles even if this should require the necessity of supplying separatly certain bacteria or dissolved substances. We have remarked that on the contrary, live prey can feed on compound food which has not been treated in any particular way so as to avoid its desintegration. In all cases, microparticles must have a slightly higher density than that of sea-water so that the disturbance caused by the aeration will ensure that they are kept in suspension.

Fish larvae and shrimp post-larvae have a predator behaviour. This behaviour must be stimulated by the microparticles, to which is added appetizing ingredients and colour solutions (red or orange more often). This results in a new constraint: a same microparticle must contain a complete and balanced food, if not, the preference for one type of particle in the case of a mixture, would entail, and unbalance in the feeding. This would mean a great difference with regard to filterers who can receive separatly an emulsion, solid particles, and dissolved substances. Finally, its water stability and its maintenace in suspension must be ensured for as long as possible. Several technological treatments permit the ensurance of these requirements (Diagram 5).

2.1. Pressure cooking methods

Its stability in the water is obtained through the action of vapour pressure compaction, Cooking also permits the better digeatibility of starch and the desturction of contain undesirable substances such as avidine found in the hen'egg. Unfortunatly, certain vitamins can be destroyed on parallel (ascorbic acid and thiamine for example).

2.1.1. Flakes

Compaction is carried out at more than 100°C on a rotative drum which permits the obtention of flakes. This particular form facilities their maintenance in suspension in the water (MAYERS and BRAND, 1975).

2.1.2. Cooking extrusion

A die is employed for compaction. Stable food is obtained with temperatures of below 100°C, but the main advantage of this method lies in the expansion provoked at higher temperatures, which permits to rehydrate the particles just before their distribution with an emulsion containing labile substances (Vitamins, appetizers, oil - METAILLER et al., 1983).

2.2. Gels

Certain food gels are very effective and permit to obtain very stable microparticles without the need of any specialized equipment: the small scale manufacture of microparticles is thus possible at hatchery level. The most widely used gels are agar, kappa-carragheen (TESHIMA et al., 1982) which are insoluble in cold water and need to be heaten to 80°C, and the, alginate of sodium which is soluble when cold and precipated by the calcium ions (L'HERROUX et al., 1977). This last technique is most interesting for the preservation of thermolabile substances.

2.3. Microencapsulation (by coacervation)

There are numerous methods in which to obtain particles with a protecting wall covering. We will give here a description of two: Microencapsulation by coacervation which consists in precipitation a polymer in liquid phase around the microparticles or the micella and the microcoating where wall is obtained by the evaporation of the external phase. This classification is not conventional as on general a spray is employed for microencapsulation. Our objective is to insist on the essential difference between the two methods when they are employed for compound food. Indeed the external liquid phase necessary for the coacervation-either water or an organic solvent-causes the loss of soluble substances: It is therefore difficult to obtain a complete food (JONES and GANNOTT, 1976). On the contrary, this is the only technique through which will be obtained graded particles of a little micron dimension without the use of specilized equipment. TESHIMA et al., (1982) give a list of the principal methods employed: nylon protein capsules, gelatine gum arabic, chitosan, zein. LANGDON (1983) Proposed a technique using emulsion to cover hydrosoluble substances in a coating which contains fish oil: It is unfortunatly impossible to isolate these capsules from the external liquid phase: This is however a method which could be very useful so as to ensure the soluble ingredients for filterers while not having to dissolve great quantities.

2.4. Microcoating

With this technique, the external phase is evaporated, which hinders all losses of soluble elements during its manufacture and permits the incorporation of appetizing substances in the coating (GATESOUPE and LUQUET, 1977). The use for particles of some hundreds of microns does not require specialized equipment: the coating then consists of zein or of a cholesterol mixture-lecithin (TESHIMA et al., 1982). The spray (atomization, etc...) permits obtaining much finer particles (BALASSA and FANGER, 1971).

3. THE CHOICE OF MICROPARTICLES DEPENDING ON THE SPECIES

Feeding tests, using compound microparticles have been tried out on nearly all the species employed in aquaculture. However, few of these tests have been applied on large scale in hatcheries: Most of the tests were promising, but the growth performances and survival have rarely been as good as that obtained with live prey. An economy can be made through compound microparticles if there is a prolonged larval rearing duration, moreover as a low growth is a sign of a bad nutritional state which will irremediably endanger the ulterior growth performances. This is why we can have but a rather restrictive vision of the use of microparticles in the actual state of affairs.

3.1. Live prey

We have seen that there exists a large possible variety of food. The determinant factor for these prey will be their food value. when the climatic conditions permit so, the production of selected unicellular algae in outdoor large volumes is the most rational solution to adopt. Moreover as it is always possible to complete the ration with baker's yeast for example. In the cold regions, baker's yeast is the safest basic food to employ, while ensuring a minimum quantity of live algae regularly and that prey are enriched with a fish oil emulsion before being distributed to the larvae.

3.2. Filterer molluscs

The larval and post-larval stages of these species being particularly sensitive to the physiochemical and microbiological quality of their environment, it is preferable to feed them on algae cultivated in controlled conditions. There is but one link in the food chain here, thus the cost of an algae room is quite acceptable for a mollusc hatchery. The use of microparticles is however promising for the future (TESHIMA et al., 1982).

It is necessary to carry out the fatteneing of these species in an open environment. We shall thus limit the use of compound microparticles to the experimentation in nutrition: except in particular cases, intensive rearing having a food supply has little chance of being more profitable than extensive rearing.

3.3. Shrimp

Alginated microparticles are already widely used in hatcheries of Macro brachium rosenbergii (AQUACOP, 1983). Likewise, particles linked with carragheen seem to answer the requirements of Penaeus japonicus when zoe stage is reached (TESHIMA and KANAZAWA, 1983) along with those of tropical peneids (GALGANI-TURIN, 1986). The preservation of ther molabile substances is an important factor for success (TESHIMA and KANAZAWA, 1983): the employment of fresh raw materials is thus strongly advisable, especially as squid flesh contains a growth factor (CRUZ and GUILLAUME, 1983).

3.4. Fish

If the consumption of compound microparticles by fish larvae as their first food is frequently observed, the results for growth and survival are extremely disappointing. For certain species, it seems possible to obtain good results 10 days after hatching: this applies for japanese red sea-bream (TESHIMA et al., 1982) and for sole (GATESOUPE, 1983). on general, weaning is carried out at the end of the first month, and research is still necessary so as to reduce the quantities of Artemiaconsumed, by advancing the date for weaning. This seems possible for sea-bass for example (PERSON-LE RUYET, 1986).

The most current reason put forward for this difficulty in rearing fish larvae, with compound food is the inadaptation of the stomach less digestive system. The absorption of proteins and their intercellular digestion seems to be of considerable importance, even with the trout fry which has a stomach (GEORGO-POULOU et al., 1986). It is most probable that the quality of the macromolecules present in the food plays an essential part in this absorption-digestion. On the other hand, the distribution of proteic fractions is very different in live prey (HAYASHI et al., 1985), and in the muscle of fish (HASHIMOTO et al., 1979) (Diagram 6): the proportion of free amino-acids, peptides and soluble proteins is indeed much greater in the former. These are evidently the most difficult fractions to be retained in compound microparticles. The method developed by PIGOTT et al., (1982)conferring to the proteins of fish bindery proporties through hydrolyses could be an effective way to risolve this problem.

CONCLUSION

A lot remains undone for the generalization of the employment of compound microparticles in hatcheries. This is however an inevitable evolution, at first for economic reasons: the Artemia cysts constitute a limited resource and it is preferable not to waste them, if we wish to increase the production of juveniles. Microparticles should permit to avoid a too considerable increase of the demand for Artemia, but is must not be hoped that they themselves constitute a low costing food. Indeed, the quality requirements of raw materials-sometimes mean for human consumption such as squid-and the use of expensive machinery risk to maintain high prices, moreover as the delays of conservation do not permit the manufacture, at real industrial scale, of a product of limited demand. Secondly, compound microparticles should permit the improvement of the nutritional state of juveniles, with regard to what is obtained with Brachionus plicatilis and Artemia which when even enriched are not necessarily the ideal food. The first results are already available: CHEN et al., (1986) have obtained better growth of peneid post - larvae by distribution simultaneously Artemia and compound food.

Diagram 1 - The food chains employed in hatcheries (the arrows indicate the trophic relations

Diagram 2 - The unicellular organisms used in marine aquaculture

Diagram 3- Food for Rotifers and Artemia according to their nutritive quality

Diagram 4: Quality required for the compound food

Diagram 5 - The different types of compound microparticles

Diagram 6 Distribution of the proteic fractions in live prey, compared with that of the sardine muscle.

REFERENCES

ANDREEV L..V, M.A.BOBYK at A.GUELIN, 1979. Sur la composition en acides gras des microvibrions marins. C.R.Acad. Sc. Paris, 288 D, 173–175.

AQUACOP, 1983. Production of feeds to support shrimp farming in Tahiti (French Polynesia). 1 st Int. Biennal Conf. Warm Water Aquacult. Crustasea, Feb. 9–11, 1983, Hawai (sous presse).

BALASSA L.L.et G.O.FANGER, 1971. Microencapsulation in the food industry. CRC critical reviews in food technology 2 (2), 245–265.

BARNABE G., 1986. Aquaculture (volume 1 ) Tech. et Doc. Lavoisier, Paris, 522 pp.

BRANDENBURG A.M., M.S.RAY et W.M.LEWIS, 1979. Use of carp eggs as feed for fingerling largemouth bass. Progress. Fish cult. 41 (2), 97–98.

CHEN C.S., 1977. Chlorella Industry in Taiwan. JCRR Fish. Ser. 25 B, 68–74.

CHEN H.Y., D.V.ALDRICH et Z.P.ZEIN-ELDIN, 1986. Improved growth of early postlarval penaeids through feeding artificial diets as complements to Artemia nauplii. WMS Ann. Conf., Reno (Nevada), Janv. 19–23, 1986 (sous presse).

CIFERRI O., 1983.Spirulina, the edible microorganism.Microbiol.Rev. 47 (4), 551–578.

CLAUS C., N.De PAUW et E.JASPERS, 1981. Nursery culturing of Bivalve Molluscs. EMS Spec. Pulb. 7, 35–69.

COVES D., P.AUDINEAU et J.L.NICOLAS, 1986. Les rotifères - technologies d'élevage In: BARNABE (1986), 223–238.

CRUZ E.et J.GUILLAUME, 1983. Facteur de croissance inconnu de la farine de calmar pour la crevette japonaise: localisation de ce facteur. CIEM, Cté Maricult. F:14, 12 pp, ronéo.

De PAUW N., 6, H.VERLET et L.De LEENHEER Jr, 1980. Heated and unheated outdoor cultures of marine alage with animal manure. In: Algae Biomass, G. SHELEF and C.J. SOEDER (Eds), Elsevier/North Holland Biomed. Press, 315–341.

FONTAINE C.T. et D.B.REVERA, 1980. The mass culture of the rotifer, Brachionus plicatillis, for use as foodstuff in aquaculture. Proc. World Maricul. Soc. 11, 211–218.

GALGANI-TURIN M.L., 1986. Reproduction contrôlée et élevage larvaire de crevettes pénéides en milieu torpical. Thèse 3è cycle, Univ. Aix-marseille II, 127 pp.

GATESOUPE F.J., 1983. Weaning of sole, Solea Solea, before metamorphosis, achieved with high growth and survival rates. Aquaculture 32, 401–404.

GATESOUPE F.J. and P.LUQUET, 1977. Recherche d`une alimentation artificielle adaptée à l`elevage des stades larvaires des poissons. I- Comparison de quelques techniques destinées à améliorer la stabilité à l'eau des aliments. Actes Coll. CNEXO 4, 13–20.

GETESOUPE F.J.et P.LUQUET, 1981. Practical diet for mass culture of the rotifer Brachionus Plicatilis: application to larval rearing of sea bass, Dicentrarchus labrax. Aquaculture, 22, 149–163.

GEORGOPOULOU U., M.F. SIRE et J.M. VERNIER, 1986. Absorption intestinale des protéines sous forme macromoléculaire et leur digestion chez la truite Arc-en-ciel. Etude ultrastructurale et biochimique en relation avec la premiére prise de nourriture. Can. J. Zool. 64, 1231–1240.

GOLDMAN J.C., 1979. Out door algal mass cultures - I.Applications. Water Research, 13, 1–19.

HASHIMOTO K., S. WATABE, M. KONO et K. SHIRO, 1979. Muscle protein composition of sardine and mackerel. Bull. Jap. Soc. Fish. 45 1435–1441.

HAYASHI T., Y. SUITANI, M. MURAKAMI, K. YAMAGUCHI et S. KONOSU, 1985. Nitrogen distribution in marine zooplankton as diets for fish larvae. Bull. Jap. Soc. Sci. Fish. 51 (6) 1047.

HIGASHIHARA T., S.FUKUOKA, T.ABE, I.MIZUHARA, O.IMADA et R.HIRANO, 1983a. culture of marine yeasts using alcohol fermentation slop and its taxonomic characteristics. Bull. Jap. Soc. Sci. Fish. 49 (7) 1015–1023.

HIGASHIHARA T., S.FUKUOKA, T.ABE, I.MIZUHARA, O.IMADA et R.HIRANO, 1983b. Culture of the rotifer Brachionus Plicatilis using a microbial flock produced from alcohol fermentation slop. Bull. Jap. Soc. Sci. Fish. 49 (7) 1001–1013.

HIRATA H., 1979. Rotifer culture in Japan. EMS Spec. Publ., 4, 361–375.

HIRAYAMA K.et H.FUNAMOTO, 1983. Supplementary effect of several nutrients on nutritive deficiency of baker's yeast for population growth of the rotifer Brachionus Plicatilis. Bull. Jap. Soc. Sci. Fish. 49 (4), 505–510.

IMADA O., Y.KAGEYAMA, T.WATANABE,C.KITAJIMA, S.FUJITA et Y.YONE, 1979. Development of a new yeast as a culture medium for living feeds used in the production of fish seed. Bull. Jap. Soc. Sci. Fish. 45 (8), 955–959.

JONES D.A.et P.A.GABBOTT, 1976. Prospects for the use of microcapsules as food particles for marine particulate feeders. In: Microencapsulation, R.J.NIXON (Ed.), M. DEKKER Inc., New-York, 215 pp.

KANDATSU M.et H.KAWAGUCHI, 1979. Nutritive value of green algal protein of Chlorella ellipsoidea. Part 2. Charges of artificial digestibilities of Chlorella Proteins in the course of their life cycle. Bull. Azabu Vet. Coll. 4 (1)9–15.

KAWANO T.et S.M. KAMEL, 1980. A bibliography on marine yeast. Symp. Coast. Aquacult., Jan. 12–18, 1980, Cochin (India).

KITAJIMA C., 1973.Experimental trials on mass culture of copepods. Bull. Plankton Soc. Japan, 20 (1), 54–60.

LANGDON C.J., 1982. Now techniques and their application to studies of bivalve nutrition. Proc. 2nd Int. Conf. Aquacult. Nutr., Biochem. Physiol. Approaches Shellfish Nutr., Louisiana State Univ., 305–320.

LE BORGNE Y., 1986. La Culture des micro-algues. In BARANABE (1986), 182–199.

LEGER P., D.A. BENGSTON. K.L. SIMPSN et P. SORGELOSS, 1986. The use and nutritional value of Artemia as food Sourco. Mar. Biol. Ocanogr. Ann. Rev. (sous presse).

L'HERROUX M., R.METAILLER et L.PILVIN, 1977. Remplacement des herbivores proies par des microparticulars inertes: une application àl'élevage larvaire de Penacus japonicus. Actes Coll. CNEXO, 4, 147–155.

LITCHFIELD J.H., 1983. single-cell proteins. Science, 219, 741–746.

MANZI J.J.et M.B.MADDOX, 1980. Requirements for Artemia nauplii in Macrobrachium rosenbergii (de Man) larviculture. In: The Brine Shrimp Artemia Vol. 3, G. PERSOONE, P. SORGELOSS, O. ROELS and E. JASPERS (Eds) Universa Press, weteren (Belgium), 313–329.

MELCION J.P., J.GUILLAUME, J.MEHU, R.MATAILLER et G.CUZON, 1983. Preparation d'aliments pour animaux marins par cuisson-extrusion. Revue de l'Alimentation animale, 371, 26–31.

METAILLER R., M. CADENA ROA et J. PERSON, 1983. Attractive chemical substances for the weaning of dover sole (Solea vulgaris): qualitative and quantitative approach. Proc world Maricul. soc. 14, 679–684.

MEYERS S.P. et C.W. BRAND, 1975. Experimental flake diets for fish and crustacea.progr.Fish cult., 37 (2), 67–72.

MOSTERT E.S. et J.U. GROBBELAAR, 1981. Protein manipulation of mass cultured algae.UOFS Publ., Series c, 3, 86–90.

NELL J.A. et b.wisely, 1983. Experimental feeding of Sydney rock oysters (Saccostrea commercialis). II.Protein supplementation of artificial diets for adult oysters. Aquaculture 32, 1–9.

ORO J., T.G. TORNABENE, D.W. NOONER et E. GELPI, 1967. Aliphatic hydrocarbons and fatty acids of some marine and freshwater microorganisms, J. Bacteriol., 93 (6), 1811–1818.

PERSON-LE RUYET J., 1986. Le sevrage du bar (Dicentrarchus labrax) avant un mois: resultats préliminaires. CIEM, Cte Maricult., F: 32, 13 pp., ronéo.

PIGOTT G.M., N.E.HECK, R.D.STOCKARD et J.E. HALVER, 1982. Engineering aspects of a new process for producing dry larval feed. Aquacult. Eng., 1 (3), 215–226.

POURRIOT R., 1986. Les rotifères - Biologie. In: BARNABE (1986), 201–221.

RUBIO RINCON E.A., 1986. Evolution de la compostion en acides gras de l,ovocyte a la larve du turbot (Psetta maxima l.) en fonction du régime alimentaire de reproducteurs at des larves, ainsi que de la temperature d'incubation. These 3e cycle, Univers. Bretagne Occidentale, 183 pp.

SCOTT J.M., 1983. Rotifer nutrition using supplemented monoxenic cultures. Hydrobiologia, 104, 155–166.

SETO A., H.L.WANG et C.W. HESSELTINE, 1984. Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima. J.Ann. Oil Chem. Soc., 61 (5), 892–894.

SORGELOOS P., D.A. BENGSTON et W. DECLEIR (Eds) 1986. Proc.2nd Int, Symp, the Brine Shrimp Artemia, Vol. 3, Ecology culturing and use in Aquaculture. Universa Press, Wetteren (Belgium) (sous presse).

STEPHENS G.C., 1982. Dissolved organic material and the nutrition of marine bivalves. In: Proc. 2nd Int. Conf. Aquacult. Nutr. Biochem. Biophys. Approach. Shellfish Nutr., Louisiana State Univ., 338–357.

STOTTRUP J.G., K. RICHARDSON, E. KIRKEGAARD et N.J. PIHL, 1986. The cultivation of Acartia tonsa Dana for use as a live food source for marine fish larvae. Aquaculture, 52, 87–96.

TESHIMA S., A. KANAZAWA et M.SAKAMOTO, 1981. Attempt to culture the rotifers with microencapsulated diets, Bull. Jap. Soc. Sci. Fish., 47 (12), 1575–1578.

TESHIMA S., A. KANAZAWA et M. SAKAMOTO, 1982. Microparticulate diets for the larvae of aquatic animals. Min. Rev. Data File fish. Res.,2, 67–86.

TESHIMA S. et A. KANAZAWA, 1983. Effects of several factors on growth and survival of the prawn larvae reared with microparticulate diets. Bull. Jap. Soc. Sci Fish., 49 (12), 1893–1896.

URBAN E.R. Jr et C.J. LANGDON, 1984. Reduction in costs of diets for the american Oyster, Crassostrea virginica (Gmelin) by the use of non-algal supplements. Aquaculture, 38, 277–291.

URBANI E., 1959. Proridi, glucidi e lipidi nello sviluppo di Artemia salina Leach. Acta Embryol. Morphol. Experiment, 2 171–194.

WATANABE T.,KITAJIMA C. et s. FUJITA, 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34, 115–143.

YAMAMOTO M., Y. SERIU, S. GOTO, R. OKAMOTO et T. INPUT, 1978. Growth characteristics of marine methanol utilizing bacteria. J. Ferment. Technol., 56 (50, 459–466.