4.5 Optimal Levels and Ratios of Dietary n -3 and
4.5.1.2. Larvae and Early Postlarvae
Marine fish larvae pose the aquaculturist great problems, due primarily to their small size and their often poorly developed digestive system, which has hampered the development of appropriate fabricated first feeds. Ten years ago, the technical problems associated with presenting defined micro diets to marine fish larvae had prevented the accurate determination of lipid and EFA requirements in these crucial early life stages. Even now, recent stud- ies on the development of fabricated, defined microdiets for first-feeding marine fish larvae are relatively few (Salhiet al.,1994, 1999; Bessonartet al., 1999; Halfyardet al.,1999). Poor acceptance of fabricated first feeding diets may be related to the particles presented having an unsuitable size spec- trum or to their aggregation or settling properties prior to consumption.
It is also likely that the larvae prefer to capture and ingest moving prey or require specific gustatory stimuli to ingest captured prey. As a result, live feeds are currently still the option of choice in most situations until the lar- vae are large enough to be maintained on a fabricated diet. However, the use of live feeds has presented significant problems in accurately defining lipid and EFA requirements in marine fish larvae, although this is an area that has received considerable attention in recent years (Brownet al.,1989,
1997; Brown and Jeffrey, 1992; Izquierdo, 1996; Rainuzzoet al.,1997; Reitan et al.,1997; Shansudinet al.,1997; Sargentet al.,1997, 1999a, b; McEvoy and Sargent, 1998; Dhertet al.,1998).
Several types of live feeds of variable sizes have been used, with their spe- cific utilization depending on the size of the larvae at emergence and dur- ing the subsequent growth period. The principal live feeds used have been rotifers (Brachionussp.) and brine shrimp (Artemiasp.) nauplii. Rotifers, be- ing smaller, are fed to small larvae including very early larval stages.Artemia nauplii, being larger, are fed to larger larvae including later developmen- tal stages. The major factor in the choice of these organisms has been their ready availability and ease of culture, withArtemiabeing particularly useful in that they can be packaged, stored, and transported easily in an arrested form of development as dry cysts. However,Artemiais a nutritionally poor diet for marine fish larvae in that it generally lacks then-3 HUFA essential for ma- rine larvae, being rich instead in 18:3n-3. Because many different strains of Artemiaexist, one option has been to seek strains that are nutritionally more suitable. Strains with significant levels of 20:5n-3 have been reported, but none so far with significant levels of 22:6n-3 (Navarroet al.,1991, 1992a, b, 1993a). This limitation is related, at least partly, to the propensity ofArtemia to retroconvert 22:6n-3 to 20:5n-3 (Evjemoet al.,1997; Navarroet al.,1999).
Therefore before use,Artemiamust be “enriched” withn-3 HUFA prior to feeding to marine fish larvae and several different strategies can be employed (Dhertet al.,1998; McEvoy and Sargent, 1998). Phytoplankton are the natural food of rotifers andArtemia, and provide one method of enriching the animals. Judicious choice of algal species, including a marine Chlorellaor diatoms such asTetraselmis,which are rich in 20:5n-3, or prymne- siophytes such asNannochloropsissp. orIsochrysis galbana,which are rich in 22:6n-3, can readily increase the levels of both 20:5n-3 and 22:6n-3 in rotifers fed algae (Brownet al.,1989, 1992, 1997; Reitanet al.,1993, 1997; Tamura et al.,1993). Such procedures, however, are much less effective in increasing the 22:6n-3 levels ofArtemianauplii. Nonetheless, the use of “green water” in larval production systems, i.e., marine fish larvae cultured in the presence of one or more specific algal species together with rotifers initially andArtemia nauplii later, remains common practice (Navarro and Amat, 1992; Brown and Jeffrey, 1992; Reitanet al.,1993; Mourenteet al.,1993; Kashiwakuraet al., 1994; Olsenet al.,1997). The procedure can be effective and may involve nutritional and environmental benefits other than EFA nutrition. However, it remains to some extent empirical in that it is difficult to control the levels ofn-3 HUFA in both the algae and the live feed organisms, which can vary both with time and developmental stage, during culture of the larvae.
Early work supplemented rotifers andArtemianauplii withn-3 HUFA used baker’s yeast, which had been cultured in media containing fish oil, so-called ω-yeast (Sargentet al.,1989). In recent years, methods for directly enriching
Artemianauplii and rotifers usingn-3 HUFA-rich oils have been extensively developed (Dhontet al.,1991; Olsenet al.,1993; Coutteau and Sorgeloos, 1997; Coutteau and Mourente, 1997; Sargentet al.,1997; Dhertet al.,1998;
McEvoy and Sargent, 1998; Garaet al.,1998). The general enrichment pro- cess involves incubating theArtemianauplii or rotifers with a water-miscible preparation of ann-3 HUFA-rich oil for a relatively short period of time, so that the enriching preparation is ingested into the gut of the live animals, which are then immediately fed to the larvae. The procedure is, in effect, a “bioencapsulation” of the enriching oil (Coutteau and Sorgeloos, 1997) (see also Section 4.6). With rotifers it is also possible to use a long-term enrichment period which is combined with growth of the rotifer (Planas and Cunha, 1999). The most commonly used enriching preparations are micellar emulsions of marine fish oils and, as such, are basically triacylglyc- erol micelles (Izquierdoet al.,1992; Perezet al.,1994; Sargentet al.,1997;
Andoet al.,1997). Increased understanding of the lipid and EFA require- ments of larval marine fish in recent years has resulted in a trend toward the use of a speciality oil, tuna orbital oil, which has, respectively, high and low levels of 22:6n-3 and 20:5n-3 (Bell et al., 1996a; McEvoy et al.,1997;
Ando et al.,1997; Gara et al., 1998), and ethyl and methyl ester concen- trates of 20:5n-3 and 22:6n-3 (Takeuchiet al.,1992; Rainuzzoet al.,1994).
Phospholipid rich preparations have also been used, including triacylglyc- erol oils emulsified with marine and soya oil lecithins (Rainuzzoet al.,1994;
McEvoyet al.,1996, 1997; Tocheret al.,1997; Salhiet al.,1999). Commercial products designed to enrich live feeds for larval culture have included oil emulsions (e.g., Selco, SuperSelco INVE Aquaculture, Lochnsri, Belgium), microcapsules (e.g., Frippak Booster), and dried marine fungi (AlgaMac 2000 and 3000 Aquafauna Biomarine, Hawthorne, CA). The use of live feed enrichment procedures is now widespread both in research on nutritional requirements and in commercial marine larval fish production systems (Ostrowski and Kim, 1993; Mourente et al.,1993; Naesset al., 1995; Nery et al., 1995; Fernandez-Reirezet al., 1995; McEvoyet al., 1996; Blairet al., 1998a, b; Garaet al.,1998).
Using a combination of fabricated microdiets and, predominantly, en- riched live feeds, the EFA requirements of the larval and very early juvenile stages of a number of marine fish species have been determined (Table 4.1).
The exact levels of EFA reported can vary between studies, dependent upon the precise parameter measured, such as survival, growth, and vitality (Furita et al.,1996), as well as the overall dietary lipid level (Salhiet al.,1994). Even so, one overall impression of the data to date is that larval requirements for n-3 HUFA are generally greater than those of juveniles and preadult fish (cf. Table 4.2), with the caveat that there are relatively few species where the requirements at larval and later juvenile stages can be compared directly (Takeuchiet al.,1990, 1992c, 1996; Ibeaset al.,1994a, b; Rodriguez et al.,
1994, 1998a; Furuitaet al.,1996a; Salhiet al.,1999). A second impression is that the requirement of marine fish larvae for 22:6n-3 is usually higher than that for 20:5n-3, which means that the EFA requirements are usually satisfied by a lower level of 22:6n-3 than can be achieved with 20:5n-3 alone (Watanabe, 1993). This may also be the case with later juveniles, but again, direct comparative data are few, although with gilthead sea bream it ap- pears that the optimal dietary ratio of 22:6n-3 to 20:5n-3 in the larval stage is about 2 (Rodriguezet al.,1994, 1997, 1998a), whereas it is less than 1 in older juveniles (Ibeaset al.,1997). Red drum larvae fed a diet with a ratio of 22:6n-3 to 20:5n-3 of almost 4 exhibited significantly superior performance in a salin- ity challenge test (Brinkmeyer and Holt, 1998). Moreover, stress resistance was correlated with levels of 22:6n-3 but not with levels of 20:5n-3 or total n-3 HUFA in mahimahi larvae (dolphin fish,Coryphaena hippuus) (Kraul et al.,1993). One reason for the higher dietary requirement for 22:6n-3 is for rapidly developing visual and neural tissues, which account for a rela- tively greater proportion of total body mass in larval stages. The importance of 22:6n-3 for the proper development of these tissues has been demon- strated in larval herring (Navarroet al.,1993b, c; Bellet al.,1995a) and in sea bass (Navarroet al.,1997; Bellet al.,1996a). Thus, deficiency of dietary 22:6n-3 resulted in larval herring having an impaired ability to capture prey at natural light intensities, presumably due to impaired rod function in their eyes (Bellet al.,1995). The avidity of neural tissues for 22:6n-3 was shown by the dramatic increase in this fatty acid in the brains of both turbot and sea bream when larvae of both species were weaned from a diet deficient in 22:6n-3 to one rich in 22:6n-3 (Mourenteet al.,1991; Mourente and Tocher, 1992, 1993c). Therefore, the delivery of sufficient 22:6n-3 to developing marine fish larvae is of major importance and is not without problems. Spe- cific issues such as the peroxidation of 22:6n-3 in highly aerated live feed cultures (McEvoyet al.,1995) and its retroconversion to 20:5n-3 inArtemia (Evjemoet al.,1997; Navarroet al.,1999), both of which limit the ultimate level of 22:6n-3 that can be delivered to larvae by these techniques, remain to be solved.
In turbot, the early supply of 22:6n-3 was found to be essential for correct pigmentation (Reitanet al., 1994) and in Japanese flounder (Paralichthys olivaceus) pigmentation success was related to dietary 20:4n-6 and also to HUFA levels, including 20:4n-6, in neural tissues (Estevez and Kanazawa, 1996; Estevezet al.,1997). Subsequent work on turbot confirmed the essen- tiality of 22:6n-3 for normal pigmentation but also showed that 20:4n-6 levels in neural tissue lipids were negatively correlated with pigmentation and that the optimum dietary 20:5n-3 level was more dependent on the dietary 20:4n-6 than the dietary 22:6n-3 levels, indicating the importance of feeding the correct dietary ratio of 22:6n-3:20:5n-3:20:4n-6 (Estevezet al., 1999).
Arachidonic acid, 20:4n-6, had previously been shown to influence growth in larval gilthead sea bream (Rodriguez et al., 1994) and excess 20:4n-6 (4% of the dry weight ofArtemianauplii) was shown to inhibit growth and in- crease mortality in larval yellowtail (Ishizakiet al.,1998). In contrast, Zheng et al. (1996) found that dietary 20:4n-6 had no effect on the growth or vitality of larval cod. However, at a fixed dietaryn-3 HUFA level and fixed ratio of 22:6n-3 to 20:5n-3, a level of 20:4n-6 of up to 1.5 and 1% of the dry weight of the diet was found to improve growth, respectively, in larval sea bream (Bessonartet al.,1999) and larval Japanese flounder (Estevezet al., 1997). Therefore, there is increasing qualitative evidence pointing to the importance and probable essentiality of dietary 20:4n-6 for optimal growth and development of marine fish larvae (Rodriguezet al.,1994; Ishizakiet al., 1998; Estevezet al.,1999; Bessonartet al.,1999). However, there are as yet no hard data for the optimal quantitative requirements of 20:4n-6 for marine larval fish.
A possible alternative to enrichment of nutritionally deficient live prey such as rotifers andArtemianauplii is to use zooplankton species that nat- urally have more favorable PUFA and HUFA compositions (Virtue et al., 1995; Shansudinet al.,1997; Evjemo and Olsen, 1997). In recent years this has become an increasingly important area of endeavor. Zooplankton can either be harvested directly from the sea by filtration or be extensively cul- tured in ponds or tanks, or fish larvae can be introduced into seawater mesocosms enriched with nutrients to stimulate phytoplankton growth and thereby contain natural zooplankton in abundance. In all cases, there are problems associated with the use of essentially wild zooplankton in that their naupliar and early copepodite stages (which are the size required for ma- rine fish larvae culture) generally contain relatively low levels of total lipid (and therefore have a low energy content) whose fatty acid composition can vary seasonally, due largely to changes in the dominant phytoplankton prey species present. In addition, the use of wild zooplankton can intro- duce potentially pathological organisms. The problem of energy supply can be overcome by cofeeding with energy-richArtemianauplii and by feeding zooplankton at specific critical periods of development (Naesset al.,1995;
McEvoyet al.,1998). Cultured marine copepods have been successfully used to raise larval mahimahi (Kraulet al.,1993) and also in rearing turbot and halibut larvae, where they have been shown to improve pigmentation success significantly (Naesset al.,1995; McEvoyet al.,1998). The increased pigmen- tation was associated with higher levels ofn-3 HUFA in the zooplankton-fed larvae, suggesting that pigmentation in flatfish appears to be influenced by dietary fatty acids as well as overall nutritional status (Garaet al.,1998; Olsen et al.,1999). Recent pilot-scale intensive culture of the harpacticoid cope- pod,Tisbesp., was shown to be more successful than conventional rotifer
feeding for growth and development of larvae of the American plaice (Hippo- glossoides platessoides) (Nanton and Castell, 1998). However, Tisbe was less successful as a first feeding organism for larvae of haddock than rotifers (Nanton and Castell, 1998). This may have been due to the benthicTisbe being unavailable to the pelagic haddock larvae.