Broodstock and larval nutrition in successful aquaculture

Introduction

The future of aquaculture production, as in other animal production systems, is towards greater control of the physical, chemical and biological variables surrounding the production system. The trend from collection of wild fry to the development of hatcheries for fry production, from the use of wild spawners to the use of maturation systems and ultimately closing the reproductive cycle to use domesticated and selected stocks is clear. At the same time, there is an increasing trend towards production efficiency and cost-effectiveness, not least in the hatchery stage, particularly where the supply – demand situation progresses from quantity production to quality production. These trends are becoming more evident in all forms of commercial aquaculture.

Broodstock and larval nutrition are key elements underpinning this progress towards greater control and domestication. However, there is substantial work to be done if rearing of aquaculture species is to approach the level of control and understanding which are evident in the poultry, swine and ruminant sectors.

This paper gives a brief introduction to some of the key areas and issues in broodstock and larval nutrition.

Constraints of time and space mean that the paper is largely confined to crustacean, and particularly, penaeid shrimp culture. However, many of the same gaps are found when looking at the broodstock and larval nutrition of most fish species.


Broodstock nutrition

Research into shrimp broodstock nutrition is gaining importance with the increasing use of domesticated and genetically selected stocks for aquaculture. Also,given the high cost of feed in a captive broodstock maturation facility, estimated at around 50% of total cost, as well as a heavy reliance on fresh feed items such as squid, mollusk meals and polychaete worms which may vary in quality and availability, there is a demand for feeds specifically formulated for use in maturation diets. It has also been pointed out (Doyle, personal communication) that the development of genetically improved lines of shrimp may benefit from a simultaneous development of feeds specifically tailored to individual strain requirements.

During penaeid shrimp maturation, nutrient reserves, mainly from the hepatopancreas, are mobilised to support ovarian and testicular maturation, gametogenesis and vitellogenesis (Harrison, 1990; 1997). Tissue reserves in the hepatopancreas can be depleted rapidly so that the diet becomes the most important contributor of nutrients to the developing egg.

This is particularly true when, as is the case in most captive maturation facilities, eyestalk ablation is used to accelerate the process of gonadal maturation. The hormonal and metabolic changes that come around during such forced maturation may take place when the nutrient reserves are insufficient to support rapid ovarian development placing an even larger burden on the diet as a source of essential nutrients.

The nutrition of penaeid shrimp broodstock has recently been reviewed (Wouters et al., 2001a). The authors recognize that there is a dearth of information in the scientific literature on the subject, possibly due to the expense and complexity of running sufficiently rigorous nutrition experiments on shrimp broodstock. In addition, much of the research has been carried out with fresh feeds, either alone or in combination with formulated diets or dietary supplements.


LIPIDS

Due to the importance of lipids in crustacean maturation, much of the work carried out to date has focused on this aspect, particularly the requirement for highly unsaturated fatty acids (HUFA) and phospholipid. During maturation, lipids are mobilized from the hepatopancreas in many species and dietary lipids rapidly processed for transport to the developing ovaries.

Total lipid does not appear to be important although Wouters et al. (2001b) reported that excessively high total lipid in the diet had an adverse effect on ovarian maturation and feed consumption, possibly due to satiation. However, the average lipid level in commercial broodstock diets (10%) appears to be around 3% higher than in grower feeds used in commercial culture ponds.

Highly unsaturated fatty acids (HUFA), especially 20:5n-3 and 22:6n-3, are abundant in ovarian tissues and are believed to be an important component of live and formulated maturation diets. Diets deficient in n-3 HUFA have been found to have a negative effect on ovarian development, fecundity and egg quality (Teshima et al., 1988; Alava et al., 1993; Xu et al., 1992; 1994; Cahu et al., 1994; Wouters et al., 1999a).

Arachidonic acid (20:4n-6; AA) has been detected at high levels in the ovaries of wild shrimp and is also abundant in some of the best fresh feed items such as polychaetes (bloodworms), clams and mussels (Harrison, 1997; Wouters et al., 2001a). The n-6 HUFA are known to be precursors of the prostaglandin hormones, which act in reproduction and vitellogenesis. According to Wouters et al. (2001a), formulated maturation diets appear to be deficient in AA as well as relatively low eicosapentanoic acid (EPA) levels. It has been proposed that the ratio of n-3 to n-6 levels in the diet is important (Lytle et al., 1990) and that it should be around 2–3 to 1 (Ravid et al., 1999; Wouters et al., 1999b).

Phospholipids, mainly phosphatidylcholine and phosphatidylethanolamine appear to be predominant in the shrimp ovary and there seems to be a requirement for phospholipids in the diet. Several studies (Alava et al., 1993; Cahu et al., 1994; Ravid et al., 1999; Wouters et al., 1999b) have demonstrated the effects of phospholipid levels in the diet and it has been suggested that broodstock diets should contain more than 2% phospholipid to ensure that 50% of total egg lipid is in this form (Cahu et al., 1994).

Cholesterol is the precursor of steroid hormones and it is known that shrimp have a requirement for cholesterol in the diet. Cholesterol is stored in the hepatopancreas and is mobilized during maturation. The role and mobilization of cholesterol during shrimp maturation has been reviewed by Harrison (1990). Some of the live feed organisms used in maturation diets have relatively high cholesterol levels (e.g. squid, clams) although to date there has been limited research into the effects of dietary cholesterol on maturation and reproduction (Harrison, 1997; Wouters, 2001).

During maturation, the level of triacylglycerides (TAG) in the ovaries increases as they are incorporated into the egg and decrease after spawning (Ravid et al., 1999; Wouters et al., 1999b). Triacylglycerides appear to be the principal energy source in eggs and nauplii and their importance in reproduction, and egg and postlarval quality has been shown (Palacios et al., 1998; 1999).


PROTEIN AND AMINO ACIDS

Maturation is a time of intense protein synthesis and it is likely that the requirement for protein is higher at this time (Harrison, 1990; 1997). Wouters et al. (2001a) report that the protein content of formulated diets in their studies was around 50% but that this was still low compared to the level in fresh feeds.

Detailed studies into protein requirements for shrimp broodstock are still lacking and it has been proposed that the amino acid profiles should mimic those found in fresh feeds (Deshimaru, 1982).

Some studies have shown changes in protein content of the ovaries associated with egg development and spawning, and with spawning success. Animulkar (1980), reported in Harrison (1997), found an increase in ovarian protein levels associated with ovarian development followed by a sharp decrease after spawning in the shrimp Paratelphysa hydrodromaus and several authors have noted a similar increase in farmed penaeid shrimp (Read and Caulton, 1980; Castille and Lawrence, 1989). A marked difference has also been noted in the protein content of the hepatopancreas and ovaries of wild and domesticated females of Litopenaeus vannamei with good repeat spawning performance which have been found to have significantly higher protein content than females with poorer spawning performance (Palacios et al., 2000).


CARBOHYDRATES

Carbohydrates do not appear to be essential for shrimp broodstock diets although Palacios et al. (1998; 1999)
related egg glucose levels with larval quality and broodstock condition. Carbohydrates can be used as cost-effective ingredients to contribute to glycogen accumulation in the hepatopancreas (Harrison, 1997) as well as providing other benefits in the broodstock diet, acting as binders and possibly playing a role in transport of nutrients in the hemolymph (Harrison, 1997).


VITAMINS AND MINERALS

Detailed vitamin and mineral requirements for shrimp broodstock diets are relatively unknown with only a few studies on vitamins A, C and E. Alava et al. (1993) found that ovarian maturation was slower when fed a diet deficient in either vitamins E, A and C. Vitamin E appears to be important in crustacean broodstock nutrition.

Chamberlain (1988), reported in Harrison (1997), found a correlation between vitamin E deficiency and the percentage of abnormal sperm in Litopenaeus setiferus and Cahu et al. (1991) found an improvement in hatching rate with increasing dietary vitamin E correlated to increasingα−tocopherol levels in the egg.

Wouters et al. (1999b) found a similar correlation to that observed by Cahu et al. (1991) between spawn and hatch quality with α-tocopherol levels in wild spawners and nauplii of L. vannamei. They found that mature
ovaries and nauplii contained higher levels of α−tocopherol than immature and spent ovaries. Harrison (1997) also speculated that vitamin E in the egg yolk may also act as a natural antioxidant.

Work conducted by Fisher and Kon (1958), reported in Harrison (1997), suggested the importance of dietary vitamin A due to its accumulation in the ovaries of crustaceans during maturation. Vitamin C content of eggs of Fenneropenaeus indicus are also influenced by the levels in the diet, and high hatching rate has been related to high ascorbic acid levels in the eggs (Cahu et al., 1995). Harrison (1997) assumes that vitamin D is important in broodstock diets due to its probable role in calcium and phosphorus metabolism in crustaceans.

Harrison (1990) discussed the possibility that mineral deficiencies or imbalances could have a negative impact on crustacean reproduction and may play a role in oocyte resorption, reduction in reproductive performance and egg quality. Studies into mineral requirements are rare due to several complications (Wouters et al., 2001a). Where studies have been conducted, diets were formulated with mineral mixes with added calcium, phosphorus, magnesium, sodium, iron, manganese and selenium (Chamberlain, 1988; Marsden et al., 1997; Mendoza et al., 1997; Xu et al., 1994).

Spent broodstock of L. vannamei had lower levels of calcium and magnesium in the muscle and lower magnesium levels in the hepatopancreas (Mendez et al., 1997), possibly due to a combination of dietary deficiencies and losses through moulting and transfer to the eggs. Copper also decreased in the hepatopancreas, possibly through transfer to the ovaries, although it increased in the muscle tissue. It is clear that more studies need to be undertaken into mineral nutrition in broodstock diets.


CAROTENOIDS

Crustaceans cannot synthesise carotenoids de novo, and a dietary source of these pigments is required. During sexual maturation, most crustaceans accumulate carotenoids in the hepatopancreas and during vitellogenesis, these are transported in the hemolymph as carotenoglycolipoproteins to accumulate in the eggs as part of the lipovitellin protein. Dall (1995) found that free astaxanthin levels in the developing ovaries of Penaeus esculentus increased from 2 to 34 ppm and in the digestive gland, from 20 to 120 ppm.

Carotenoids, especially astaxanthin, are strong antioxidants and probably play a role in protecting the broodstock nutrient reserves and developing embryos from oxidation (Dall et al., 1995; Merchie et al., 1998). It has also been suggested (Harrison, 1997) that they act as pigment reserves in the embryos and larvae for the development of chromatophores and eyespots, and as a vitamin A precursor (Dall, 1995).

Wyban et al. (1997) noted a decrease in nauplius quality with successive spawns associated with a loss of pigmentation in the ovary of L. vannamei. Addition of paprika, an inexpensive source of carotenoids, to the fresh diet at a rate of 2% (2 g paprika per 100 g squid meat), resulted in a significant improvement in nauplius quality (measured as survival to zoea 2 stage).

Pangantihon-Kühlmann and Hunter (1999) found that astaxanthin supplementation (50 mg/kg) of the diet resulted in increased egg production in Penaeusmonodon but could not demonstrate any benefit of astaxanthin supplementation on either hatching rate or metamorphosis to zoea 1 stage.


MISCELLANEOUS FACTORS

Hormones

It has been suggested that some of the more successful live feed organisms may provide benefits through the provision of hormones or their precursors. Naessens et al. (1997) speculated that part of the reason for the success of reproductive adult Artemia biomass supplementation of the diet of L. vannamei broodstock could be due to the presence of specific hormones or analogous peptides in the Artemia that provoked a response in the shrimp. Bloodworms used in maturation have also been found to contain methyl farnesoate, an ecdysone hormone that has been shown to increase reproductive performance in the spider crab Libinia emarginata (Laufer et al., 1987), L. vannamei (Laufer et al., 1997), P. monodon (Hall et al., 1999) and the crayfish Procambarus clarkii (Laufer et al., 1998). In P. clarkii, the hemolymph titer increased from basal levels during early vitellogenesis, peaked during mid-cycle and then returned to basal levels when the ovaries were in late vitellogenesis.


Nucleotides

Nucleotides, the basic building blocks of nucleic acids, are recognised as important elements in mammalian nutrition especially during periods of rapid growth or physiological stress (Uauy, 1989; 1994; Barness 1994; Van Buren, 1994) and also appear to play a key role in the immune system. Traditionally, nucleotides have not been considered essential nutrients although de novo synthesis and salvage pathways are thought to be costly processes in metabolic terms. Several studies have demonstrated that dietary sources of nucleotides can have beneficial effects and the term‘conditionally essential’ has been used to describe their role in nutrition (Carver and Walker, 1995).

Exogenous sources of nucleotides are thought to optimise the functions of rapidly dividing tissues, such as those of the developing embryo and young, and the reproductive and immune systems.

Most aquaculture diet ingredients of animal and plant origin contain nucleotides although there are differences in the concentration and availability. Nucleotide content is particularly high in ingredients such as fish solubles, animal protein solubles, fishmeal, legumes (adenine is particularly high in blackeyed peas), yeast extracts and unicellular organisms such as yeasts and bacteria that are rich in RNA or DNA (Carver and Walker, 1995; Devresse, 2000).

Devresse (2000) noted that the low digestibility of whole yeast compared to yeast extract may be related to the protein (nitrogen) solubility as yeast extract has much higher protein solubility than whole yeast. He also noted that, although fish solubles are highly digestible, they leach easily in water affecting availability.

Reproduction and egg development have a high requirement for RNA and DNA and it may be expected that increasing the availability of nucleotides in broodstock diets may have a beneficial effect on egg development. Recently, research has demonstrated the effect of a nucleotide-enriched diet for broodstock nutrition in aquaculture (Gonzalez-Vecino, 2002; Gonzalez-Vecino et al., 2003).

Nucleotide enrichment of broodstock diets for Atlantic halibut (Hippoglossus hippoglossus) and haddock (Melanogrammus aeglefinus) resulted in a general trend towards better spawning performance and egg quality with the nucleotide diet. Total egg yield was 30% higher in the halibut fed with the nucleotide diet and the relative fecundity, mean egg density, hatching rate and survival of yolk-sac larvae were also significantly improved. Haddock fed on the nucleotide-enriched diet also had significantly higher fertilization and hatching rates. To date, no work has been published on nucleotide supplementation of broodstock diets for shrimp but it would be interesting to conduct some trials to determine if broodstock diets enriched with nucleotides might offer similar benefits in shrimp maturation and breeding. Similarly, the potential for nucleotide supplementation of diets for shrimp larvae should also be investigated.


Larval feeds

Typically, marine larvae are fed with live feeds such as algae, zooplankton, rotifers and Artemia. In some cases, especially with rotifers and Artemia, the fatty acid profile is inadequate (Léger et al., 1986), especially with regard to the HUFA profile. The practice of enrichment has been developed as a means of overcoming this nutritional deficiency.

Enrichment usually involves enhancing the docosahexanoic acid (DHA) and EPA content of the natural feed through ‘bioencapsulation’. This involves feeding the live organism with a DHA/EPA enriched formulation to boost the levels in the tissues and then feeding the enriched organism to the larvae.

In the case of shrimp larvae, the use of enriched Artemia is restricted to the post-larval stages due to the size of the first feeding instar stages of the Artemia (Sorgeloos et al. 1998).


FORMULATED FEEDS

Although live feeds provide an excellent source of nutrition, there are several drawbacks associated with their use. Algal cultures require considerable expertise to maintain them in peak nutritional condition and facilities for their mass production can be expensive to operate. Rotifers also require considerable expenditure in time and effort to maintain, especially if they, in turn, need to be provided with live feed.

Live Artemia nauplii suffer from inconsistent supply and quality as they are obtained from cysts collected in the wild environment. The bulk of cysts come from the Great Salt Lake in Utah in the US where annual fluctuations have been shown to cause wide fluctuations in yield. As a result, price and quality can vary unpredictably (D’Abramo, 2002).

Such problems with live feeds have led to the development of diets specifically formulated for their replacement. However, the development of formulated larval diets to completely replace live feeds has been an elusive goal, despite considerable effort (Langdon, 2003). The difficulties inherent in providing a complete nutritional package in a sufficiently small particle to be ingested and digested by the small larvae of many marine species are clear.

Loss of nutrients from such diets can be rapid and result in loss of nutritional value and fouling of the culture medium and should be used to determine the effectiveness of microparticulates as nutrient delivery systems.

On the other hand, provision of a sufficiently impermeable coat to prevent leaching may result in poor digestibility and availability of the nutrients to the developing larvae. Microbound and cross-linked protein-walled capsules may be used to deliver lipids and high-molecular weight, water-soluble nutrients such as proteins and carbohydrates whereas lipidbased particulates, including liposomes, could be useful in delivering low-molecular weight, watersoluble nutrients, such as amino acids and water-soluble vitamins.

Although the use of formulated larval feeds as partial replacements for microalgae is common in commercial hatcheries, total replacement of algae has proved to be more difficult. Complete replacement has only been achieved using ocean quality seawater that is partly filtered to retain the natural bacterial community (Ottogali, 1991; 1992) and reports of complete replacement in commercial hatcheries appears restricted to those located in the oceanic waters of the Pacific islands (Chim, 2003). Alabi et al. (1997) also showed that total replacement requires the establishment of a balanced bacterial community from either the filtered seawater or following conditioning by microalgae. As a result Jones et al. (1997; 1998) suggested the inoculation of a single dose of live algae (SDLA) before use of artificial feeds to condition hatchery water when it is taken from coastal water of variable bacterial quality.

Wouters and Van Horenbeek (in press) summarize the various types of commercial larval feeds available in the market. These include microbound diets, flakes, granulated feeds, microencapsulated feeds, liquid feeds (lipid-walled capsules).

Microbound feeds are bound using a variety of different binders and produced as a small particle, or as a pellet, cake or flake, which is then crumbled to the appropriate size. They are inexpensive to produce but leach rapidly. Crumbles are usually used during postlarval stages.

Flakes are commonly used in Asia and the Americas. Dietary ingredients are added to water to obtain a dense soup. An appropriate binder is added and the resulting suspension is sprayed onto a steamdrum dryer.

Temperatures can exceed 100°C. and significant nutrient loss can occur unless passage times are kept short.

Large flakes can be crushed and passed through an appropriate mesh screen immediately prior to use. They are generally used for the postlarval stages of the shrimp.

Granulated feeds are produced using liquid binder and water sprayed onto the feed mix, resulting in granules with a raspberry-like structure.

Microencapsulated feeds have an outer coat (capsule) that retains the ingredients inside the particle. They can be designed to have a slow release of the material or to totally prevent leaching of watersoluble nutrients.

Some techniques encapsulate using a cross-linked protein-wall that can be digestible yet capable of  withstanding drying.

Liquid feeds are essentially a slurry of particles in a suspension medium. Although expensive, they are claimed to cause less fouling and can be continuously dosed into larviculture tanks using peristaltic pumps.


CONSIDERATIONS IN DEVELOPMENT OF FORMULATED LARVAL FEEDS

The nutrition of marine larvae involves an understanding of the behavioural, mechanical and physiological processes of feeding in the target animal.

These are likely to be very different in the larval stages compared to the adult form. Feeding habits in many species show a distinct change as the larvae develop. In penaeid shrimp, many species change from a primarily herbivorous diet in the zoea stages to a more omnivorous diet in the postlarval stages (Lemos and Phan, 2001). During the postlarval and early juvenile stages, further changes may involve a switch to a more carnivorous or detritivorous diet depending on the species. There are also changes from a planktonic existence to a benthic one and from a filter feeder to an active predator to be considered, all within a few days of hatching.

One of the key considerations is the development of the gut structure and function. Larval crustaceans have a simple gut structure, which gradually becomes more complex. The physiology of the gut and gut enzymes also changes and, since transit times may be quite short (Jones et al., 1997; Jones and Kurmaly, 1987), designing a nutritious, easily digestible diet is a challenge.


Digestive physiology of marine larvae

The development of the digestive system of marine larvae plays a fundamental role in larval nutrition. In particular the development of gut function and ontogenetic changes in enzyme function are of critical importance.

The structure of the larval gut in crustaceans has been studied in only a few species (Factor, 1981; Lovett and Felder, 1989; 1990a; 1990b; Abubakr and Jones, 1992). The development of gut structure appears to follow similar patterns in most of the species studied. The gut of early larval stages of penaeids is relatively simple and the gastric mill and hepatopancreas are either not present or not yet functional. Feed is mixed with enzymes through expansion and contraction of the midgut gland and there is free fluxing of food between the midgut gland and the midgut, especially in time of food scarcity (Lovett and Felder, 1990b). As the larva grows, the hepatopancreas proper develops, the lobes increasing in number and elongating during the mysis stages.

At this point, the rudimentary gastric filter begins to develop. The postlarval stages are marked by the rapid development of the hepatopancreas, which increases in size and complexity, and the development of the gastric mill and gastric filter to the adult form.

Until the gastric mill becomes functional, the shrimp larvae are restricted to feed items that are relatively easily digestible. In the case of the herbivorous stages, they generally filter the feed from the water and digestion takes place in the gut. As the appendages and mouthparts become stronger and more complex, larger feed particles and live prey are held and torn apart before ingestion, rendering them more easily digestible.

There has been relatively little research into the digestive enzymes of crustacean larvae. Jones et al. (1997) noted that larval enzymes differ in range and level of activity from those that are present in adults and that there exist species differences which may relate to the feeding strategy of the larvae. There are also changes in enzyme activity patterns between different larval stages and, overlaid on these, the possibility of diet-induced changes in enzyme levels.

In a brief review of research into enzyme activity of decapod larvae, Jones et al. (1997) reported that all appeared to have strong protease activity, predominantly trypsin, non-specific esterase activity and amylase. In the few reports available, pepsin appears to be absent in larval and most adult decapods (Glass et al., 1989) although it has been reported in the freshwater prawn, Macrobrachium rosenbergii (Lee et al., 1980). Similarly, lipase activity was only reported in larval stages of the black tiger prawn, Penaeus monodon and the lobster, Homarus americanus although this may be open to debate.

Collagenase, elastase and chymotrypsin all seem to be rare or absent in larval stages.

Changes in enzyme activity patterns with larval stage have been clearly demonstrated in penaeids (Lovett and Felder, 1990a, b; Glass et al., 1989; MacDonald et al., 1989; Ribeiro and Jones, 2000; Ngamphongsai, 2000; Puello-Cruz et al., 2002). MacDonald et al. (1989) noted that the general pattern of amylase, protease and lipase in P. monodon larvae was similar with a maximum of enzymatic activity at zoea 3, possibly coinciding with the change from predominantly filter feeding to mechanical digestion.

They also noted a minimum level of all three enzymes in the early postlarval stage, similar to that noted by Lovett and Felder (1990a) (the so-called ‘enzyme crisis’), which was attributed to a change in gut development associated with changing feeding habits as the postlarvae become more benthic. This coincides with a critical period in postlarval development whenpoor postlarval condition and high mortality may be observed in commercial hatcheries. Puello-Cruz et al. (2002), on the other hand, found that trypsin levels in Litopenaeus vannamei larvae were significantly highest at the Zoea 1 stage, and declined thereafter to the PL1 stage. They also noted that trypsin content in zoea 2 and zoea 3 stage larvae feeding on Artemia was significantly lower than in those feeding on algae and suggested that L. vannamei may be physiologically adapted to transfer to a more carnivorous diet during the zoeal stages than more herbivorous species such as P. monodon or P. indicus (Ngamphongsai, 2000). This accords well with commercial hatchery observations that L. vannamei is capable of feeding on Artemia from the zoea 3 stage whereas P. monodon larvae do not become fully capable of feeding on live Artemia until at least mysis 2 or 3. Changes in the sequence of enzyme activity consistent with a carnivorous diet were also noted in early larval stages of Macrobrachium rosenbergii (Kamarudin et al., 1994).

Several authors have suggested that fish larvae may benefit from the assistance of exogenous digestive
enzymes of their live food organisms, either through autolysis or by the action of zymogens that activate larval endogenous digestive enzymes (Kolkovski et al., 1993; 1997; Munilla-Moran et al., 1990) and the same may hold for crustacean larvae.


LIPIDS

Much work on larval feeds has centred on the lipid requirement, in particular the requirement for highly unsaturated fatty acids (HUFA) and phospholipids.

The n-3 highly unsaturated fatty acids (HUFA) docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) are essential for normal growth and development of many species of marine fish and crustaceans (Sargent et al., 1999; Jones et al., 1997; Suprayudia et al., 2004). Marine fish are also unable to synthesize arachidonic acid (20:4n-6; AA).

Quantitative requirements for essential fatty acids have been rarely reported for larvae of penaeid shrimp. It has been suggested (Kanazawa et al., 1979, in González-Félix and Pérez-Velazquez, 2002) that 1% n-3 HUFA in the diet could be considered as a minimal value for postlarvae although Chen and Tsai (1986) indicated a requirement for HUFA at 0.5-1% of the diet for P. monodon and Xu et al. (1994) suggested a requirement of between 0.7% and 1% of the diet in Fenneropenaeus chinensis.

The dietary effects of phospholipids on fish and crustacean larvae have been reviewed by Coutteau et al. (1997). Although there appears to be little doubt that crustacean larvae can synthesise phospholipid from HUFA, the addition of dietary sources of phospholipid has been shown to be beneficial, possibly through enhancing the absorption of dietary cholesterol and triacylglycerols (Jones et al., 1997).

Gonzalez-Felix et al. (2000), for example, demonstrated that L. vannamei postlarvae fed diets containing phospholipid demonstrated improved growth and enhanced muscle phosphatidylcholine and phosphatidylethanolamine concentration. Coutteau et al. (1996) however, showed that the other phospholipids in lecithin could not compensate for phosphatidylcholine deficiency in the diet of postlarval L. vannamei and that, as dietary phosphatidylcholine increased, significantly higher levels of total HUFA, 20:1n-9 and 20:5n-3 were present in the shrimp.

In experiments carried out with larval P. japonicus, optimal metamorphosis was obtained with diets containing 15-30 g/kg soybean phosphatidylcholine and the lower level (15 g/kg) was more beneficial to postlarvae (Camara et al., 1997). Results also suggested that there was no requirement for phosphatidylethanolamine or phosphatidylinositol in the presence of adequate dietary phosphatidylcholine. The phospholipid requirement of crustacean larvae from this study appears to be within the range of 1-3% given by Coutteau et al. (1997) as the range for most marine larval species and agrees with the earlier figure of 3% given by Kanazawa (1990).

As with adult crustaceans, larvae are unable to synthesise cholesterol and have an absolute requirement for cholesterol in the diet (Teshima et al., 1983).


PROTEIN AND AMINO ACIDS

The optimal dietary protein level in a larval diet can be expected to vary with species, larval stage (Durruty et al., 2002), protein source, digestibility (Le Vay et al., 1993) and amino acid composition. Kanazawa (1990) recommended protein levels in larval feeds between 23% and 57%. Durruty et al. (2002) reported differences in protein requirement according to the larval stage of L. vannamei and L. setiferus. They estimated that protein requirement increased from 30% in the zoea stages up to 50% or 60% for mysis stages. Data on optimal protein:energy ratiosor amino acid profiles for larval shrimp feeds have not been reported (Wouters and Van Horenbeek, in press).

The essential amino acids for shrimp are thought to be methionine, arginine, threonine, tryptophan, histidine, isoleucine, leucine, lysine, valine and phenylalanine (Akiyama, 1992). There is no reason to believe that shrimp larvae have any specific amino acid requirement that differs from the adult shrimp (Jones et al., 1997). Although no single protein source appears to satisfy the complete requirements for amino acids (Wouters and Van Horenbeek, in press), Cahu (1999) stated that lower nutritional value protein sources can be fortified by the addition of essential amino acids (Cahu, 1999).


CARBOHYDRATES

As with adult crustaceans, there appears to be no specific requirement for carbohydrates in the diet. Carbohydrates can be used to reduce feed costs through protein or lipid sparing and are frequently used as binders in larval feeds.


VITAMINS AND MINERALS

Fat-soluble and water-soluble vitamins as well as carotenoids are essential for shrimp larvae (Wouters and Van Horenbeek, in press). Kanazawa (1986; 1990) determined the vitamin requirements for P. japonicus (Table 1). These levels provide a practical baseline, but Kanazawa (1990) also admits that they may be affected by leaching of the vitamins from the test diet. In practice, formulated feeds for shrimp larvae will contain a complete vitamin and mineral premix as used in feeds for older shrimp.





Kanazawa’s work on vitamin C requirements was conducted using sodium ascorbate. More recently, vitamin C has been available in the form of Lascorbyl-2-polyphosphate. Although there is little work on larval nutrition using this form of the vitamin, work on the early postlarval stages of L. vannamei has shown that high dietary levels (40 mg/kg)can increase resistance to salinity stress. The benefit of elevated dietary vitamin C levels in
increasing stress and disease resistance has also been noted by other authors (Kontara et al., 1997; Merchie et al., 1997; 1998).


OTHERS

Nucleotides have demonstrated much potential when fed to the young or juvenile stages of vertebrates (Carver and Walker, 1995). The benefits of adding dietary nucleotides to feeds for the rapidly developing larval stages of crustaceans would appear to be clear. However, there are no research data available on the application of exogenous nucleotides for crustacean larval culture.

Hatchery operators often use a mixture of additives in a typical feeding regime including additives such as immune stimulants or probiotic bacteria. There is still little or no scientific data to support the use of such additives in shrimp larval culture.


Conclusion

The current state of knowledge of broodstock and larval nutrition of crustaceans contains many gaps. This is partly the result of the complexity and expense of conducting research into these two areas. However, given the increasing importance of domestication in shrimp aquaculture particularly, there is a need for an increased focus on these areas. The role of nutrition in broodstock and maturation performance and in increasing larval survival and quality will be fundamental to obtaining optimal performance from domesticated stocks. Even in species where domestication is not an issue, the improvement of maturation performance and larval production remains a key goal in improving the efficiency of production systems.

It is not clear how far the goal of complete replacement of live feeds may be. It is likely that this will be attained in broodstock diets long before larval feeds. Indeed, given the complexities inherent in supplying a complete nutritional package in a small particle, it may be that this goal is never reached.

However, it may be possible to supply a range of diet particles (e.g. high lipid particles, high protein particles, carbohydrate particles etc.) that are aimed specifically at providing the right mix of nutritional elements in the culture tank that will expose the larvae to the appropriate nutritional mix. Alternatively, it may be that, as expressed by D’Abramo (2002), the complexity of the ontogeny of larval nutritional physiology may mean that technical success will be based on a compromise between the desire to provide a complete diet package and the need to strive for simplicity in formulation and manufacture.


Acknowledgements

I would like to acknowledge the debt owed to Roeland Wouters of INVE Technologies, Belgium and Prof. Patrick Sorgeloos of the University of Ghent for their kind help and provision of access to their publications for use in writing the paper.


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