From rearing day 10 onwards in a recirculation hatchery, Daniels et al. (1992) used a supplemental diet, consisting of 85% cleaned squid, 10% chicken eggs, 3% beef liver powder and 2% cod liver oil bound with an alginate hardened by CaCl2. This semi-moist feed was separated through 250, 425, 600, 850 and 1000 | m sieves and partially air dried before use. In an effort to produce a more uniform feed particle, a field trial was conducted (M. Frinsko, pers. comm. 2008), which included cooking the squid (1kg for 40 min) in an 8 L stove-top pressure cooker. Cooking broke down the connective tissues, reducing the elastic quality of the meat. After cooling, the meat was removed and minced using a commercial spice grinder, producing a much smaller and more uniform ingredient. After mixing with the other ingredients, formation into patties, air-drying and grinding, the resultant microbound diet was significantly more uniform and consistent in size and shape. When administered, this diet was efficiently utilised by stage IV larvae as they were attracted to it and actively began consuming it immediately upon placement into the culture tanks. Particles of semi-moist feeds have a disadvantage because they tend to stick together, forming larger than desired particles. Another field test was conducted (M. Frinsko, pers. comm. 2008) to further improve particle size, separation and uniformity. Following the previously detailed incorporation of production changes, the diet was spread out on trays and then placed on racks in a conventional electric oven. After drying at 60°C for 15 minutes, the moisture content was reduced to approximately 5%. The feed was then ground as a dry product, which was effectively sieved to sizes as needed. This 'dry' feed was consumed well by the larvae and was fed at smaller particle sizes than normal due to water absorption caused from the alginate in the diet. This diet remained buoyant or semi-buoyant for approximately 15 minutes, eventually settling to the tank bottom.
Bell (1994) postulated that the problem for early feeding stages did not reside in nutritional inadequacy of the micro-encapsulated diets (MED) but rather a lack of assimilation capacity. Unlike penaeid shrimp, first-feeding freshwater prawn larvae do not have anterior midgut di-verticulae, which are the main sites for digestive enzyme production during the early larval stages in marine shrimp (Lovette & Felder 1990; Abubakr & Jones 1992). The hep-atopancreas (midgut gland) of freshwater prawns is also relatively small during early larval stages. A considerable increase in the level of trypsin activity coincides with the expansion of the hepatopancreas after stage V (Jones et al. 1993), when larvae are able to survive on formulated diets (Deru 1990). On the other hand, Kamarudin (1992) found that amylase levels in the herbivorous protozoeal stages of Marsupenaeus japonicus were much higher than those observed in the early stages of carnivorous shrimp larvae. Specific amylase content in M. rosenbergii larvae can only be observed from stages VI to VIII to PL, suggesting that prawn larvae become less exclusively carnivorous as they grow (Kamarudin et al. 1994). Thus, they may have specific nutritional requirements during the first-feeding period. It is postulated that in previous experiments (Ohs et al. 1998) egg albumen may have been a less digestible protein ingredient than assumed. Kovalenko et al. (2002) fed a high moisture micro-bound diet exclusively to stage V to XI of M. rosenbergii. After 14 days, larval growth using this diet was 90% of that found for larvae fed Artemia nauplii exclusively. The greater assimilative efficiency of this diet compared to that of Ohs et al.(1998) maybe due to the higher digestibility of casein or the egg yolk protein, which was not present in the earlier tests.
Additional problems could be attributed to ingestion and gut retention time. Deru (1990) found that early larval stages of M. rosenbergii rapidly accept and consume microcapsules; however, Kumlu (1995) demonstrated that after a short period of consumption, the microcapsule is totally rejected. Sick & Millikin (1983) examined the effect of adding selected attractants to a porous gel diet consisting of fish, soybean and shrimp meals on M. rosenbergii. These workers found that the addition of ovalbumin, in combination with certain amino acids (proline, arginine, glycine, taurine), resulted in significantly higher ingestion rates by all larval stages. However, Teshima et al. (2000) stated that protocols to test rates of ingestion may need re-evaluation. These authors indicated that use of inert marking tools, such as cholestane, dotriacontane and other long chain hydrocarbons would improve the accuracy of the data. Daily data collection was recommended to derive more effectively meaningful experimental results, i.e. to ensure that nutrients are being consumed at known rates.
Early attempts at exclusive replacement with MED throughout the larval cycle have generally not been effective.
Table 6.6 Ingredient composition (% dry weight) and results of proximate analysis of the experimental microbound diet. (Reproduced from Kovalenko etal. 2002, copyright 2002 with permission of Elsevier.)
Ingredient Amount (%)
Egg yolk 38.45
Casein (vitamin free) 14.69
Fish protein hydrolysate 15.38 Rice starch 7.69 Refined soy lecithin 1.92 Wheat gluten 3.85 Menhaden oil 5.63 Canthaxanthin (10%) 2.31 Cholesterol 0.12 Ascorbyl palmitate 0.04 Vitamin premix BML #2a 1.15 Betaine 0.15 KH2PO4 1.15 Choline chloride 0.38 Mineral premix 1.54 Glucosamine 0.15 Alginate 5.38
Lipid 37.4 Ash 5.6
Nitrogen free extract (NFE) (by difference) 10.9
a Composition not specified.
Ohs et al. (1998) evaluated a spray-dried artificial diet for prawns and found that the survival and growth rates of larvae were poor when larvae were fed the artificial diet exclusively. Deru (1990) demonstrated that M. rosenbergii larvae could survive on MED from zoea VI to VII to metamorphosis. This finding was observed again at stages VI to VII to PL by Barros and Valenti (2003a) when particle sizes of the wet diet ranged from 250 to 1190 ^m.
Kovalenko et al. (2002) developed and tested a micro-bound, micro-particulate formulated diet that served as the first complete replacement for live Artemia nauplii from stage V through PL. Survival and growth achieved with the micro-bound diet were similar to that achieved with live food. The formula for this diet is found in Table 6.6. In 2007, a few kilograms of a micro-bound diet described by Kovalenko et al.(2002) were produced as a dry feed (12% moisture) using a proprietary manufacturing protocol (L.R. D'Abramo, pers. comm. 2008). During a 30 day culture period, this semi-purified diet was fed exclusively to larvae from stage II to X, in a clearwater recirculating culture system (M. Frinsko, pers. comm. 2008). Each ofthree separate 750 L tank systems were stocked with stage I larvae at 125/L. This field trial was conducted at a commercial hatchery. In the absence of Artemia supplementation, projected growth
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