Exogenous nucleotides, i.e. IMP, AMP and GMP, are flavour enhancer and commonly used as chemoattractant in fish feed (Mackie 1973; Kiyohara et al. 1975; Ishida & Hi-daka 1987). In the present study, the feeding rate was fixed (3% of body weight) and all fish finished their ration within 1-2 min after fed. Thus, the flavour-enhancer effect of nucleotides for grouper should not be a factor.

Phagocytes possess a unique membrane enzyme, NADPH oxidase, capable of oxidation of molecular oxygen into superoxide anion (O2) during a process termed the respiratory burst. Superoxide anion production is considered to be one of the most important microbicidal components in the armoury of phagocytes (Secombes 1990). The immunoglob-ulin (Ig) constitutes the antibodies that are used in the immune defense against pathogens in all vertebrates (Pilstrom & Bengten 1996). It is well-known that fish treated with immunostimulants show increased immune response parameters (Sakai 1999). In the present study, all these three non-specific immune responses, i.e. head kidney O₂ production ratio and plasma total Ig concentration were enhanced in grouper fed diet supplemented with 1.5 g mixed-NT kg^-1 (Table 3). The O₂^-1 production ratio and Ig concentration in fish fed diet supplemented with 1.5 g kg^-1 of individual NT were generally comparable with those fed diet with 1.5 g mixed-NT kg^-1 (Table 5).

Nucleotides have been generally considered non-essential nutrients because deficiency signs have not been observed (Carver & Walker 1995; Li & Gatlin 2006). It is known that tissues such as intestinal mucosa, bone marrow hematopoi-etic cells, lymphocytes and the brain have limited capacity for de novo nucleotide synthesis and depend on the supply by the salvage pathway in terrestrial animals (Yamauchi et al. 2002). Yamauchi et al. (2002) raised the hypothesis that endogenous supply of nucleotides may not be adequate for optimum functioning, especially of the immune system, under stressful conditions. Information pertaining to the synthesis and metabolism of NT in lower vertebrates such as fish is limited to date. While the nutritional value of nucleotides to human and other animals is still in some dispute, the results from studies on fish appear to be rather consistent that supplement exogenous nucleotides to the diet of aquatic species seem to be favourable.

Increasing amount (≥1.5 g kg^-1) of mixed-NT and single nucleotides (i.e. AMP and GMP, Table 5) increase total plasma Ig concentration is of interest. The discovery of the toll-like receptors (TLRs) in many mammalian species has partly shed light on the mechanisms that different immunostimulants induce (Bricknell & Dalmo 2005). Several TLRs genes have been identified in fish species. The TLRs have been demonstrated that altered the innate and adaptive immune responses of fish (Werling & Jungi 2003). This may explain AMP and GMP, the TLRs-like compounds, enhancing the plasma total Ig concentration offish. Whereas, in the case of UMP and CMP, their growth performance was similar to that of fish fed mixed-NT (Table 4) but with lower plasma Ig concentration (Table 5), this could be that nucleotides exert their major effects via the immune system. Weight gain may not be observed in healthy individuals, perhaps it would be more profound in starving individuals. The commercial NT product also contains RNA and other components as RNA has profound effect on the immune system. Future study in this regard is needed.

Acknowledgements

This work was supported by a grant from Center for Marine Bioscience and Technology of National Taiwan Ocean University, grant number 95529001C3.