¹ Values are mean ± SD from three groups of fish (n = 3) with the remaining 10 fish per group.
² NT-free control, basal diet without nucleotides; IMP, inosine monophosphate; AMP, adenosine monophosphate; GMP, guano-sine monophosphate; UMP, uridine monophosphate; CMP, cytidine monophosphate; Mixed-NT, mixture of IMP, AMP, GMP, UMP and CMP (1:1:1:1:1).
³ FE = Final body weight (g) ) initial body weight (g)/feed intake (g).

Different superscripts in the column indicate significant (P < 0.05) difference between different dietary treatments.

¹ Values are mean ± SD from three groups of fish (n = 3) with three fish per group.
² NT-free control, basal diet without nucleotides; IMP, inosine monophosphate; AMP, adenosine monophosphate; GMP, guano-sine monophosphate; UMP, uridine monophosphate; CMP, cytidine monophosphate; Mixed-NT, mixture of IMP, AMP, GMP, UMP and CMP (1:1:1:1:1).

The head kidney leucocyte Ог production ratio was higher in fish fed the diet with 1.5 g AMP kg)1, followed by fish fed diets with 1.5 g UMP and 1.5 g mixed-NT kg)1, and lowest in fish fed the NT-free control diet (Table 5). Plasma total Ig concentration was higher in fish fed diets with 1.5 g AMP and GMP kg)1 than those in fish fed diets with 1.5 g IMP, CMP and UMP kg)1 and the NT-free control diet.

Discussion

It is clear from the present study that dietary supplementation of 1.5 g mixed-NT kg^-1 exerts a positive effect on growth performance of grouper. Growth promoting effect was also observed in fish fed diet supplemented with 1.5 g kg^-1 of each individual NT which exhibited lower WG and FE in fish as compared with the mixed-NT group (Table 4). The basal diet used in the present study provided complete nutritional composition based on our past research. While grouper fed the NT-free control diet recorded the lowest weight gain in both Experiments 1 and 2, the growth performance of this group compared favourably with those of previous studies in our laboratory (Shiau & Lin 2002; Lin & Shiau 2003, 2005a,b,c) in which 200-300% WG were in a similar size grouper given nutritionally adequate diet for 8 weeks. This indicates that the lower weight gain of the control group was not due to inadequate formulation of diet. It also provides an evidence of growth-promoting effect of dietary NT supplementation for the grouper.

Atlantic salmon fed diet with 2 g kg^-1 diet of commercial nucleotide product (containing 0.3 g NT kg^-1 diet) enhanced growth and resistance to Vibio anguillarum (Burrells et al. 2001a,b). Li et al. (2004) reported that hybrid striped bass fed diet with 5 g kg^-1 diet of commercial nucleotide product (containing 0.75 g NT kg^-1 diet) raised non-specific immune responses and resistance to Streptococcus iniae. The commercial nucleotides additive extracted from yeast commonly contains impure components such as trace element and polysaccharides. These components have been reported to provide additional immunostimulative effect for fish (Sakai 1999; Li & Gatlin 2006). Recently, red drum (Li et al. 2007a) and Pacific white shrimp (Li et al. 2007b) were fed diets with 0.3-3 g kg^-1 and 0.4 g kg^-1 of purified nucleotide mixture, respectively, both enhanced the growth performance. However, it should be noted, in all of these studies, fish meal-based diet was used, fish meal is rich in nucleotides component. Carver & Walker (1995) reported that the content of nucleotides, total purine and RNA of fish meal was 2, 4 and 3.4 g kg^-1, respectively. In the present study, by using pure NT, our result suggests that supplementation of mixed-NT at 1.5 g kg^-1 diet maximize grouper growth. The NT content of a typical fish-meal based commercial grouper feed (contains about 50% fish meal) is 1 g kg^-1 (Carver & Walker 1995). This amount is lower than 1.5 g NT kg^-1 obtained from our present study for the grouper optimum growth. Thus, dietary NT supplementation in commercial grouper feed is suggested.