Blood was collected from the caudal vein by syringe with heparin as the anticoagulant from three fish selected randomly per aquarium and pooled for humoral immune response analysis. The blood sample was centrifuged at 3000 g for 15 min. Plasma was collected and stored at -80°C until analysis. Plasma total immunoglobulin (Ig) content was determined following the method of Puangkaew et al. (2004). The assay was based on the measurement of total protein content in plasma using the Lowry method (Lowry et al. 1951) prior to and after precipitating the Ig molecules employing a 12% solution of polyethylene glycol (Sigma Chemical). The difference in the protein content is considered as the Ig content for grouper.

For respiratory burst activity determination, head kidney was removed from three fish, and the leucocytes were isolated as described previously (Plytycz et al. 1989) with minor modification suited for grouper. Cell suspensions were prepared by pushing the tissue through stainless steel mesh (mesh size 100 lm) and layered onto 34% and 51% Percoll density gradient. The cells were centrifuged at 400 g for 30 min at 25°C. After centrifugation, the bands of leucocytes above the 34-51% interfaces were collected, washed twice, counted and adjusted to the required cell number for the assay. Respiratory burst activity [intracellular superoxide anion (O₂^-) production ratio] of leucocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to for-mazan as a measure of superoxide anion production as described by Secombes (1990). All analyses were conducted in triplicates.

Statistical analysis

Each experimental diet was fed to three groups of fish according to a completely randomized design. Results were analysed by one-way analysis of variance (anova) using the SAS/PC statistical software (SAS Inst. Inc., Cary, NC, USA), and significance was set at P < 0.05. Multiple comparisons among means were made with Duncans new multiple range test.


Results

In Experiment 1, weight gain (WG) and feed efficiency (FE) were higher (P < 0.05) in fish fed the diet supplemented with 1.5 g mixed-NT kg^-1 than those in fish fed the NT-free control diet (Table 2). Survival of fish did not differ among the dietary treatments. Fish fed diets with 1.0 g and 1.5 g mixed-NT kg^-1 had higher head kidney leucocyte superoxide anion (O₂) production ratio than that in fish fed diets with £0.5 g mixed-NT kg^-1 (Table 3). Plasma total immunoglobulin (Ig) concentration was higher in fish fed diets with 1.5 g and 2.0 g mixed-NT kg^-1 than that in fish fed diets with £0.5 g mixed-NT kg^-1. In Experiment 2, fish fed diets with 1.5 g AMP and mixed-NT kg^-1 had highest WG and FE, followed by fish fed the diet with 1.5 g IMP kg^-1, and lowest in fish fed the NT-free control diet (Table 4). Survival of fish did not differ among the dietary treatments.

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 the remaining 10 fish per group.
² Mixture of inosine monophosphate (IMP), adenosine mono-phosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP) and cytidine monophosphate (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 Mixture of inosine monophosphate (IMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP) and cytidine monophosphate (CMP) (1:1:1:1:1).