Two feeding trials were conducted to study the requirements of juvenile cobia for dietary methionine and lysine. Experiment I compared both fishmeal-based (fish meal and soybean meal as protein sources) and casein-based (casein and gelatin as protein sources) diets. All diets were isonitrogenous and isoenergetic. Crystalline amino acids was pre-coated with carboxymethylcellulose (CMC) and supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except methionine. Five methionine levels of 0.45, 0.70, 0.95, 1.20 and 1.45% (1.07, 1.66, 2.26, 2.86 and 3.45% of protein) were studied. The test diets contained 0.16% cystine. Juvenile cobia (initial weight 35g) were fed test diets for 8 weeks. The results showed that the cobia used fish meal more effectively than casein. Final weight, percent weight gain (PWG), feed conversion ratio(FCR), hepatosomatic index (HSI), condition factor (CF), protein efficiency ratio (PER), protein retention (PR), carcass proximate composition and free methionine concentration in the serum were significantly enhanced (p<0.05) by increasing dietary methionine level. But profile of essential amino acids in the muscle and serum superoxidase and lysozyme activity were not significantly affected. Weight gain of the cobia fed the fishmeal-based diet containing 1.2% methionine was significantly higher than the other dietary groups. Methionine content of 0.45% resulted in reduced growth. No significant difference in weight gain was found in the casein-containing diets when methionine was 0.7% and over. The cobia fed diet containing 0.45% methionine started show apparent skin lesions in their heads on the sixth week of the growth trial and significant mortalities were observed since the seventh week (p<0.05). The optimum dietary level of methionine for cobia, estimated by the broken-line regression analysis on weight gain, was 1.10% (2.62% of protein) based on the fishmeal-based diet results and was 0.70% (1.67% of protein) based on the casein-based diet results. Experiment II used fish meal and wheat gluten as the protein sources. Pre-coated crystalline amino acids were supplemented to simulate the amino acid pattern of juvenile cobia muscle protein except lysine. Lysine levels including 1.49, 1.75, 2.00, 2.25, 2.50 and 2.75% (3.54, 4.16, 4.76, 5.35, 5.95 and 6.54%) were studied. Juvenile cobia (initially 12g) were fed test diets for 8 weeks. The results showed that the final weight, PWG, FCR, CF, PER, PR, carcass proximate composition and free lysine concentration in the serum were significantly enhanced (p<0.05) by increasing lysine levels. But HSI and essential amino acid profile of fish in the muscle were not significantly affected. Final weight and percent weight gain of the cobia fed diets containing 1.75% lysine or over were significantly higher than fish fed diet contained 1.49% lysine. FCR was lower for cobia fed diets containing 1.75% lysine or over than the 1.49% lysine group. The optimum dietary level of lysine, estimated by the broken-line regression analysis on weight gain was 1.76% (4.19% of protein), and was 1.95% (4.64% of protein) on serum free lysine concentration. There was no overt deficiency symptom, such as caudal fin rot in rainbow trout, in the cobia during the growth trial.

With the aim to enhance the efficiency of utilization of crystalline methionine supplemented in the high plant-protein diet for grouper (Epinephelus coioides), this study used Bacillus subtitlis spore as a probiotic additive in the diet to shorten the absorption time difference between protein-bound amino acid and crystalline methionine. The study was conducted in two parts. In the first part, juvenile groupers were fed for 14 weeks with 5 experimental diets: fish meal diet, high plant-protein diet with/without crystalline methionine, as well as with/without B. subtitlis spore separately. Growth performance, PER, protein digestibility, amino acid digestibility, non-specific immune responses, and free amino acid concentration in both muscle and serum were assessed. The second part was a time-series study on serum free amino acids concentration after a force-feeding experiment. The results showed that crystalline methionine supplementation in the high plant-protein diet effectively improved the growth of E. coioides (P<0.05). However, B. subtitlis spore supplementation did not affect fish growth performance significantly (P>0.05). A delay in the appearance of peak serum amino acid concentration was observed when fishmeal was partially replaced by soy protein. On the other hand, the force-feeding experiment showed that serum essential amino acid (include methionine) concentrations droped drastically after they reached the peak concentrations from being forced-fed with the B. subtilis containing diet. Supplementation of crystalline methionine seemed to ease the drop of serum methionine concentration. Based on these results, it is concluded that addition of B. subtitlis spore in high plant-protein diet for the grouper does not enhance the utilization of crystalline methionine, but supplementation of crystalline methionine significantly improve the growth performance of the grouper.