The aim of the present research was to investigate the expression profiles of GnRH-R and neuropeptide Y (NPY) genes in brain and pituitary of Orange-spotted Grouper, Epinephelus coioides and also to understand the regulatory mechanism by administering different sex steroids. GnRH-R (TMD2 to TMD6) was partially involved cloned in this study. Tissue distribution analysis revealed a significant expression of GnRH-R in pituitary compared to others tissues. The expression of GnRH-R in brain and pituitary of groupers at different ages showed a significant increased during the fourth year, probably indicating the time of maturation. However, there was no significant difference in the expression of GnRH-R during different seasons. Treatment of two and three year old groupers with different sex steroids revealed an increase in the expression of GnRH-R in pituitary by E2 in both the age groups tested, while T could induce the expression of GnRH-R only in three year old groupers. The result, thus, indicates that the sensitivity of grouper to sex steroid is dependent on the age and the kind of steroid adminstered. In different sections of brain, the GnRH-R expression was in general lower in the group treated with E2 or T compared to the control group. The expression of the gene was more or less the same in two year and three year old control groups. This result may have been caused by suppression of GnRH-R expression in forebrain and midbrain after sex steroid injection. Administration of T induced a significant increase in the expression of GnRH-R in forebrain and midbrain, while E2 treatment did not have a similar effect. In hindbrain, the expression profile GnRH-R was not affected by sex steroid treatment in both two year and three year old groupers. The results suggest that sex steroids can only regulate the expression of GnRH-R in the forebrain and midbrain, probably due to the wide distribution of steroid¡¦s receptor in these regions. LHRH and pimozide injections to two year old groupers showed an increase in the expression of GnRH-R in pituitary after LHRH treatment while there was no stimulatory effect on other sections of the brain. In contrast, treating the fish with pimozide alone or pimozide together with LHRH did not stimulate GnRH-R expression in brain. Thus, the study suggests that LHRH can significantly increase the expression of GnRH-R in pituitary while dopamine has no stimulatory effect. Studies on NPY showed that the gene was distributed in different sections of brain especially in the forebrain but it was also present in gills, liver, intestine ¡K etc. The presence of NPY in gills, though less compared to that in brain, suggests that NPY might play an important role in osmosis regulation. The expression of NPY decreased with increase in age which may be due to the effect of other regulatory factors. Treatment of two and three year old groupers with different sex steroids did not effect the expression of NPY significantly in brain, which is different from other published reports. This may be due to the difference in the zoning of brain regions. In the present study, forebrain and midbrain were sampled together for analyses. The expression of NPY in brain did not change by treating the fish with LHRH or pimozide.

Grouper (Epinephelus coioides) is one of the important farmed fish in the southern Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detecting the presence of pathogen is critical for preventing epidemic outbreak. Interleukin-1 (IL-1) is one of proinflammatory cytokines that form a feedback control loop with anti-inflammatory cytokines to maintain the homeostasis of host immune response. The increase of IL-1 expression could be an indicator of pathogenic insult. In this study, total RNA of Epinephelus coioides fertilized egg was extracted for reverse transcription-polymerase chain reaction (RT-PCR) to amplify cDNA of IL -1£]. The cDNA amplified was then cloned into pGEX4T-3 for the expression and purification of GST-IL-1£] fusion protein. GST-IL-1£] fusion protein purified was then used to immunize New Zealand white rabbit for generation of antiserum against IL-1£]. Western blot result confirmed the specificity of antiserum as the immune serum, but not the preimmune serum, detected the immunogen GST-IL-1s. Further experiments using live Epinephelus coioides injected with or without lipopolysarcharides (LPS) further confirmed that this antiserum could detect a massive increase of IL-1£] protein after the injection of LPS in either protein lysate by western blotting or in frozen tissue section of head kidney by immunohistochemistry. In summary, we successfully generated a rabbit specific antiserum against IL-1£] of Epinephelus coioides , which could be a useful reagent for future analysis of fish immune response upon pathogen infection.

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.

Dietary effects of a nucleotide mixture that contained AMP¡BGMP¡BCMP¡BUMP and IMP in equivalent amount were evaluated in 4 supplemental levels of 0, 0.05, 0.2, and 0.4 g/100g diet. Weight gain (519-616%) of juvenile groupers fed for 14 weeks with the test diets was not significantly affected by the supplements. Serum concentrations of total protein, albumin, and cholesterol, A/G ratio and activities of AST were also not significantly affected. Serum total bilirubin concentration in the 0.4 g/100g group was significantly higher than the 0 or 0.05 g/100g group. Head-kidney macrophage phagocytosis and intracellular superoxide anion production as well as serum lysozyme activity were also not different among treatments. Histological sections of fish gut indicated that villous heights of intestinal fold were significantly greater in the 0.2g/100g group than the 0.4 g/100g group, muscle layer thickness, in contrast, was not affected. When challenged with Photobacterium damsela subsp. damselae after fed the test for 3 weeks, relative percent survivals of the groupers were 87.8, 91.1, 90.0 and 94.4% (0, 0.05, 0.2, and 0.4 g/100g diet, respectively). The survivals were 74.5, 82.4, 86.3, and 85.3%, respectively when the feeding trial was extended to 18 weeks. The results show that a diet containg 38% protein and asupplemented with nucleotides at the tested levels was not effective in promoting growth, but was moderately effective in enhancing immune responses and pathogen resistance of juvenile grouper.