The effects of dietary lipids on the growth and body composition of young cobia, Rachycentron canadum Ho-shin Huang Advisor¡GDr. Houng- Yung Chen Institute of Marine Biology, National Sun Yat-sen University Kaohsiung 804, Taiwan, ROC Two experiments were conducted to study the effects of dietary lipid on the growth and body composition of young cobia , Rachycentron canadum. In the first experiment, cobia (mean body weight 69.67g) were fed fishmeal-based (contained 5% lipid) supplemented with 0, 5, 10, 15 and 20% oil mixture ( corn oil/ cod liver oil, 2:1) for 8 weeks. Results indicated that cobia fed diets containing 10% or 15% lipid had a significantly higher weight gain than those fed diets containing 5%, 20% or 25% lipid. Food conversion ratio (FCR) and protein efficiency ratio (PER) were better for fish fed the lipid diets containing 15% and 25% than those fed with 5% lipid diet. Regression analysis indicates that dietary lipid level for maximal growth of young Rachycentron canadum is about 14.6%. No significant changes were observed in hepatosomatic index. Changes in body composition were not significant with respect to ash and protein content of liver and muscle. High dietary lipid level caused a significant increase in the level in liver and muscle lipid. Aspartate transaminase (AST) was significantly higher for fish fed diets containing 20% and 25% than those fed with 5% and 10% lipid. Alanine transaminase (ALT) was significantly higher for fish fed diets containing 25% than those fed the other diets. In the second experiment, Rachycentron canadum were fed fishmeal-based diets supplemented with lard oil, canola oil, corn oil, soybean oil, soybean + 1% DHA or cod liver oil. Fishmeal together with fish oil supplement contributed 8% lipid¡Fwhile the other oil sources were supplemented at 8% levels. Results show that fish fed diets with canola oil had a growth performance significantly lower than the other groups; while no difference was found among all other diets. The fish fed diets supplemented with corn oil, soybean oil or soybean + 1% DHA showed better food conversion ratio and protein efficiency ratio than the canola group. There was no difference among the canola, lard and fish oil groups. No significant changes were observed in hepato-somatic index. Liver lipid content was higher in fish fed diets with lard oil than fish fed other diets.

This research examined the quantitative effects of dietary conjugated linoleic acid (CLA) on juvenile cobia Rachycentron canadum with a mean initial weight of 10g and fed for 8 weeks. Four experimental diets that contained 16% crude lipid and 0%, 0.5%, 1.0% or 2.0% CLA were compared. At the end of the growth trial there was no significant differences in growth parameters. With increased CLA supplement, the body lipid content tended to reduce and protein content was significantly decreased. Increased dietary CLA caused significantly decreased hepatic FAS activity. Hepatic ME activity was decreased significantly in 0.5%, 0.75% and 1% CLA treatment, while no significant effect was detected on hepatic G6PDH activity. Total lipid contents of the liver and visceral fatty depot were significant decreased in 0.5%, 0.75% and 1% CLA treatment. There was a significant decrease in lipid content in the ventral muscle when CLA was supplemented at 0.75%, 1% and 2%. Fish fed 0.5% CLA had a lower adipocyte density in dorsal muscle, while fish fed 2% CLA treatment had a larger size of adipocytes in the liver and dorsal muscle. Gene expression of PPAR-£\ and PPAR-£^ showed no definite trend. Significant deposition of CLA was found in the liver, ventral muscle, dorsal muscle and visceral fatty depot of the cobia. The deposition ratio of cis-9, trans-11 CLA was slightly higher than that of trans-10, cis-12 CLA. Dietary CLA also affected the deposition of other fatty acids. Increased dietary inclusion of CLA significantly increased the total percentages of saturated fatty acids (SFA) but significantly decreased polyunsaturated fatty acids (PUFA). However DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) contents of the visceral fatty depot were significantly increased. In terms of immune response, dietary CLA had no effect on respiratory burst activities of head kidney phagocytes. Serum of fish fed 0.5% CLA had significantly increased lysozyme activities. In conclusion, dietary CLA affected the metabolism of lipid and fatty acid composition of cobia juveniles, and enhanced slightly immune responses. As CLA can deposit in the tissues of cobia, production of CLA-containing fish as a functional food is thus possible in cobia through dietary manipulation. An supplemental rate of 0.5% CLA is the optimal concentration considering fish nutrition and cost effectiveness.

The present study cloned full-length genes of peroxisome proliferators activated receptors (PPARs) of the cobia Rachycentron canadum and investigated their expressions in association with cobias¡¦ body adiposity and lipid-metabolism related physiological parameters. In addition to gene cloning, several studies evaluating the roles of PPARs were carried out, including: a time-series study on cage-farmed cobias from week 5 to week 52 post-hatching, a study comparing fish groups with contrasting growth performance and a study elucidating the effects of dietary fatty acids. Three isotypes, PPAR £\, PPAR£] and PPAR£^, that were cloned from cobia¡¦s cDNA contained 2046 bp, 2702 bp and 1943 bp, respectively. Their open reading frames encode 476, 510 and 531 amino acids, respectively. The identity in amino acid sequences between the PPARs are 52% (between PPAR£\ and PPAR£]), 52% (between PPAR£\ and PPAR£^), and 44% (between PPAR£] and PPAR£^), respectively. RT-PCR and real-time PCR (qPCR) analyses showed that expression of PPAR£\ mRNA predominated in red muscle, heart and liver, and at a lower level in the head kidney and dorsal muscle. PPAR£] transcripts were particularly abundant in the heart, liver, brain, and pyloric caeca. In contrast, PPAR£^ mRNA was detected primarily in the adipose tissues, liver, and pyloric caeca. In the time-series study, the PPARs expression was related to the body adiposity and lipid-metabolism related physiological parameters of the cobias that were raised for one year to approximately 4.5 Kg in a commercial cage-culture farm. Ten samplings were conducted on weeks 5, 7, 9, 14, 18, 23, 29, 34, 41, and 52 post-hatching. The cobias were raised in an outdoor nursery to 88 g before being transferred to an offshore cage on week 9. The adipocytes in the liver and ventral muscle showed a hypertrophic (increase in cell size) increase towards the end of the nursery phase. Their cell size decreased significantly after the cage transfer and was maintained afterwards a size spectrum dominated by small cells until week 34. The cobias grew rapidly after the offshore transfer and reached 330 g on week 14. They showed a concurrent increase in fat deposition in the liver and ventral muscle and a concurrent hyperplasia increase in density of adipocytes in the ventral and dorsal muscle. Adipocyte hypertrophy was obvious on week 41 and regressed afterwards. As the fish grew, serum phospholipids concentration increased significantly from approximately 380 to 750 mg/dL. Time-series pattern for the specific activity of two NADPH-generating enzymes, malic enzyme and glucose-6- phosphate dehydrogenase, were reciprocal and compensatory. The expression of liver PPAR£\ mRNA was negatively correlated to fat deposition and adiposity. There was a significant increase in body lipid deposition and hepatic PPAR£^ expression as the fish grew. Hepatic PPAR£^ expression could be a sufficient parameter describing its expression in whole body. These results showed that PPAR£^ and PPAR£\ played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia. In the study comparing differential fish growth, two groups of cobias were selected based on their growth performance from a same batch of fish raised in a nursery. The large-size group that was regarded as superior grower was 54.1 cm in total length and 1,287 g in weight; while the small-size fish (inferior grower) was 36.8 cm and 386 g. Compared to large cobias, small cobias showed a similar hepatosomatic index and viscerasomatic index, but a significantly (p ¡Õ 0.05) smaller mesenteric fat index (MFI).The levels of crude lipid in the liver (35% vs. 26%) and the proportions of neutral lipids in lipid were higher in large cobias than in small cobias. Concentrations of serum phospholipids, free fatty acids and total protein of large cobias were significantly higher than those of small cobias. Adipocyte density of liver and ventral muscle was increased with increasing fish size. The PPAR£\ mRNA expression in the liver of small cobias was significantly higher (p ¡Õ 0.05) than large cobias, ascribing to possible stress effect from their inferior growth. The growth superiority obviously affected PPAR£\ mRNA expression and fat deposition in the liver. In general, the expression of liver PPAR£\ mRNA was negatively correlated to body weight, body length, MFI, and serum NEFA, as well as lipid concentration, adiposity (adipocyte density and adipocyte size), G6PDH enzyme activity in the liver. The PPAR£^ mRNA expression in the liver was positively correlated to size of the adipocytes size. The effects of dietary fatty acids on PPARs expression were evaluated in a 10-week growth trial, in which cobias with an initial weight of 80 g were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were fish oil (rich in EPA and DHA), perilla oil (C18:3n-3), safflower oil (C18:2n-6), olive oil (C18:1n-9) or palm oil (C16:0). Significant difference was detected in PPARs mRNA expression among dietary treatments and among tissues. In the liver, among the dietary treatments, significantly higher expression levels of PPAR£\ mRNA were detected in perilla oil and olive oil group, PPAR£] mRNA in palm oil group and PPAR£^ mRNA in fish oil group. Linear regression analysis showed that liver PPAR£\ mRNA expression was positively (p ¡Õ 0.05) correlated with dietary C18:3n-3 levels and negatively with dietary C18:0 levels. Liver PPAR£] mRNA expression was positively correlated to C16:0 or C18:0 levels in diets. The PPAR£^ expression was positively (p ¡Õ 0.001) correlated to dietary levels of C20:1n-9, C20:5n-3 and C22:6n-3. In summary, the mRNA expression pattern of PPARs was tissue or organ-specific with the expression of PPAR£\ occurred predominantly in the liver and PPAR£^ in the adipose tissues. The expressions of PPARs in the liver were more related to their physiological roles than in other tissues or organs studied in the present study. The expression of PPAR£\ in the liver was shown correlated negatively to body fat deposition; and reciprocally, expression of PPAR£^ was positively correlated to fat deposition. PPARs mRNA expression was also associated with major dietary fatty acids. Increased dietary C18:0 levels down-regulated PPAR£\ and up-regulated PPAR£]. Up-regulation of PPAR£^ was significantly related to increased levels of highly (C>20) unsaturated fatty acid in diets. Dietary C16¡VC18 fatty acids on the other hand were more related to expressions of PPAR£\ and PPAR£]. These results suggest that fish oil could be partially replaced by plant oils as the lipid source in the diet of the cobia. In addition to highly unsaturated fatty acids, reduction in dietary C18:3n-3 and increase in C18:0 lead to increased fat deposition, implicating a possible strategy to modulate body lipid contents of the cobia through dietary manipulation.

This research studied the effects of dietary lipids unsaturation or oxidation on growth, body composition, tissue oxidative status, and health of the juvenile cobia. Five isonitrogenous and isoenergetic experimental diets containing 20% crude lipid, among which 9% was fish oil and 11% was various supplemental oils, were evaluated. The supplemental oils included palm oil (FP), soybean oil (FS), fish oil (FO), oxidized fish oil (OF) and fish oil supplemented with 300 mg/kg diet vitamin E (FE). The degree of lipid oxidation in FE, FO and OF was increased progressively, and so was the degree of unsaturation in FP, FS and FO. The results of a 10-week growth trial showed that lipid unsaturation didn¡¦t affect fish groeth significantly, but lipid oxidation significantly reduced growth. Decrease in lipid unsaturation and increase in lipid oxidation significantly reduced the crude lipid level of ventral muscle but not that of dorsal muscle and liver. Decreasing lipid unsaturation significantly affected serum TC and LDL concentration. The tissue fatty acid composition was significantly affected by the dietary fatty acid composition. Degree of dietary lipid Oxidation was negatively correlated with tissue n-3 PUFA and HUFA concentrations. Muscle TBARS concentrations was significantly affected by dietary lipids oxidation, but not was liver TBARS. Muscle TBARS was decreased as dietary lipid unsaturation was decreased, especially in dorsal muscle. The degree of dietary lipid oxidation of was positively correlated with tissue TBARS. The supplement additional of vitamin E reduced muscle TBARS. Liver catalase activity was decreased when dietary lipid unsaturated was decreased. Even though lysozyme activity in serum, SOD activity in erythrocyte, oxidative burst activity and phagocytic activity in neutrophils, and intracellular superoxide anion ratio in head kidney macrophages were not significantly affected by dietary lipids unsaturation or oxidation, these parameters showed the lowest levels in the group of fish fed the OF diet, indicating the adverse effects of oil oxidation on fish immunity. The results of this research show that oxidation of dietary lipids was more prominent than unsaturation in affecting growth, body lipid composition, peroxidative status, and health of cobia.

Two feeding trials were conducted to evaluate the effects of dietary phytic acid contents and removal of phytate from plant protein sources on growth and utilization of phosphorus, zinc and iron in juvenile cobia. In experimental I, test diets were formulated by adding phytic acid, 0, 5, 10, 15 and 20 g/kg diet to the basal diet that used fish meal and wheat gluten as the protein source. Juvenile cobia with an initial weight of 20g were fed the test diets for 8 weeks. No significant difference among fish groups was found in percent weight gain, feed conversion ratio, protein efficiency, net protein utilization and body composition. Dietary phytic acid level significantly affected zinc concentrations in body, vertebra, serum and feces. Body zinc concentration in fish fed diet containing 2% phytic acid was 10.2% lower than the control group. Vertebra and serum zinc concentrations decreased with increasing dietary phytic acid levels, vertebra zinc concentration in fish fed diet containing 2% phytic acid was 22.3% lower than the control group. The dietary phytic acid concentration was positively related to the fecal zinc concentration. In experimental II, nine isonitrogenous, isolipid and isocaloric diets were formulated including control diet that contained 421g/kg fish meal, and four test diets with fish meal protein in control diet being replaced by 40 or 50% with soybean meal or by 30% or 40% with canola meal respectively. Another four test diets used dephytinized soybean or canola meal after phytase treatments removed 90.9~94.6% of phytate. Juvenile cobia with an initial weight of 94g were fed the test diets for 8 weeks. Growth, feed conversion ratio, protein efficiency and net protein utilization of fish fed diets containing plant proteins were poorer than control group. Better weight gain, feed conversion ratio and net protein utilization were observed in those groups fed diets contained soybean meal rather than canola meal. Ash, phosphorus, zinc and iron contents of whole body and vertebra in cobia fed phytase-pretreated plant protein were not significantly different from groups of fish fed raw plant protein. In conclusion, dietary phytic acid reduced the zinc bioavailability. Performance of cobia as well as diet quality indicated that soybean meal as the better alternative protein source for fish meal than canola meal. Dephytinization had no positive effect on utilization of phosphorus, zinc and iron in juvenile cobia.

A fifteen-year plan has been developed for cobia (Rachycentron canadum) aquaculture in Brazil. Guidelines are recommended for implementing reachable goals within a reasonable time frame, taking into consideration current status of technology development, environmental parameters, logistics and market. This plan reports potentialities, constraints and outlines the infrastructure and operational needs of a vertically integrated (hatchery and growout) cobia aquaculture industry in Brazil. Fingerlings availability, manpower, feeds among others essential requirements are evaluated and discussed. A pilot-scale operation plan including risk and financial analysis is also presented for the early development stages of the project. Asian countries, especially China, Taiwan and Vietnam, where approximately 50,000 tons are produced per year, currently dominate cobia aquaculture. Although production is expanding rapidly, combined yielding is still incipient. By 2025, an increasing global population is expected to raise seafood demand on 37% (FAO, 2010) and this plan is intended to profit from it. Therefore, the aim is to supply 2.5% of the global aquaculture marine fish demand by 2025, which will be approximately 66,750 tons of cobia per year according to our projections.

As concerns regarding the growing human population, rising seafood demand, and up to present limited success of fisheries management intensify, aquaculture is increasingly posited as a means to more efficiently produce seafood commodities. However, aquaculture expansion raises contentious issues itself. The current study addresses a number of these issues in attempts to improve hatchery production and related activities. Investigation of less-harmful chemotherapeutants for disease reduction at the egg stage could lead to improved hatchery microbial management, increased survival during early larval stages, increased efficacy of bacterial probiotic incorporation, and reductions in disease transfer between hatcheries and locations. Attempts to surface sterilize and disinfect cobia Rachycentron canadum eggs with 3 and 2% hydrogen peroxide solutions significantly reduced survival through the yolk-sac larvae stage. Furthermore, timing of treatment application at differing stages of egg development was found to significantly impact survival, highlighting the importance of this compounding factor. Bacterial probiotics can remediate water quality, reduce target host stress, and improve survival and population growth rates of live feed organisms. Two of the following studies investigated the benefits of incorporation of a Bacillus spp. probiotic blend in aquaculture activities. During closed container mock shipment of yellowfin tuna Thunnus albacares yolk-sac larvae, significant reductions in total ammonia nitrogen resultant from probiotic incorporation were observed. Furthermore, significantly reduced dissolved oxygen utilization might have resulted from stress reduction, as suggested by a non-significant lower degree in the breakdown of osmoregulation of the larvae. Incorporation of probiotics within rotifer Brachionus plicatilis cultures resulted in significantly higher daily mean populations, significantly lower population variability among replicates, and non-coincident logistic population growth regressions which yielded higher population growth rates and system carrying capacities. Broodstock management should not only maximize quantity of reproductive output, but quality as well. Significant increases in cobia egg diameter with increasing broodstock female age were detected, as well as significant increases in egg diameter with decreasing salinity. Confirmation of potentially increased larval growth rates resultant from increased energy stores of larger eggs when produced by older females, as demonstrated in other species, is warranted. Attempts to find production parameters which maximize cobia larval growth and survival would lead to increased sustainability via reducing demand for wild-collected Artemia and economic efficiency. Gaps in knowledge for achieving this goal continue to exist, but conclusions drawn from analysis of multiple production scale trials indicate temperatures of 29-31 °C maximize growth and survival. Improvement in sampling design and data analysis would increase statistical rigor and ease comparability of larvicultures outcomes across ranging influential factors. Further investigation of all of these matters is certainly warranted, although conclusions drawn could be effectively utilized to improve success of hatchery operations.

This research investigated the changes in body adipocyte size and density as well as parameters relating to adipogenesis in growing cobia, Rachycentron canadum. The cobia from a same batch were sampled 5 , 7 , 9 , 13 , 17 , 21 , 26 and 31 weeks after hatching, Liver activities of fatty acid synthase, (FAS), mailc enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PDH) as well as serum total triacylglycerol (TG), total cholesterol (TC), phospholipid (PL), non-esterified fatty acid (NEFA) were assayed. Adipocyte abundance and size spectrum in liver, ventral muscle and dorsal muscle were determined. The cobia attained an average weight of 13.6 ¡Ó 0.5 g at week 5, and were cultured in nursery outdoor ponds until an average weight 87.5 ¡Ó 3.8 g when moved to open ocean cages. They grew to an average weight 1690.8 ¡Ó 106.6 g at week 31. The crude lipid content in liver, ventral and dorsal muscle increased significantly with fish age, and were 36.1 ¡Ó 3.2 g, 14.8 ¡Ó 0.2g, 4.6 ¡Ó 0.0 g at week 31. No significant change was found in hepatic FAS activity. Hepatic ME activity increased significantly after the fish were stocked in the cages. Whilr hepatic ME activity reduced with age, TG and PL were both increased significantly with age. While adipocyte diameter in liver showed a decreasing trend as the fish grew its abundance increased significantly at week 13 and maintained between 3070¡ã3356 x 104 cell/g tissue during week 17-31. Similarly, adipocyte abundance in ventral muscle increased significantly at week 13, and maintained between 255¡ã269 x 104 cell/g tissue afterwards. There was no significant change in size of ventral muscle adipocyte as fish grew. These results indicate that the adipocyte hyperplasia occurred during week 7- 13, when body weight was 100- 400 g. Adipocyte hypertrophy was observed when the fish reached week 31, there was a sign of adipocyte hypertrophy in liver when the fish were at the last phase of nursery outdoor ponds before being moved to the offshore cages.

This research studied the dietary effect of supplemental plant oils on growth, adiposity and lipid metabolism-related enzyme activity of juvenile cobia. The isonitrogenous and isoenergetic basal diet contained 15% crude lipid, 6% fish oil and 9% supplemental oils. The supplemental oils were varied among 5 dietary treatments, including fish oil (HUFA, n-3)(FO), perilla oil (18:3 n-3)(PE), safflower oil (18:2 n-6)(SA), olive oil (18:1 n-9)(OL), and palm oil (16:0)(PA). Results of the 10-wk feeding trial show that fish fed diet containing palm oil had the highest final weight, and was significant higher than fish fed SA diet. SA group had highest crude lipid concentration. OL group had the least crude protein concentration. PA group had the highest ash concentration. FO group had the highest crude protein and moisture content, the least crude lipid and ash content. Adipocyte density in various, tissues did not vary with time, except the ventral fat depot. Tissue adipocyte density of FO group was the least. Adipocyte density of PE group was higher than FO group, and its mean adipcoyte diameter in dorsal muscle was great than the other groups. Fatty acid synthase (FAS), as measured by specific activity, decreased with times in the PE group. Adipocyte density of SA group the highest of all groups, and its mean adipocyte diameter in dorsal muscle was also the greatest. Adipocyte density in the ventral fat depot of OL group was the highest, and its mean diameter in dorsal muscle was the smallest. Adipocyte density of PA group was only slightly lower than SA group. The tissue acid composition of the cobia was influenced by the supplemental plant oils. Tissue HUFA concentration and n-3/n-6 ratio was decreased, MUFA, PUFA and SAF composition was increased when the plant oils were supplemented. The results show that the supplementation of the plant oils could affect the density , size and tissue distribution of adipocytes, fatty acid synthesis pathway in the liver and tissue fatty acid composition. Feeding the cobia diet containing supplemental safflower or palm oil significantly increased density and cell size of adipocytes in the tissue of the cobia.

The present study cloned successfully two lipid-metabolism genes, lipoprotein lipase (LPL) and fatty acid binding protein (FABPs) from cobia and studied the mRNA expressions of the two genes and their upstream gene PPARs when the cobia were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were supplemented by fish oil (FO, rich in n-3 HUFA), perilla oil (PE, rich in 18:2 n-6), safflower oil (SA, rich in 18:2 n-6), olive oil (OL, rich in 18:1 n-9) or palm oil (PA, rich in 16:0). The whole sequences of LPL, liver-FABP (L-FABP) and muscle-FABP (M-FABP) encode 520, 126 and 133 amino acids, respectively. RT-PCR and real time PCR analyses based on these gene sequences show that the mRNA expressions of L-FABP and M-FABP in the tissue of the cobia were diet-specific. The mRNA expression of LPL, on the other hand, did not respond to the treatments, except in visceral fat depot. Linear regression analysis shows that the mRNA expression of LPL in the liver and muscle was positively (P<0.05) related to dietary fatty acids and ther concentration, but that in the visceral fat depot was negatively related. The mRNA expression of FABPs was also positively correlated with dietary fatty acid levels. Among all fatty acids, the levels of C14:0, C20:1 n-9, EPA and DHA were positively correlated with the mRNA expression of PPAR£^and also with FABPs mRNA expression in the visceral fat depot and LPL mRNA expression in the muscle. Thus, LPL, L-FABP and M-FABP mRNA expression of the cobia were highly influenced by the kind and amount of dietary fatty acids. The role of PPARs was not clearly demonstrated.