Determination of suitable protein and lipid levels in diets for Pacific bluefin tuna, Thunnus orientalis at grow-out stage

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1 Aquacult. Sci Determination of suitable protein and lipid levels in diets for Pacific bluefin tuna, Thunnus orientalis at grow-out stage Amal BISWAS 1,, Masashi NAKAJIMA 2, Takahiro NAKAO 2, Osamu TAKAOKA 1 and Kenji TAKII 1 Abstract: Three test diets were formulated using non-enzyme treated fish meal (FM) and fish oil with protein and lipid levels ( ) of 55 and 18 (D1), 51 and 22 (D2), and 47 and 26 (D3) to determine the suitable dietary level as well as the utility of non-enzyme treated FM at grow-out stage of Pacific bluefin tuna (PBT), Thunnus orientalis. Raw sand lance, Ammodytes personatus (D4) with 64 protein and 11 lipid was used as the control. Duplicate groups of 100 young PBT (mean weight 82.9 g) were stocked into m 3 net cages and reared for 35 days. Although there was no significant difference in mean body weight between D1 and D4, D4 showed significantly higher value than that of D2 and D3 (P < 0.05). The survival was significantly higher in fish fed with D1 (59.0 ) than that of D4 (47.5 ) (P < 0.05). Dietary protein/energy ratio and protein content showed strong positive linear correlation with final mean weight. Relative weights of viscera, pyloric caeca and intestine in fish fed with D1-D3 were significantly higher than those of D4 (P < 0.05). The increasing level of dietary lipid in D1-D3 resulted in significantly lower retention efficiency than that of D4 (P < 0.05). Results suggest that the non-enzyme treated FM can be utilized and dietary level of 55 protein and 18 lipid could improve the growth performance as well as the survival of juveniles PBT at grow-out stage. Key words: Pacific bluefin tuna; Grow-out stage; Lipid; Protein While the contribution of marine species to the total world fish production from the capture fisheries sector has either declined or reached its maximum sustainable yield, aquaculture production has steadily expanded, albeit at a low rate (FAO 2014). Among a total of 24.7 million tonnes (mt) of marine species produced from aquaculture sector in 2012, tunas, bonitos and billfishes contributed only mt (0.069 of total aquaculture production from marine species) (FAO 2014). In recent years, aquaculture production of Pacific bluefin tuna (PBT) Thunnus orientalis, which has the highest market value with great consumer demand because of high meat quality and excellent taste, has tendency to increase using cage culture in some countries (Nakahara 2004). However, the cage culture of this species has developed depending on mainly wild caught young tuna, which poses great threat to the natural resource. To realize the importance of PBT sustainable aquaculture, the Aquaculture Research Institute of Kindai University has started the research on aquaculture of this species in 1970 and successfully accomplished the full-cycle culture in captivity in 2002 for the first time in the world (Sawada et al. 2005). Immediately after this success, a lot of efforts have been taken by government agency, feed company and universities to establish a suitable diet for this species with limited success. After a series of studies seeking for protein source and its digestibility (Takii et al. 2007a, 2007b), it was Received 11 May 2016; Accepted 28 July Aquaculture Research Institute, Uragami Station, Kindai University, Uragami, Nachikatsuura, Wakayama , Japan. 2 Chubu Feed Co. Ltd., Nagoya, Aichi , Japan. Corresponding author: Tel, (+81) ; Fax, (+81) ; , akbiswas74@hotmail.com (A. Biswas).

2 A. Biswas, M. Nakajima, T. Nakao, O. Takaoka and K. Takii revealed that an enzyme treated fish meal (FM) instead of a non-enzyme treated FM can be a good protein source for PBT at early juvenile stage during the indoor rearing period (Ji, et al. 2008). Following this finding, a suitable nutritionally balanced formulated feed with optimal level of protein and lipid was established for early juvenile stage of PBT (Biswas et al. 2009). Although this feed has contributed to increase the survival and growth at indoor rearing, the efficiency of large scale production of young PBT at grow-out stage for aquaculture industry is yet to be achieved because of the high mortality due to transfer related stress and collision with net cage wall, and probably the lack of suitable feed. A proper nutritionally balanced formulated feed at grow-out stage may help to improve the situation. Although the enzyme treated FM is necessary as protein source at early juvenile stage, non-enzyme treated FM may be sufficient for grow-out stage, assuming that the digestive capacity is developed with growth. Moreover, the high cost and less availability of enzyme treated FM urge to investigate the utility of non-enzyme treated FM for the feed at grow-out stage of PBT. Fish has different protein and lipid requirement at different growth stages and the protein requirement usually decreased with fish growth. For example, while the protein requirement of common dentex Dentex dentex at mean initial body weight ca. 10 g is about 50-57, it is reduced to 43 with mean initial weight 90 g, but the lipid requirement increased from to 19.7 (Skalli et al. 2004). Therefore, it is necessary to determine the suitable protein and lipid levels at different sizes of fish to increase feed efficiency as well as to avoid unnecessary wastage of expensive components in feed. Therefore, this study was aimed to determine the utility of non-enzyme treated FM as well as the suitable levels of protein and lipid for diet of young PBT at grow-out stage. A suitable formulated diet for young PBT will ensure the expansion of grow-out tuna operation as well as to contribute to the true sustainability for this species. Materials and Methods Feed formulation Feed formula and proximate composition are given in Table 1. Three test diets were formulated with FM:FO ratios of 78:5, 73:10 and 68:15 in D1, D2 and D3, respectively. Vitamin and mineral mixtures were according to Halver (1957) with modification in vitamin mixture. The L-ascorbic acid content was doubled than that of original level recommended by Halver (1957) formula. FM used in this study was produced from horse mackerel (67 protein) and provided by Chubu Feed Co. Ltd., Nagoya, Japan. According to the feed formula, all diets (dry pellets) were produced at the factory of Chubu Feed Co. Ltd., and packed into air-tight bag and stored in a freezer at -20 C until feeding. Diet sizes were adjusted into two categories (3 and 5 mm diameter). Frozen raw sand lance (SL), Ammodytes personatus, which is commonly fed to young PBT in Japan, was used as reference diet (D4). The SL was chopped into small pieces to fit with fish sizes and kept frozen at -20 C until use. While look at the proximate composition of diets, the highest protein and lowest lipid contents were in D4 (Table 1). Protein and lipid levels from D1 to Table 1. Ingredients and proximate composition of experimental diets Experimental diets Ingredients D4 D1 D2 D3 (raw SL ) Fish meal Fish oil Starch Vitamin mixture Mineral mixture Soybean lecithin Proximate analysis (% of dyr matter basis) Crude protein Crude lipid Crude ash Sugar Gross energy (kj/g) Protein/energy ratio (mg/kj) Lipid/energy ratio (mg/kj) SL, sand lance, Ammodytes personatus. 1 Provided by Chubu Feed Co. Ltd. (Nagoya, Japan). 2 Halver (1957), but ascorbic acid amount is doubled. 3 Halver (1957).

3 Protein and lipid level for tuna grow-out feed D4 were 55 and 18, 51 and 22, 47 and 26, and 64 and 11, respectively. The decreasing and increasing trend of protein and lipid contents in experimental diets resulted in similar trends of protein/energy (P/E) and lipid/energy (L/E) ratios, respectively. Fish husbandry Experimental fish were obtained from the net cages of Fish Nursery Center, Kindai University, Uragami, Wakayama, Japan. Fish were transferred to the experimental net cages very carefully and minimizing the handling stress as much as possible. Duplicate groups of 100 young PBT, mean body weight 82.9 g (52 days old) were stocked into m 3 net cages and fed to apparent satiation for 35 days. The initial mean weight (82.9 g) was determined from another 100 young PBT by selecting randomly from the stocked net cage. For the first 17 days, fish were fed 4 times daily (07:30, 11: 00, 14: 30 and 18: 00) until apparent satiation with 3 mm pellets. For the remaining 18 days, fish were fed 3 times daily (07:30, 13:00 and 18:00) until apparent satiation with 5 mm pellets. The average water temperature and dissolved oxygen during the rearing period were C and mg/l, respectively. Net cages were checked once every day for dead fish and weighed, if any. To protect collision with net cage wall, one 6.4 W LED (Panasonic Corp., Osaka, Japan) was set at the middle of each net cage and one 70 mm diameter polyvinyl pipe was set as center pole just below the light so that the fish can swim around the pipe at night. Biochemical analysis At the end of rearing trial, all survived fish were sampled to compare the growth performance among the treatments. Blood was collected from 3 fish at each net cage from the caudal vein using a heparinized syringe equipped with a 22 G needle after anesthetizing in 100- ppm 2-phenoxyethanol (Wako Pure Chemical Industries Ltd., Osaka, Japan). Anesthesia, measurement and blood withdrawal took less than 2 min for three fish. Hemoglobin was measured immediately after blood collection by commercial kit (Hemoglobin B-Test, Wako Pure Chemical Industries Ltd., Osaka, Japan), and plasma was separated from the remaining blood by centrifugation at 2000 g for 15 min (4 C) and stored at -80 C for subsequent analysis of different constituents. After length-weight measurement of all survived fish, five fish from each net cage were randomly selected close to the mean body weight and dissected to measure relative organs weight. All remaining fish were frozen at -20 C and used for different analyses. Growth parameters such as survival, weight gain (WG), specific growth rate (SGR), daily feeding rate (DFR), feed efficiency (FE), protein productive value (PPV), condition factor (CF), relative organ weight of viscera (VSI), liver (HSI), stomach (SSI), pyloric caeca (PSI) and intestine (ISI), and retention efficiency of protein (PRE), lipid (LRE) and energy (ERE) were calculated to compare the growth performance among the treatments. Five fish from each net cage were used for whole body proximate composition and another five fish were used for liver proximate composition analyses by the standard AOAC method (AOAC 1995). Moisture content was determined by a dry oven at 100 C until constant weight, crude protein by micro-kjeldahl, crude lipid by Soxhlet extraction with diethyl ether for 16 h and ash content by a muffle furnace at 600 C until constant weight. Dietary sugar content was determined by the phenol-sulfuric acid method (Hodge and Hofreiter 1962). The energy contents of feed, initial and final whole body of fish were determined directly using an automated oxygen bomb calorimeter (IKA- Werke GmbH & Col KG, Germany). Plasma constituents were determined by commercial kit using Fuji Dry-chem (Fujifilm Company Ltd., Tokyo, Japan). Statistical analysis Data are expressed as the mean SE for each treatment for all parameters. Where significant differences were found by one-way analysis of variance (ANOVA), the means within each treatment and among treatments were compared using Tukey s test of multiple comparison at P < 0.05 significance level. All statistical

4 A. Biswas, M. Nakajima, T. Nakao, O. Takaoka and K. Takii Table 2. Growth performance of young T. orientalis fed diets for 35 days Parameters Final Initial D1 D2 D3 D4 Body weight (g) ab b b a Fork length (cm) Survival rate ( ) a ab ab b WG ( ) SGR ( /day) ab b b a DFR ( ) FE ( ) PPV CF Values are mean SE (n = 2). Values in a row with different letters are significantly different (P < 0.05). 1 Weight gain, WG ( ) = 100 (average weight gain / average initial body weight), where average weight gain = [{(final total weight + dead fish weight) - initial total weight} / average of initial and final number of fish]. 2 Specific growth rate, SGR ( /day) = 100 (ln final mean weight - ln initial mean weight) / rearing period (days). 3 Daily feeding rate, DFR ( ) = 100 total feed intake/{(mean of initial and final no of fish mean of initial and final body weight)/rearing period}. 4 Feed efficiency, FE ( ) = 100 (total wet weight gain / total dry feed intake). 5 Protein productive value (PPV) =(Final total whole body protein - initial total whole body protein) / protein intake from feed. 6 Condition factor, CF = 100 W/L 3, wehere W = weight (g) and L = length (cm). analyses were carried out using the Statistical Package for the Social Sciences (SPSS) program for Windows (v. 10.0, Chicago, IL, USA). Results The growth performance in fish fed with different diets is given in Table 2. The survival was significantly lower in fish fed with D4 compared to that of D1 (P < 0.05). Final mean body weight of g in D4 was significantly higher than that of g in D2 and g in D3 (P < 0.05). However, there was no significant difference in mean body weight between D1 (388.1 g) and D4. Similar trend was observed in SGR among the treatments. However, there were no significant differences in WG, DFR, FE, PPV and CF among the treatments, though D1 had higher FE than other diets. While look at the linear relationship, both dietary protein content and P/E ratio showed a significant positive correlation with final mean weight (Fig. 1). The variation in relative organ weight is given in Table 3. VSI, ISI and PSI in fish fed D4 showed significantly lower values than those observed in fish fed D1, D2 and D3 (P < 0.05). However, there were no significant differences in those parameters among the fish fed with formulated feeds (P > 0.05). There was no significant difference in HSI and SSI among the treatments (P>0.05). Fig. 1. Linear relationship between weight and protein/ energy ratio (A), and weight and dietary protein contents (B). Hemoglobin (Hb) content was significantly higher but triglyceride (TG) content was significantly lower in fish fed with D4 than those of other diets (Table 4). Total protein (TP) content in fish fed with D1 showed significantly higher value than that of D3 (P < 0.05). However, there were no significant differences in other parameters.

5 Protein and lipid level for tuna grow-out feed The whole body and liver proximate compositions are given in Table 5. In whole body, increasing level of dietary lipid resulted in significantly higher whole body lipid content in fish fed with D2 and D3 (P < 0.05). There were no significant differences in other parameters among the treatments. In liver, moisture content was significantly higher in fish fed D4 than that of other diets (P < 0.05). Crude protein Table 3. Relative organ weight of T. orientalis under different treatments at the end of 35 days rearing trial Parameters ( ) D1 D2 D3 D4 VSI a a a b HSI SSI PSI a a a b ISI a a a b Values are mean SE (n = 10). Values in a row with different superscripts are significantly different (P < 0.05). VSI, viscerosomatic index; HSI, hepatosomatic index; SSI, stomatosomatic index; PSI, pyloric caeca somatic index; ISI, intestinosomatic index. VSI, HSI, SSI, PSI or ISI ( ) = 100 (viscera, liver, stomach, pyloric caeca or intestine weight / body weight). content was significantly higher in fish fed D4 and D1 than that of D3. Again, the increasing level of dietary lipid content resulted in significantly higher crude lipid value in fish fed D3 followed by D2, D1 and D4. There was no significant difference in ash content among the treatments. Retention efficiencies of nutrients and energy are given in Table 6. There were no significant differences in PRE and ERE among the treatments. However, dietary increasing levels of lipid resulted in decreasing trend in LRE and fish fed with D4 showed significantly higher value than that of formulated diets (P < 0.05). Discussion Although the rearing period was only 35 days, the final mean body weights in different treatments were times bigger than the initial mean weight. It indicates that the formulated feeds with non-enzyme treated FM were well accepted by the young PBT to sustain their fast Table 4. Hematology and plasma constituents of young T. orientalis fed diets for 35 days Parameters D1 D2 D3 D4 Ht ( ) Hb mg/dl b b b a BUN mg/dl TP g/dl a ab b ab TG mg/dl a a a b TCHO mg/dl GLU mg/dl Values are mean SE (n = 6). Values in a row with different superscripts are significantly different (P < 0.05). Ht, hematocrit; Hb, hemoglobin, BUN, blood urea nitrogen; TP, total protein; TG, triglyceride; TCHO, total cholesterol; GLU, glucose. Table 5. Proximate composition of whole body and liver of young T. orientalis fed diets for 35 days Parameters Initial Final D1 D2 D3 D4 Whole body Moisture ( ) Crude protein ( ) Crude lipid ( ) b a a c Crude ash ( ) Gross energy (kj/g) Liver Moisture ( ) b b b a Crude protein ( ) a ab b a Crude lipid ( ) b b a c Crude ash ( ) Values are mean SE (n = 10). Values in a row with different superscripts are significantly different (P < 0.05).

6 A. Biswas, M. Nakajima, T. Nakao, O. Takaoka and K. Takii Table 6. Variation in retention efficiencies of nutrients and energy in T. orientalis fed with different diets for 35 days Parameters ( ) D1 D2 D3 D4 PRE LRE b b b a ERE Values are mean SE (n = 2). Values in a row with different superscripts are significantly different (P < 0.05). PRE, protein retention efficiency; LRE, lipid retention efficiency; ERE, energy retention efficiency. PRE, LRE or ERE ( ) = 100 (protein, lipid or energy retained in the body / protein, lipid or energy intake). growth at grow-out stage. In this experiment, each treatment was designed in duplicate due to the lack of some facilities. Although the treatment with more replicates is desirable, the lack of significant difference between D1 and D4 indicates that non-enzyme treated FM can be used as protein source to support the growth at this stage at least with the experimental design used here. Ji et al. (2008) demonstrated that the FM needs to be made more digestible by enzyme treatment to use as protein source for rearing PBT at early juvenile stage during indoor rearing period. However, the utility of non-enzyme treated FM at grow-out stage suggests that the digestion and absorption capacity of protein may be improved along with the growth of PBT. The significantly higher survival in fish fed with D1 indicates that the formulated feed with suitable protein and lipid would help to provide more young PBT to aquaculture industry. High mortality is a normal phenomenon at larval and juveniles stages of PBT. Previous rearing trials with artificial formulated feed at early juvenile stage of PBT showed survival when reared for days (Biswas et al. 2009, 2011, 2013). Therefore, the survival of in this study at the end of 35 days rearing trial is comparable considering the difficulty of PBT rearing. Similar to the previous studies with juvenile PBT (Biswas et al. 2009, 2011, 2013), about fish died during the first 3 days after commencing the experiment. This could be due to the stress caused by the transfer of fish to the experimental net cages although it was tried to minimize as much as possible, suggesting very sensitive nature and weakness of PBT on handling. Apart from the death due to transferring stress in juvenile PBT, it is demonstrated that the death due to collision has also frequently occurred (Ishibashi et al. 2009). In this study, LED light was set at the middle of net cage to reduce the death from collision with net wall. However, the dead fish with head-stature and peeling skin confirmed that the collision was still frequently occurred. Based on the growth performance, the lack of significant difference between D1 and D4 suggests that the suitable dietary levels of protein and lipid in grow-out feed for young PBT will be 55 and 18, respectively. The suitable protein level found in this study is lower than the requirement of more than 60 protein suggested for juvenile PBT ( g) during the indoor rearing period (Biswas et al. 2009). This may indicate that the protein requirement decreased with increasing body weight, which agreed with decreasing protein requirement from in mean initial body weight ca. 10 g to 43 in mean initial weight ca. 90 g in common dentex, Dentex dentex (Skalli et al. 2004). However, the positive linear relation of dietary protein and P/E ratio with final mean body weight observed in this study urges to clarify whether more increase in dietary protein can further increase the growth performance. Since the protein and lipid sources of D1-3 and D4 (raw fish) are different, it may not proper to draw the regression line combining all treatments. However, regardless of sources, the P/E of D4 provided more plot to draw the regression line. In this study, the results clearly demonstrated that the increasing levels of dietary lipid or L/E ratio resulted in lower growth performance during the rearing period. As mentioned earlier, the utility of lipid increased in common dentex from 10.6 or 13.5 in mean initial body weight ca. 10 g to 19.7 in mean initial body weight ca. 90 g (Skalli et al. 2004). Therefore, it was expected that the PBT of >80 g body weight could utilize more lipid compared to that of g body weight (Biswas et al. 2009). However, the lipid requirements are similar for both sizes.

7 Protein and lipid level for tuna grow-out feed Considering the maximum weight of around 450 kg (Bayliff 2001), body weight from 0.2 to 430 g may be a narrow range for the PBT to vary in lipid requirement. The higher growth from feed with lower lipid contents also agreed with Hebb et al. (2003), who demonstrated that the growth performance was significantly improved in juvenile winter flounder, Pleuronectes americanus fed diet with 10 lipid compared to that of 20. Wang et al. (2005) also found significantly higher growth performance in juvenile cobia, Rachycentron canadum when fed diet with 5 lipid compared to that of 25. While compared the digestive organs among the treatments, pyloric caeca and intestine weights in fish fed D1-D3 were higher than those of fish fed D4. Similar results were observed in other studies with PBT (Ji et al. 2008) and yellowtail Seriola quinqueradiata (Shimeno et al. 1993). The digestibility of protein may be reduced little bit due to the heat treatment while producing FM from raw fish. Therefore, the higher weight of those digestive organs may be due to one of the adaptive behaviors of fish when fed with less digestible protein sources. That means, the fish might enlarge their digestive organs either to increase enzymes secretion or to keep more feed in it for long time to enhance the digestibility. Although the whole body content was not affected in this study, it showed the enhancement of liver protein content by increasing dietary protein levels. It agreed with the demonstration that the protein content of liver is dependent on the dietary protein intake (Schuchardt et al. 2008). It has also been shown that liver is the major site for fat deposition in fishes (Péres et al. 1999; Hemre et al. 2002). In this study, both whole body and liver lipid contents were significantly affected by dietary lipid levels, which agreed with the findings in orange spotted grouper, Epinephelus coloides where increasing dietary lipid resulted in significant enhancement of liver lipid content (Luo et al. 2005). The enhancement of whole body lipid by increasing dietary lipid levels is also agreed with the results reported in brown-marbled grouper, Epinephelus fuscoguttatus (Shapawi et al. 2014) and hybrid grouper, Epinephelus fuscoguttatus E. lanceleolatsu (Rahimnejad et al. 2015). In conclusion, the lack of significant difference in the growth performance between D1 and D4 indicates that the non-enzyme treated FM can be utilized as protein source in growout feed for young PBT and the suitable level of protein and lipid will be around 55 and 18, respectively. Moreover, the higher survival from D1 would ensure to supply more PBT to aquaculture industry. However, the significant relation between final body weight and P/E ratio urges further studies to clarify the effect of diet with more protein and less lipid contents on the growth performance at grow-out stage. Acknowledgements The expenses of this study were defrayed in part by a grant from Chubu Feed Co. Ltd., Nagoya, Japan. References AOAC (1995) Official Methods of Analysis of AOAC International. vol I. Agricultural Chemicals; Contaminants, Drugs. 16th edition. AOAC International, Arlington, VA, USA (1298 pp.). Bayliff, W. H. (2001) Organization, functions, and achievements of the Inter-American Tropical Tuna Commission. Special Report 13, pp. 14 (DOI: Biswas, B. K., S. C. Ji, A. Biswas, M. Seoka, Y. S. Kim, K. Kawasaki and K. Takii (2009) Dietary protein and lipid requirements for the Pacific bluefin tuna Thunnus orientalis juvenile. Aquaculture, 288, Biswas, A., B. K. Biswas, J. Ito, O. Takaoka, N. Yagi, S. Itoh and K. Takii (2011) Soybean meal can partially replace enzyme-treated fish meal in the diet of juvenile Pacific bluefin tuna Thunnus orientalis. Fish. Sci., 77, Biswas, B. K., A. Biswas, J. Ito, Y. S. Kim and K. Takii (2013) The optimal dietary level of ascorbic acid for juvenile Pacific bluefin tuna, Thunnus orientalis. Aquacult. Int., 21, FAO (2014) The State of World Fisheries and Aquaculture, Rome, 223 pp. Halver, J. E. (1957) Nutrition of salmonid fish-iii. Watersoluble vitamin requirements of Chinook salmon. J. Nutr., 62, Hebb, C. D., J. D. Castell, D. M. Anderson and J. Batt (2003) Growth and feed conversion of juvenile winter flounder (Pleuronectes americanus) in relation to different protein-to-lipid levels in isocaloric diets. Aquaculture, 221,

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