EPA and DHA Requirements in Early Juvenile Red Sea Bream Using HUFA Enriched Artemia Nauplii*1

Transcription

1 Fisheries Science 62(2), (1996) EPA and DHA Requirements in Early Juvenile Red Sea Bream Using HUFA Enriched Artemia Nauplii*1 Hirofumi Furuita,*2 Toshio Takeuchi, Masanori Toyota,*3 and Takeshi Watanabe Department of Aquatic Bioscience, Tokyo University of Fisheries, Konan, Minato, Tokyo 108, Japan *2National Research Institute of Aquaculture, Nansei, Mie , Japan *3Animal Health Research Laboratory, Agro Division, Takeda Chemical Industries, Ltd, Juso-honmachi, Yodogawa, Osaka 532, Japan (Received April 24, 1995) Feeding experiments were conducted to determine the requirements of early juvenile red sea bream (Pagrus major) for eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) by feeding Arte mia nauplii containing different levels of EPA or DHA for 12 days Juveniles fed EPA-enriched Arte mia had a high survival rate but very poor vitality, while those fed on Artemia enriched with DHA had both a high survival rate and vitality The vitality of fish was effectively improved by elevation of DHA levels in Artemia In the DHA-enriched Artemia, the EPA content increased from the initial value together with the appearance of docosapentaenoic acid (DPA), indicating retroconversion from DHA to EPA through DPA in the nauplii The requirement of early juvenile red sea bream for DHA was esti mated to be % in Artemia on a dry matter basis when vitality was used as an index, and was satisfied with 225% EPA or 095% DHA (205% n-3 HUFA) on a dry basis when based on the survival rate Key words: essential fatty acid, EPA, DHA, red sea bream, larvae, docosahexaenoic acid It has been demonstrated that one of the principal fac tors affecting the dietary value of food organisms such as rotifers and Artemia is their concentration of n-3 highly unsaturated fatty acids (n-3 HUFA) such as eicosapen taenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential fatty acids (EFA) for marine fishes1,2) Recent studies on EFA of marine fish have shown that the EFA value of DHA is superior to EPA for larval and juvenile red sea bream, larval yellowtail, juvenile striped jack, etc3) Feeding larval red sea bream with EFA deficient rotifers resulted in high mortality, a low growth rate and poor vitality together with a high percentage of hydrops These abnormalities were effectively improved by feeding rotifers containing EPA or DHA; however, DHA was far more effective than EPA4) In juvenile red sea bream of approximately 3 g fed semi-purified diets, DHA was superior to EPA though growth and feed efficiency were effectively improved by elevating dietary EPA and DHA levels5) However, in these studies the physiological function of EPA and DHA was suggested to differ depend ing upon the growth stage This study compared the EFA value between EPA and DHA for early juvenile red sea bream, and determined their requirements for both fatty acids using Artemia nauplii enriched with different levels of EPA or DHA Materials and Methods Two feeding experiments were designed in this study; Ex periment I compared EFA efficiency between EPA and DHA for early juvenile red sea bream, and Experiment II determined their requirements for EPA and DHA as EFA Feeding of Artemia Oleic acid (OA), EPA and DHA, the ethyl ester type,6) were used as the experimental lipids in Experiment I (Treat ment Nos 1 to 3) In Experiment II, four levels of EPA or DHA, respectively, were prepared by mixing OA with EPA or DHA Namely, OA (Treatment No 4), 1/4EPA (OA:EPA= 3:1, Treatment No5), 1/2EPA (OA:E PA = 1: 1, Treatment No 6), EPA (Treatment No 7), 1/ 4DHA (OA:DHA=3:1, Treatment No 8), 1/2DHA (OA: DHA=1:1, Treatment No 9), DHA (Treatment No 10) The purities of OA, EPA and DHA were 95%, 99% and 99%, respectively EPA and DHA were purchased from Idemitsu Petrochemical Co, Ltd Newly hatched Artemia (Utah strain, Shoei Trading Co, Ltd), were stocked into 10 1 tanks at a density of 100 in dividuals/ml and an emulsion prepared by mixing 1 ml lipid with 01 g raw egg yolk and 100 ml water was added to the tanks Enrichment was conducted for h at a water temperature of Ž with vigorous aeration *1 This study was presented in part at the Annual Meeting of the Japanese Society of Scientific Fisheries, held in Tokyo, in April 1991 and in Shimonoseki, in October 1992

2 Requirements of juvenile red sea bream for EPA and DHA 247 Table 1 Rearing conditions for red sea bream in Expts I and U of enriched Artemia for 12 days At the end of the feeding trials, an activity test was con ducted to check the fish's response to stress (vitality) Thir ty fish were taken out of the water, held in the air in a scoop net for 30 s or 2 min and moved to 301 tanks to check their survival after 12 h The analytical procedures for lipids were all described in previous papers 7,8) Tricosanoic acid methyl ester (>99% purity, Sigma Chemical Co) as an internal standard was added to total lipids of Artemia when preparing the fatty acid methyl esters in Experiment 11 *1 Mean }SD (n=30) *2 Mean }SD (n=50) Samples of Artemia were taken after enrichment for analy sis of lipids Feeding of Fish with Artemia The rearing conditions of the fish in Experiments I and II are shown in Table 1 The initial total length of fish was approximately 10 mm in both experiments The fish, which had been reared with S-type rotifers fed on Nan nochloropsis oculata and omega-yeast (Kyowa Hakko Kogyo Co, Ltd), and Artemia nauplii enriched with Es ter 85 (Nippon Chemical Feed Co, Ltd), were given non enriched Artemia for 3 days before the feeding experiment to reduce n-3 HUFA content in the fish The experimental fish were divided into 3 and 7 tanks in Experiments I and U, respectively; each group contained 200 fish in a 1001 polycarbonate tank The fish were fed one of various kinds Results Lipid and Fatty Acid Compositions of Artemia The contents of EPA and DHA or n-3 HUFA in the nauplii in Experiment I were almost the same as in Experi ment 11, both enriched by the same lipid Table 2 shows proximate and fatty acid compositions of Artemia in Ex periment II The lipid content was the highest in the EPA fed Artemia The concentration of EPA in Artemia en riched with EPA was higher than that of the DHA-fed Ar temia at any level of treatment Furthermore, EPA con tents in nauplii which received DHA-treatment were higher than that of initial ones, and docosapentaenoic acid (22:5n-3, DPA) was detected, though it was not detected before enrichment This result indicates that Artemia con verted DHA to EPA through DPA This might be a reason why incorporation of DHA by Artemia is always lower than that of EPA The contents of EPA or n-3 HUFA in Artemia (dry mat ter basis) enriched with various levels of EPA were 206 Table 2 Proximate and fatty acid compositions of total lipids in Artemia nauplii enriched with the experimental lipids*1 in Expt U *1 OA, oleic acid ethyl ester (purity, 95%<); EPA, eicosapentaenoic acid ethyl ester (purity, 99% <); DHA, docosahexaenoic acid ethyl ester (purity, 99%<) *2 ND, not detected

3 248 Furuita et al 844 g/ 100 g and g/ 100 g, respectively The con tents of DHA and n-3 HUFA in Artemia enriched with DHA were g/ 100 g and g/ 100 g, re spectively Growth, Survival, and Vitality of Fish The results of the feeding experiments are shown in Fig 1 and Table 3 In Experiment I, fish fed Artemia contain ing 06% n-3 HUFA (Tank No 1) showed poor swimming activities and a low survival rate When they were submit ted to the activity test, they exhibited the so-called shock syndrome described in a previous paper2) Fish which received EPA- or DHA-Artemia (Tank Nos 2 and 3) showed high survival rates In the activity test, however, the enrichment of DHA gave a better result than EPA In Experiment U, feeding Artemia without enrichment (Tank No 4) resulted in a low survival rate and poor vitali ty as observed in Experiment I The fish which received n-3 HUFA showed a high survival rate In contrast, fish fed Fig 2 Effect of EPA and/or DHA contents in Artemia nauplii on vital ity of early juvenile red sea bream in Expt U Artemia enriched with EPA showed poor vitality irrespec tive of EPA levels in the nauplii The vitality of fish was effectively improved by elevation of DHA levels in the nauplii (Fig 2) Table 3 Growth, survival and vitality of early red sea bream after 12-day feeding trials in Expts I and U Lipid Classes and Fatty Acid Composition of Fish Lipid content and lipid classes of fish are shown in Ta bles 4 and 5 The lipid content of fish after preliminary feeding decreased from 181% to 159% in Experiment I and 171% to 155% in Experiment U The lipid content of fish was highest in those fed EPA-Artemia in both ex periments The content of polar lipids, however, was highest in the fish fed DHA-Artemia in Experiment I, and in those fed 1 /2DHA-Artemia in Experiment U The fatty acid compositions of polar and/or neutral lipids in fish are shown in Tables 6 and 7 The percentages of n-3 HUFA of polar lipid in fish after preliminary feed Table 4 Lipid content and lipid classes*1 of early juvenile red sea bream fed Artemia nauplii enriched with the experimental lipids in Expt T (%) *1 Survival rate at activity test Number of fish, 30 *2 Mean }S D (n=30) *3 -, Not tested *1 Abbreviations: SE, sterol esters; TO, triglycerides; FFA, free fatty acids, FS, free sterols; DO, diglycerides; MG, monoglycerides; PE, phosphatidyl ethanolamine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholi ne; Sph, sphingomyeline; LPC, lysophosphatidylcholine *2 Fig 1 Survival rates of early juvenile red sea bream fed Artemia nauplii enriched with the experimental lipids in Expt U Fish fed the newly-hatched Artemia nauphi without enrichment for 3 days *3 Mean }SD (n=3) *4 Phosphatidylinositol and phosphatidylserine are included

4 Requirements of juvenile red sea bream for EPA and Dl-IA 249 Table 5 Moisture and lipid compositions of early juvenile red sea bream fed Artemia nauplii enriched with the experimental lipids in Expt U (%) * See the footnote of Table 4 Table 6 Fatty acid compositions of neutral lipids (NL and polar lipids (PL) in early juvenile red sea bream fed Artemia nauplii en riched with the exprimental lipids in Expt I (area%) *1 Seethe footnote of Table 4 *2 Mean }SD (n=3) *3 Not calculated ing decreased to two-thirds of the initial value in both ex periments, while DHA decreased to half of the initial The levels of EPA or DHA of polar lipid in fish increased in proportional to the contents of these fatty acids in the naupllj The percentage of DPA of polar lipid in the fish fed EPA-Artemia increased from 15% to 57% and from 26% to 42% in Experiments I and II, respectively Discussion In the present study, fish growth was not different among fish fed OA, EPA or DHA enriched Artemia However, fish which received EPA-enriched nauplii showed poor vitality, although fish fed 1 / 2EPA- or EPA Artemia were receiving more than 30% n-3 HUFA, which is the requirement of larval red sea bream91 In contrast, the fish fed on the DHA-enriched Artemia showed a quite strong vitality, with 57% of the fish surviving after 2 min exposure to the air This indicates a difference of EFA value between EPA and DHA These phenomena are in good agreement with the fact that a high mortality fre quently occurs during transportation of juvenile red sea bream from rearing tanks on land to net cages in the sea This might be due to the lack of DHA in live foods such as rotifer cultured with the so-called "marine Chlorella", mostly N oculata, or non-enriched Artemia In some way, DHA seems to be able to alleviate various forms of stress in fish larvae Recently, Watanabe et al4) have shown that the dietary value of rotifers containing DHA for larval red sea bream is higher than those containing EPA as the main n-3 HUFA They also showed that the elevation of DHA level

5 250 Furuita et al Table 7 Fatty acid composition of polar lipids in early juvenile red sea bream fed Artemia nauplii enriched with the experimental lipids in Expt II (area%) * See the footnote of Table 4 in nauplii improved the survival and activity of fish in agreement with the former experiments4,9) The importance of the DHA content in nauplii has also been suggested by Gatesoupe and Le Milinaire10) for turbot The importance of DHA was also shown by the fatty acid composition in fish The level of DHA in polar lipid in the fish fed DHA Artemia was higher than that of EPA in the fish fed EPA Artemia, though DHA contents in Artemia were lower than EPA contents, suggesting a selective incorporation of EFA in biomembranes The result indicates that the n-3 HUFA requirement of red sea bream larvae depends on the EPA/DHA ratio in the diet as suggested by Rodriguez et al for gilthead seabream,11) and Watanabe et al for red sea bream,41 Further experiments are required to clarify this point Fujii et al12) showed that the requirement of juvenile red sea bream for EFA was satisfied if the ratio of 18:1n-9 (OA)/n-3 HUFA of polar lipid in fish (EFA index) was lower than 1 The growth and/or survival improved as the ratio of polar lipid or total lipid in fish decreased in some larval fish4,7,9,11,13) In the present study, the ratio of OA/n 3 HUFA of polar lipid in fish was lower than 1 except for the fish in tank Nos 1, 4 and 5 (Tables 6 and 7), though the vitality of fish which received EPA (Tank Nos 3, 6 and 7) was poor Especially, the ratio in fish fed EPA-Artemia was as low as those fed DHA-Artemia, which showed ex cellent vitality This result suggests that the OA/n-3 HUFA ratio used as an EFA index is not adequate for ear ly juvenile red sea bream when they are receiving EPA as a main n-3 HUFA In this context, the ratio of OA/DHA of polar lipid or total lipid may be more adequate for evaluating the EFA status in marine fish larvae and juveniles in terms of the en hancement of vitality by DHA The ratio of OA/DHA of polar lipid in the experimental fish is shown in Tables 6 and 7 It was less than 1 in the fish showing a high survival rate in the activity test (Tank Nos 3, 9 and 10), and was higher than 1 in the fish showing poor vitality In the previ ous studies9,14) the ratio was also lower than 1 in larval red sea bream which showed excellent vitality The present results have shown that the EFA value of DHA is superior to that of EPA for early juvenile red sea bream at the Artemia-eating stage, as already reported by Watanabe et al4) who conducted a similar experiment on larval red sea bream using DHA-enriched rotifers The EFA requirement of early juvenile red sea bream is esti mated to be % for DHA in Artemia on a dry mat ter basis in terms of vitality, and may be satisfied with 225% EPA or 095% DHA (205% n-3 HUFA, on a dry matter basis of Artemia) in terms of the survival rate Acknowledgments We express our sincere gratitude to the staff of the Nagasaki Prefectural Institute of Fisheries for their help in the feeding ex periments This work was supported in part by a research grant from the Fisheries Agency and Grant-in-Aid for Scientific Research from the Mi nistry of Education, Science, Sports and Culture References 1) T Watanabe, F Oowa, C Kitajima, T Fujita, and Y YOne: Relationship between the dietary value of rotifer, Brachionus plicatilis, and their content of w3 highly unsaturated fatty acids Nippon Suisan Gakkaishi, 45, (1979) 2) T Watanabe, F Oowa, C Kitajima, and S Fujita: Relationship be tween dietary value of brine shrimp Artemia salina and their con

6 Requirements of juvenile red sea bream for EPA and DHA 251 tent of n-3 highly unsaturated fatty acids Nippon Suisan Gak kaishi, 46, (1980) 3) T Watanabe: Importance of docosahexaenoic acid in marine larval fish J World Aquaculture Soc, 24, (1993) 4) T Watanabe, M S Izquierdo, T Takeuchi, S Satoh, and C Kitajima: Comparison between eicosapentaenoic and docosahex aenoic acids in terms of essential fatty acid efficacy in larval red seabream Nippon Suisan Gakkaishi, 55, (1989) 5) T Takeuchi, M Toyota, S Satoh, and T Watanabe: Requirement of juvenile red seabream Pagrus major for eicosapentaenoic and docosahexaenoic acids Nippon Suisan Gakkaishi, 56, (1990) 6) T Takeuchi, M Toyota, and T Watanabe: Comparison of lipid and n-3 highly unsaturated fatty acid incorporation between Arte mia enriched with various types of oil by direct method Nippon Sui san Gakkaishi, 58, (1992) 7) M S Izquierdo, T Watanabe, T Takeuchi, T Arakawa, and C Kitajima: Requirement of larval red seabream Pagrus major for es sential fatty acids Nippon Suisan Gakkaishi, 55, (1989) 8) T Watanabe, T Takeuchi, T Arakawa, K Imaizumi, and S Sekiya: Requirement of juvenile striped jack Longirostris delicatissi mus for n-3 highly unsaturated fatty acids Nippon Suisan Gak kaishi, 55, (1989) 9) M S lzquierdo, T Watanabe, T Takeuchi, T Arakawa, and C Kitajima: Optimal EFA levels in Artemia to meet the EFA require ments of red seabream (Pagrus major) Proc Third Intn Symp on Feeding Nutr in Fish Toba Aug 28 Sept I, Japan, 1989, pp ) F J Gatesoupe and C Le Milinaire: Dietary value adaptation of live food organisms for covering the nutritional requirements of ma rine fish larvae Coll Fr-Jp Oceanogr Marseille Sep, 8, (1985) 11) C Rodriguez, J A Perez, M S Izquierdo, A Lorenzo, and H F Palacios: The effect of n-3 HUFA proportions in diets for gilthead seabream (Sparus aurata) larval culture Aquaculture, 124, 284 (1994) 12) M Fujii, H Nakayama, and Y Yone: Effect of w3 fatty acids on growth, fed efficiency and fatty acid composition of red sea bream (Chrysophrys major) Rep Fish Res Lab Kyushu Univ, 3, (1976) 13) W M Koven, A Tandler, G W Kissil, and D Sklan: The impor tance of n-3 highly unsaturated fatty acids for larval Sparus aurata and their effect on survival, lipid composition and size distribution Aquaculture, 104, (1992) 14) T Takeuchi, M Toyota, and T Watanabe: Dietary value of Arte mia enriched with various types of oil for larval striped knifejaw and red sea bream Nippon Suisan Gakkaishi, 58, (1992)