(Received August 20, 1993)

Transcription

1 Fisheries Science 60(3), (1994) Effects of Dietary Oxidized Oil for Penaeus japonicus Shunsuke Koshio, Shin-ichi Teshima, and Akio Kanazawa Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima 890, Japan (Received August 20, 1993) Larval and juvenile prawns P japonicus were fed test diets containing fresh or oxidized corn, squid liver, and pollack liver oils In the larval study, the peroxide values (POV) of test oils added to each of the test diets were respectively 06, 246, 698 meq/kg oil for fresh (Of), slightly oxidized (Os), and mildly oxidized (Om) oils for the corn oil-based diets, 03, 198, 376 meq/kg oil for corresponding squid liver oil-based diets, and 17, 255, 386 meq/kg oil for corresponding pollack liver oil-based diets In the juvenile study, these figures are respectively 06, 299, 443 meq/kg oil for corn oil-based diets, and 19, 353, 458 meq/kg oil for pollack liver oil-based diets Os and Om oils delayed development, reduced postlarval size, and weakened the postlarva1 although mortality was not so high A POV of more than 13 meq/kg seems to be a critical level for larvae whereas a POV of 35 meq/kg was still within the allowable degree of oxidation for juveniles The size of postlarva1 was reduced further when fed diets containing marine Os or Om oils compared to vegetable oil such as corn oil Key words: oxidized oil, Penaeus japonicus, growth, development Since harmful effects of oxidized oil on fish health has been reported in many studies,1) high-quality lipids should be used in the diet formulation Although some oxidation is inevitable during diet processing and warm storage very little is known about the effects of the degree of oxidation on the performance of aquatic animals Although heavy oxidation can result in high mortality of animals due to the toxic oxidation products, there are few studies on the effect of mild oxidation for crustaceans Since the aquaculture of crustaceans is very popular in subtropical or tropical areas, the careful maintenance of feeds quality should be a priority for the industry Thus, it is necessary to determine the allowable degree of oxidation which does not cause mass mortality of crustaceans This study was carried out to determine the effects of diets containing oxidized oil from different sources with varying degrees of oxidation on both larval and juvenile Penaeus japonicus (Kuruma prawn) experiment, anti-oxidants were not present in pollack liver oil but were in corn oil Formulation of Test Diets Microbound diets were prepared by the method described by Teshima et al3) and Koshio et al) in the larval trial The protein source of the test diets was casein, and 10% of test oils were added to the formulation (Table 3) Modified formulae of the mineral and vitamin mixtures in the study of Kanazawa et al5) and Koshio et al6) were used The composition of the test diets for the juvenile trial was similar to that used in the larval trial except for the exclusion of n-3 HUFA, Na-citrate, and Na-succinate, and activated gluten was mixed as a binder (Table 3) Diets were prepared according to the study of Koshio et al7) and 8% of test oils were added in the juvenile trial, Test diets were stored at -20 Ž until feeding Methods of Rearing and Feeding Larvae were reared with a similar method to Koshio et al4) One hun- Table 1 POV (meq/kg) of test oils for larval experiment Materials and Methods Oxidation of Oils In the larval experiment, about 100 ml of pollack liver, squid liver, and corn oils were oxidized for certain periods in a 100ml flask placed in a 40 Ž water temperature bath by inducing air-flow through a pipette until three different peroxide values (POV) were obtained POV was used as an indicator to estimate the degree of oxidation for test oils The POV of test oils and duration (hours) required to produce the necessary degree of oxidation are shown in Table 1 A total of nine oils were used: fresh corn oil (COO, slightly oxidized corn oil (COs), mildly oxidized corn oil (Corn), fresh squid liver oil (SOf), slightly oxidized squid liver oil (SOs), mildly oxidized squid liver oil (SOm), fresh pollack liver oil (Pot), slightly oxidiz ed pollack liver oil (POs), and mildly oxidized pollack liver oil (POm) The POV of the test oils was measured by the method of the Japanese Society of Lipid Chemistry2) Pollack liver oil and squid liver oil used in this study did not contain anti-oxidants but corn oil did In the juvenile experiment, the preparation and POV measurement of test oils were the same as in the larval experiment A total of six oils were used: COf, COs, Corn, POf, POs, and Porn (Table 2) Like the larval * ( ) indicates aeration time (h) under 40 Ž Table 2 POV (meq/kg) of test oils for juvenile experiment * ( ) indicates aeration time (h) under 40 Ž

2 284 Koshio et al, dred zoea1 stage larvae from wild-caught female spawners were distribut ed in 1l beakers containing 1l filtered and aerated sea water Duplicated beakers were placed in a water bath maintained at a constant temperature (27 }1 Ž) by thermostatically controlled heaters The feeding method, feed amounts, and feed size followed the study of Teshima et al8) The trial was terminated when all larvae reached the postlarval stage and data taken on the day when more than 50% of the larvae reached the postlarva1 stage In the juvenile trial, prawns (average 02 g) were randomly selected from a stock tank, and 15 animals per experimental tank with two replicate tanks per treatment (total 30 prawns) were communally housed in 54-1 rec tangular PVC tanks (60 ~ 30 ~ 30 cm) with sand bottoms (5 cm thickness) Other conditions followed the study of Koshio et al7) The trial lasted for 30 days with water temperature ranging from 23 to 28 Ž and the feeding ration was 6% of body weight, which was adjusted based on weighing every Parameter 10 days Measured The performance of larvae (initially 200 individuals) in each diet group was evaluated based on survival, developmental stages and metamorphosis (the number of postlarva1/initial number of zoea1) ~ 100, on the day when more than 50% of larvae reached the postlarva1 stage, and the total length of postlarva1 (TLP, from the tip of the rostrum to the end of the telson) In the juvenile trial, besides the survival rates, the following parameters were used to evaluate growth: final wet weight (W1, g), percent weight gain (WG)=(W1-W0) ~100/W0, feed conversion efficiency (FCE)= (W1-WO) ~ 100/D, where W0=initial wet weight (g), D=dry diet intake (g) Chemical Analysis The whole prawn body and diets were chemically analyzed as follows The dry weight was gravimetrically determined after oven-drying at 105 Ž for 24h The percentage ash was determined by ashing sample at 550 Ž for 4 h The protein content was determined by the Kjeldahl procedure as Larval Trial Results All test diets contained about 11 % crude lipids and the POVs of oils extracted from test diets ranged from 17 to 21 meq/kg for fresh, 128 to 165 for slight, and 191 to 254 for mild oxidized oils (Table 4) Although slight changes in POV between the test oils and dietary oils occurred after processing the diets, there was very little change in the POV and ƒ -tocopherol contents of the oils extracted from the test diets during the larval trial The larval performance on the 10th day after hatching is shown in Table 5 Survival was not significantly affected by either the oil source or degree of oxidation The numbers of postlarva1 which appeared on the 10th day were not significantly affected by the oil source but were affected by the degree of oxidation (p<005) In other words, larvae fed diets containing mild oxidized oil metamorphosed into postlarvae significantly later than other groups, and more than 50% of all larvae failed to reach the postlarva1 stage on the 10th day Furthermore, the postlarvae1 fed diets containing mild oxidized oil appeared weak based on visual observations For example, the postlarva stayed on the bottom without swimming around, and were easily captured by a pipette Two-factor ANOVA clearly indicated that TLP was Table 4 Lipid contents and POV of test diets for larvae previously described9) All lipids were extracted with chloroform-metha nol-water by the method of Bligh and Dyer10) and quantified by the method of Bragdon11) The fatty acid composition was analyzed according to the method of Teshima et al 12,13) Statistical Analysis The survival and growth parameters for both larvae and juveniles were compared using the chi-square test and an analysis of variance (ANOVA, Super Anova, Abacus Concepts, Inc), respectively Duncan's new multiple range test (Super Anova, Abacus Concepts, Inc) was applied to determine significant differences between individual treatments when a significance (p<005) of factors was detected by ANOVA Table 3 Composition of basal diet (g/100 g) * COf, fresh corn oil; COs, slightly oxidized corn oil; COm, mildly oxidized corn oil; SOf, fresh squid oil; SOs, slightly oxidized squid oil; SOm, mildly oxidized squid oil; POf, fresh pollack liver oil; POs, slightly oxidized pollack liver oil; POm, mildly oxidized pollack liver oil based diets Table 5 Percentages of development and survival of P, japonicus fed test diets at 10th day from the hatching *1 : Suppliers are followed by the studies of Koshio et al 4,6,7) *2 : See the text in details *1 : Symbols are the same as those in Table 4 *2: M 1=mysis1; M2=mysis2; M3=mysis3; P1=postlarval

3 Effects of Oxidized Oil for P japonicus 285 Table 6 Mean total length of postlarva1 fed test diets Table 8 Important fatty acid composition of NL in test diets *1 : Symbols are the same as those in T able 4 *2: MTLP (mean total length of p ostlarva1, mm); mean }s d The values with the same superscripts are not significantly different at 5% level *3: Number of animal s Table 7 Analytical results of juvenile diets *1: Symbols are the same as those in Table 4 *2: nd: not detected *1 : Symbols are the same as those in Table 4 *2: Dry wt basis *3: POV 1, values after processing; POV 2, values after 30 day Table 9 Important fatty acid composition of PL in test diets storage at -20 Ž significantly affected by both the oil source and degree of oxidation (p<005) In all groups, mean TLP decreased with increasing degree of oxidation (Table 6) As POV in the diet increased, the TLP of larvae which were fed diets containing both squid and pollack liver oils became smaller than those fed diets containing corn oil Juvenile Trial Since it was assumed that juveniles are less sensitive to oxidation than larvae, higher degrees of oxidation were used for the juvenile experiment The results of the test diets are shown in Tables 7 to 9 The oxidation proceeded further in pollack liver oil than in corn oil during 4 week-storage at -20 Ž (Table 7) In the neutral lipids (NL) of the test diets, the percentage of 18:2n-6 for both pollack liver oil and corn oil based diets and that of n-3 HUFA including 20:5n-3 and 22:6n-3 for pollack liver oil based diet decreased as the degree of oxidation in the diets increased (Table 8) On the other hand, the percentage of total saturated and monoenoic fatty acids for the corn oil based diet increased as the degree of oxidation in the diets increased (Table 8) In the polar lipids (PL) of the test diets, the percentage of 18:2n-6 and 18:3n-3 decreased with increased POV in the pollack liver oil based diet and a similar trend occurred in n-3 HUFA in both pollack liver oil and corn oil based diets On the other hand, the percentage increased with increased POV of the diet in all saturated and monoenoic fatty acids *1: Symbols are the same as those in Table 4 *2: nd: not detected for the pollack liver oil based diet (Table 9) The survival of juveniles in the feeding trial for 30 days was not significantly affected (p<005) by the degree of oxidation but was affected by the oil source Juveniles fed the pollack liver oil based diet showed a significantly higher

4 286 Koshio et al Table 10 Result of a 30-day feeding trial for juveniles*1 *1: Values are mean or mean }S D *2: Symbols are the same as those in Table 4 *3: Values with the same scripts are not significantly different at 5% level *4: Feed conversion efficiency=wt gain (g) ~100/feed intake (g) Table 11 Analytical results of whole juvenile body at the end of feeding trial Table 13 Important fatty acid composition of PL in whole body of juveniles *1: Symbols are the same as those in Table 4 *2: Dry wt basis Table 12 Important fatty acid composition of NL in whole body of juveniles *1: Symbols are the same as those in Table 4 *2: nd: not detected *1: Symbols are the same as those in Table 4 *2: nd: not detected survival rate than those fed the diet containing corn oil (Table 10) The highest weight gain was in the POf group but this was not significantly different from gains in the POs and POm groups In the corn oil based diet group, the COf group showed a significantly higher weight gain than the COs and COm groups (Table 10) Although significant effects of both the degree of oxidation and oil source were detected for FCE by ANOVA, the effect of the degree of oxidation was not as great as the oil source Chemical analysis of the whole juvenile body indicated that neither the degree of oxidation nor oil source affected the proximate composition of the body: average values of moisture, crude protein, lipid and ash were 78, 62, 71, and 19%, respectively (Table 11) In NL of the whole body, n-3 HUFA content decreased with increased degree of oxidation for juveniles fed pollack liver oil based diets whereas total saturated and monoenoic acid contents in creased with increased degree of oxidation for juveniles fed both pollack liver oil and corn oil based diets (Table 12) In the polar lipds (PL) of the whole body, n-3 HUFA content decreased slightly with increased degree of oxida-

5 tion for juveniles fed the pollack liver oil based diet (Table 13) Discussion There are very few studied on the effect of dietary oxi dized lipids for Kuruma prawn compared to those for fish1) The results of the larval trial in this study showed that although high mortality did not occur for the degree of oxidation used in this study, slightly and mildly oxidized oils delayed development, reduced postlarval size and caused weakness Therefore, the diet containing 11 % lipids with POV of more than 13 meq/kg seems to be a critical level for larval Kuruma prawn Since the primary products of early-stage autoxidation are hydroperoxides,14) the hydroperoxides seem to impair the phisiological condition of larvae Furthermore, the poor performance of larvae fed diets containing the pollack liver oil and squid liver oil with slightly and mildly oxidized oils may be related to the destruction of dietary n-3 HUFA by oxidation It is known that ƒ -tocopherol can function as an anti oxidant and that the contents of dietary ƒ -tocopherol may decrease as oxidation proceeds The ƒ -tocopherol require ment for animals may be increased by feeding a diet con taining oxidized lipids If 20 to 30 mg of dietary ƒ -tocoph erol per 100g diet is the safe level for the performance of Penaeid as suggested by Akiyama,15) it is likely that the short experiment (10 days) and sufficient quantity of ƒ - tocopherol (632 mg/100 g diet) in the larvvval trial did not cause the deficiency of ƒ -tocopherol even though the a tocopherol requirements for larvae increased Although it has been reported that 20:5n-3 (EPA) and 22:6n-3 (DHA) are more effective than 18:2n-6 and 18:3n-3 for juvenile Kuruma prawn, 16) the postlarva1 fed the COf based diet were even large than the postlarva1 fed SOf diet Furthermore, the survival and development between larvae fed COf, SOf, and POf were very similar Therefore, corn oil can be utilized by larval Kuruma prawn when n-3 HUFA is supplemented to meet the n-3 HUFA requirements In the juvenile trial, the 4-week storage of the test diets caused more oxidation in the pollack liver oil based diet than the corn oil based diet, possibly due to the exclusion of antioxidants and inclusion of polyunsaturated fatty acids in pollack liver oil based diets In both NL and PL of the test diets, the polyunsaturated fatty acids such as 18:2n-6, 20:5n-3, 22:5n-3 and 22:6n-3 tended to reduce as POV increased, but the reduction was not great due to the mild oxidation The pattern of dietary polyunsaturated fatty acids clearly reflected the polyunsaturated fatty acids in the whole body, particularly for prawn fed pollack liver oil based diets Similar results have been reported for rainbow trout, in which the polyunsaturated fatty acids in the liver and muscle reduced when fed a diet containing mildly oxidized oil17) The degree of oxidation used in the present study does not seem high enough to induce mortality of juveniles whereas the oil source had a significant effect on growth The slower growth of juveniles fed the corn oil based diet compared with the pollack liver oil based diet may be due to the lack of EPA and DHA, which agrees with the study of Kanazawa et al16) A significant effect of the degree of oxidation on growth was, not detected and there was no Effects of Oxidized Oil for P japonicus 287 sign of vitamine E deficiency in the present study There fore, the degree of oxidation in diets containing mildly oxidized oil in this study (POV = 38 meq/kg oil) was still below the level which damages the juvenile performance although the content of n-3 HUFA in the whole body was lowest in animals fed the POm based diet Several studies on the effect of oxidized oil on fish have been carried out rather than on shrimp and prawns It was reported that Yellowtail Seriola quinqueradiata showed 23 % mortality and poor growth when fed a diet containing 5% oxidized oil and a POV of 26meq/kg oil18) On the other hand, the weight gain and mortality of rainbow trout were not significantly different when fed diets containing 75% herring oil and a POV ranging from 6 to 51 meq/kg oil19) Furthermore, when rainbow trout fed a diet con taining 75 % herring oil and POV 120 meq/kg oil, growth did not reduce but mortality did20) This study demonstrated that juveniles are more resistant to oxidation than larvae However, the growth and FCE of juveniles were significantly impaired by feeding a COm based diet It is likely that animals are more sensitive to oxidized oils when supplies of EPA and DHA are in sufficient Acknowledgments The authors would like to express their gratitude to Mr K Matsushita for his assistance in conducting the experiments and analyses, and to Riken Vitamin Co, Ltd for supplying squid liver and pollack liver oils References 1) J D Hendricks and G S Bailey: Adventitious Toxins, in "Fish Nutrition" (ed by J E Halver), Academic Press, Inc, San Diego, 1989, pp ) Japanese Society of Lipid Chemistry: Standard Methods of Lipid Analysis, 1986, p 392 3) S Teshima, A Kanazawa, and M Sakamoto: Microparticulate diets for the larvae of aquatic animals Mem Fac Fish, Kagoshima Univ, 2, (1982) 4) S Koshio, A Kanazawa, and S Teshima: Nutritional evaluation of crab protein for larval Penaeus japonicus fed microparticulate diets Aquaculture, 81, (1989) 5) A Kanazawa, S Teshima, and S Tokiwa: Nutritional requirements of prawn Z Effect of dietary lipids on growth Nippon Suisan Gakkaishi, 43, (1977) 6) S Koshio, A Kanazawa, and S Teshima: Search for effective protein combination with crab protein for the larval Kuruma prawn Penaeus japonicus Nippon Suisan Gakkaisshi, 58, (1992) 7) S Koshio, S Teshima, A Kanazawa, and T Watase: The effect of dietary protein content on growth, digestion efficiencies and nitrogen excretion of juvenile Kuruma prawns, Penaeus japonicus Aquaculture, 113, (1993) 8) S Teshima, A Kanazawa, H Sasada, and M Kawasaki: Requirements of the larval prawn, Penaeus japonicus, for cholesterol and soybean phospholipids Mem, Fac Fish Kagoshima Univ, 31, (1982) 9) S Koshio, A Kanazawa, and S Teshima: Nutritional evaluation of dietary soybean protein for juvenile freshwater prawn Macrobrachium rosenbergii Nippon Suisan Gakkaishi, 58, (1992) 10) E G Bligh and W J Dyer: A rapid method of total lipid extraction and purification Can J Biochem Physiol, 37, (1959) 11) J H Bragdon: Colorimetric determination of blood lipids J Bio Chem, 190, (1950) 12) S Teshima, A Kanazawa, and H Okamoto: Analysis of fatty acids of some crustaceans Mem Fac Fish, Kagoshima Univ, 25, (1976) 13) S Teshima, A Kanazawa, and R Shimamoto: Anatomical

6 288 Koshio et al distribution of sterols and fatty acids in the bivalve Mactra chinensis Nippon Suisan Gakkaishi, 54, (1988) 14) E N Frankel: Recent advances in lipid oxidation J Sci Food Agric, 42, (1991) 15) D M Akiyama, W G Dominy, and A L Lawrence: Penaeid Shrimp Nutrition, in "Marine Shrimp Culture: Principles and Practices" (ed by A W Fast and L J Lester), Vol 23, Elsevier, Amsterdam, 1992, Chapter 25, pp ) A Kanazawa, S Teshima, S Tokiwa, M Kayama, and M Hirata: Essential, fatty acids in the diet of prawn-il Effect of docosa hexaenoic acid on growth Nippon Suisan Gakkaishi, 45, (1979) 17) C B Cowey, E Degener, A G J Tacon, A Youngson, and J G Bell: The effect of vitamin E and Oxidized fish oil on the nutrition of rainbow trout (Salmo gairdneri) grown at natural, varying water temperatures Brit J Nutr, 51, (1984) 18) T Murai, T Akiyama, H Ogata, and T Suzuki: Interaction of dietary oxidized fish oil and glutathione on fingerling Yellowtail Seriola quinqueradiata Nippon Suisan Gakkaishi, 54, (1988) 19) S S, 0 Hung, C Y Cho, and S J Slinger: Measurement of oxidation in fish oil and its effect on vitamin E nutrition of rainbow trout (Salmo gairdneri) Can J Fish Aquat Sci, 37, (1980) 20) S S O Hung, C Y Cho, and S J Slinger: Effect of oxidized fish oil, DL-ƒ -tocopheryl acetate and ethoxyquin supplementation on the vitamin E nutrition of rainbow trout (Salmo gairdneri) fed practical diets J Nutr, 111, (1981)