Lipid nutrition in fish

Comp. Biochem. Physiol. Vol. 73B, No. 1, pp. 3 to 15, 1982 Printed in Great Britain

0305-0491/82/090003-13503.00/0 Pergamon Press Ltd

LIPID N U T R I T I O N IN FISH T. WATANABE Laboratory of Fish Nutrition, Tokyo University of Fisheries, Konan 4, Minato-ku, Tokyo, Japan (Received 2 February 1981)

INTRODUCTION Dietary lipids play important roles in the energy production processes of animal tissues and as the source of essential fatty acids (EFA). Besides these functions they do have other important dietary roles as carriers of certain non-fat nutrients, notably the fat-soluble vitamins A, D and K. Recent studies on EFA in fish have demonstrated that EFA requirements of fish differ considerably from species to species. On the other hand, the results of studies on the energy requirements of fish during recent years have indicated that in the carnivorous fish such as rainbow trout, eel, yellowtail and plaice which have limited ability to utilize carbohydrates of high mol. wt as an energy source, dietary lipids play an important role in this respect and have a sparing action on dietary protein. ESSENTIAL FATTY ACID REQUIREMENT OF FISH During the last few years, the nutritional aspects of EFA in fish have been extensively studied. Among various species saimonid fish have been chosen as the first experimental animals in several studies, and many investigators have demonstrated that rainbow trout (Salmo gairdneri) require dietary fatty acids of the linolenic family (093 series) for maximal growth, feed conversion and freedom from pathology (Lee et al., 1967; Higashi et al., 1964, 1966; Castell et al., 1972a,b,c; Watanabe et al., 1974a,b,c). Nicolaides & Woodall (1962) initially found impaired pigmentation in chinook salmon fed diets deficient in linoleic (18:2096)* and linolenic (18:3093)* acids. Castell et al. (1972a) found that the requirement of rainbow trout for 18:3093 is 1~o in the diet and no combination of 18:3093 with 18:2096 resulted in as fast a growth rate or as efficient a feed conversion ratio as 1~o of 18:3093 alone in the diet. Inclusion of 18:2096 in the diet resulted in some improvement in growth and feed conversion compared with EFA-deficient diet, however, the 096 fatty acid did not prevent some EFA deficiency symptoms such as the "shock syndrome". Later experiments of Watanabe et al. (1974a,c) place the linolenic requirement of this spe* A short hand designation for fatty acids will be used throughout where the co number identifies the position of the first double bond counting from the methyl end. Linoleic and linolenic acids would be written respectively 18:2o96 and 18:3o93. The first number identifies the number of carbons, the second number, the number of double bonds and the last number the position of the double bond.

cies at between 0.8 and 1.6~o in the diet. Fish fed on diets containing less than 0.5~o 18:3093 exhibited retarded growth, erosion of the caudal fin, and a shock syndrome caused by physical irritation of the fish (Castell et al., 1972a). In a series of papers, the authors described the physiological symptoms of EFA deficiency (Castell et al., 1972b) and the effect of EFA deficiency on the lipid metabolism and fatty acid composition (Castell et al., 1972c). They concluded that 18:3093 has an essential role in nutrition of trout similar to that assigned to linoleic acid in higher animals. The EFA requirements of rainbow trout reported by these authors were obtained by using diets containing lipids in relatively low amounts (2-5Yo). Takeuchi & Watanabe (1977b) investigated whether in trout there is an increased requirement for dietary 18:3o93 as the lipid levels in the diet are increased. They found that feeding a diet containing 1~o 18:3093 with 4~o 12:0 resulted in a good growth as demonstrated by Castell et al. and Watanabe et al., while the same diet containing 1~o 18:3093 with 9~o or 14~ 12:0 resulted in reduced growth. With such elevated lipid levels, more than 2~ 18:3093 was required for maximal growth, indicating that the elevated dietary lipid levels increase the requirement of rainbow trout for 18:3093. They suggested consequently that the EFA requirement should be expressed as percentage of dietary lipids and that of rainbow trout for 18:3093 can be postulated to be around 20~ of dietary lipids. This fact is very important for the practical diet for rainbow trout which require a relatively high amount of lipid as an energy source. The channel catfish (lctalurus punctatus), one of the most important warm water fish in North America, is found not to utilize 18:3093 as efficiently as do salmonids (Stickney & Andrews, 1972). Dupree (1969) at first showed that corn oil added to the basal diet resulted in a positive growth response and protein sparing effect, but it appeared in their later experiment that 18:2co6 of corn oil inhibited growth as both beef tallow and hydrogenated corn oil were superior to untreated corn oil. The same results were obtained by Stickney & Andrews (1972), that the highest average weights occurred when the diets were supplemented with beef tallow, olive oil and menhaden oil triglycerides and the lower gains were obtained from groups fed diets containing safflower oil high in 18:2096 and linseed oil high in 18:3093. Furthermore, fish fed the fat-free diet revealed no EFA deficiency syndromes other than a reduction in growth and elevated level of 20:3099 in the liver and carcass. These

4

T. WATANABE

results suggest that the requirement of catfish for EFA is much lower than that of rainbow trout. Another warm water fish, the carp (Cyprinus carpio), one of the most important cultured fish in Japan, is found to be similar to catfish in EFA requirement (Watanabe et al., 1975a). Initially they conducted feeding experiments using carp fingerlings weighing around 2.5 g and found them able to grow on a diet with no fat for a fairly long period without any appreciable problems, differing considerably from the case for rainbow trout. Adding saturated lipid (5°~, methyl laurate) resulted in a positive growth response and supplements of 18:2~,~6 or 18:3~o3 to the diet for 22 weeks resulted in little further improvement in growth and feed conversion. The relatively low growth rate obtained in the fat-free group may be attributable to the lower caloric content due to the absence of fat. When the feeding trials were done with very young carp weighing about 0.65 g which had been kept on a fat-free diet for 4 months before the initiation of feeding trial, it was clearly shown that these fish had an EFA requirement for both 18:2o~6 and 18:3~o3 (Watanabe et al., 1975b: Takeuchi & Watanabe, 1977a). The best weight gain and feed conversion were obtained in fish receiving a diet with both 1'~ 18:2~o6 and 1°,, 18:3~)3. These results obtained in both catfish and carp seem to indicate that the requirement of these warm water fish for EFA are far less than those of the cold water fish such as rainbow trout as suggested by Castell (1978). The eel (Anguilla japonica), another important warm water fish, has also a requirement for both 18:2(J~6 and 18:3(o3. Arai et al. (1971) found that a mixture of corn oil and cod liver oil in a 2:1 ratio, containing both ~6 and ~,)3 fatty acids was the most favorable for growth of eels. Later Takeuchi et al. (1980) found that the eel required 18 : 20)6 and 18 : 3~o3 in the same proportion as the carp but at a lower level in the diet, namely 0.50~,~,of each rather than 1°,, of each. Freshwater salmon parr undergo a characteristic transformation to smolts before embarking on their seaward migration. It is of interest whether there is a change in EFA requirement along with distinct morphological, physiological and behavioral changes in salmonids undergoing the parr smolt transformation. Takeuchi et al. (1979c, 1980b) investigated the need for EFA of chum salmon (Oncorhynchus keta) held in both freshwater and seawater and found that the requirement of chum salmon for EFA did not change according to their living environment. Chum salmon were also found to be the most sensitive to EFA deficiency among various kinds of fish species hitherto examined at several laboratories. The EFA-deficient diet resulted in poor growth, low feed efficiency, high mortality, and swollen pale livers from the second week of feeding. The addition of either 18:2~o6 or 18:3(~3 to the diet improved growth conditions, but the mortality was not efficiently reduced by the addition of 0.55o 18: 3o)3. The best weight gain and feed efficiency were obtained in the fish receiving the diet supplemented with both 1~o 18:2~o6 and 15~ 18:3o)3 as those obtained in carp. The optimum level of dietary ~o3 fatty acid ranged from I')o to 2.57~, and dietary (~6 fatty acids higher

than 1°~, depressed the growth of the same salmonid, coho salmon (Oncorhynchus kisutch) (Yu & Sinnhuber, 1979). Takeuchi & Watanabe (1980) also confirmed that 18:3o~3 was effective for the prevention of EFA deficiency in coho salmon. Kanazawa et al. (1980) recently examined the EFA requirement of Tilapia zillii, a tropical, herbivorous fish which is able to live in both freshwater and seawater. In Tilapia the growth-promoting effects of 18:2o)6 and 20:4096 were found to be superior to those of 18:3~o3 and 20:5~J3, indicating that this fish requires ~o6 fatty acids rather than ~,)3 fatty acids. The dietary requirement of Tilapia for 18:2~,)6 or 20:4<,)6 was about 1.0°il in the diet. On the other hand, Yone et al. demonstrated that 18:3(~)3 is not of much importance per se for the nutrition of marine fish such as red sea bream (Chrysophrys major) (Yone & Fujii, 1975a,b; Fujii & Yone. 1976a,b), black sea bream (Mylio macrocephalus), opaleye (Girella nigricans) and yellow tail (Seriola quinqueradiata) (Yone, 1978) as for that of freshwater fish. The red sea bream and yellowtail are both commercially important, cultured warm water marine fish in Japan. The yellowtail grew better, had higher red cell counts, hematocrit and hemoglobin and better feed conversion when the diet contained a fish oil such as cod liver oil containing a large amount of (~)3 fatty acids of long chain length and high unsaturation than with a vegetable oil such as corn oil high in ~06 fatty acids (Furukawa et al., 1966: Tsukahara et al., 1967). Yone et al. (1974) obtained results similar to those in yellowtail when red sea bream were also shown to grow better on diets containing pollock liver oil than on diets with corn oil. The EFA requirement was not satisfied by either 18:2~o6 or 18:3¢o3 (Yone & Fujii, 1975a,b: Fujii & Yone, 1976a). In addition supplemental 18:3~o3 at levels of more than 3'~, in diet resulted in fatty liver for red sea bream, quite different from the case of rainbow trout-which revealed fatty liver in the absence of 18:3~o3 (Fujii & Yone, 1976b). Supplementation with a mixed oil of 20:5to3 and 22:6~o3 prepared from cuttlefish liver oil was effective in improving growth and conditions of these fish. And finally Yone et al. (19781 found that (03 highly unsaturated fatty acids with more than 20 carbon atoms (~o3HUFA) play an essential role in nutrition of these marine fishes and the requirement of red sea bream for ~)3 H U F A is around 0.5°; in diet. A reason for the difference observed in the essential role of 18:3~)3 between marine and freshwater fishes can be presumed from changes in fatty acid composition noted during a long period feeding with a 18:3to3 supplemented diet. In freshwater fishes, carp, trout, eel, Ayu fish (Pteco.qlossus ahivelis), the concentration of 20:5to3 and 22:6t,)3 in body lipid increased as a result of feeding 18:3o)3, but did not in plaice (Pleuronectes platessa) (Owen et al., 1972), and red sea bream (Fujii et al., 1976). From these findings, Fujii et al. presumed that marine fishes posses lower abilities to convert 18:3,)3 to ~3 H U F A when compared with freshwater fishes. The turbot (Scophthahnus maximus) also appeared to be unable to convert dietary 18:2~o6 to 20:4e)6 when fed corn oil, or endogenous 18:1~)9 to 20:3~,>9 when fed the EFA-deficient diet (Cowey et al., t976aI. Cowey et al. (1976b1 further demonstrated that

Lipid nutrition in fish although turbot appeared to have an EFA requirement for fatty acids contained in cod liver oil, the requirement was not satisfied by 18:3093. Owen et al. (1975) confirmed by using [14C]-Iabelled fatty acids orally administered to turbot that the chain elongation and desaturation of [1-1'*C]18:1099, 18:2096 or 18:3093 was found to be very limited (3-15~) in the turbot compared to the rainbow trout where 70~o of the radioactivity from 18:3093 was present as 22:6093. Yamada et al. (1980) again confirmed in their recent experiment in which [1-14C] 18:3093 was administered to red sea bream, black sea bream, opaleye, stripped mullet and rainbow trout that the radioactivity of 22:6093 fraction exhibited 14.5~ and 3.6~ of total activity in rainbow trout, but 4.9-0.9 in marine fishes. Later Gatesoupe et al. (1977a) found that the required level of long chain 093 fatty acids for turbot was at least 0.8~o of the diet. In a subsequent trial, they demonstrated a positive growth response by turbot to dietary 18:3~3, although C2o and C22 093 fatty acids were still much more effective than the former acid (Gatesoupe et al., 1977b). HIGHLY UNSATURATED FATTY ACIDS AS EFA (NUTRITIONAL VALUE OF FISH LIVER OIL AS DIETARY LIPIDS)

As mentioned above recent studies on EFA in fish have demonstrated that EFA requirement of fish differ considerably from species to species. Rainbow trout require fatty acids of the linolenic family (093) as EFA, whereas carp, eel and chum salmon require not only linolenic but also linoleic acids for good growth. On the other hand, these fatty acids were found to be non-essential for red sea bream, plaice and yellowtail of marine origin and 093 HUFA, such as 20:5093 and 22:6093, were very effective as EFA for the fishes. It is of immediate interest that fish liver oil including the residual oil from molecular distillation of pollock liver oil for production of vitamin concentrates, were very effective in enhancing growth and improving feed conversion in all the species mentioned above which apparently require respectively different EFA. The mechanism of this growth stimulating effect is unknown but earlier workers suggested that it could be attributable to (1) fat-soluble vitamins A and D (Honjo, 1965), (2) phospholipids (Phillips, 1963), (3) polyunsaturated fatty acids (PUFA) (Higashi et al., 1964; Kaneko et al., 1967), in pollock liver oil. The recent results obtained on EFA requirements of fish, however, suggest that the nutritive value of dietary pollock liver oil in fish is probably due to PUFA, especially 093 HUFA, in the fatty acid fraction. Watanabe & Takeuchi (1976) have shown by feeding rainbow trout two important fractions, nonsaponifiables and total fatty acids, separated from pollock liver oil that the fatty acid fraction was the principal element in the nutritive value of pollock liver oil. Lee et al. (1967) obtained reduced growth when trout were fed corn oil as the sole lipid source but growth increased when linolenate or salmon oil rich in C2o and C22 093 fatty acids were added. A recent study by Trider & Castell (1980) and Castell & Covey (1976) has also shown that cod liver oil is of higher nutritive value for oysters (Crassostrea virginica) and lobsters

(Homarus americanus) than corn oil. These authors

suggest that the difference in nutritive value between cod liver oil and corn oil was partly attributable to their fatty acid patterns. Cod liver oil contains high amounts of 093 fatty acids whereas corn oil is characteristically low in 093 fatty acids. These results suggest that the effective components of pollock liver oil which play an important role as EFA in fish are 093 HUFA such as 20:5093 and 22:6093. Moreover, the result obtained by Watanabe & Takeuchi (1976) that replacing pollock liver oil with a comparable amount of methyl linolenate did not improve fish growth over that realized with the diets containing pollock liver oil, indicates that 093 HUFA may have a higher EFA value than that of 18:3093 in a manner similar to the relationship existing between linoleic and arachidonic acids in mammals (Alfin-Slater & Kaneda, 1962), although Yu & Sinnhuber (1972) reported that 18:3o93 and 22:6093 had similar EFA value for rainbow trout. It is also reasonable to predict that the 20:5093 and 22:6093 of pollock liver oil appear to act in enhancing fish growth, and that a balance of proportion of component fatty acids of these types in natural lipid is more important for growth of fish than is the total amount of 093 fatty acids in diet. In subsequent papers, Takeuchi & Watanabe (1976, 1977c) were able to demonstrate that the growthenhancing effect of the liver oil is due to an additive effect between 20:5093 and 22:6093 in its fatty acid fraction. In addition it was shown that 20" 5093 has the same growth-enhancing effect as 22:6093 and that both the highly unsaturated 093 fatty acids have a biological or EFA efficiency higher than that of 18:3093 on the same basis as 20:4096 is assigned an EFA efficiency higher than that of 18:2o96 in mammals. The EFA of 093 HUFA was also examined in many fish species such as carp, eel, chum salmon and coho salmon by Takeuchi & Watanabe (1977c, 1980) and it was found that the supplemental effect of 0.5~ 20:5093 and 0.5~o 093 HUFA mixture (20:5093:22:6093 = 1 : 1) on the growth of carp, eel and chum salmon slightly exceeded that of 1~ 18:3093 (Fig. 1). The fact that the control diets containing pollock liver oil always produced the best weight gain in various kinds of fish species in spite of its relatively low amounts of 093 fatty acids in the diets (0.4-0.6~o) could be explained by these results obtained by Takeuchi & Watanabe. There is also another fact supported by these results that the commercial diets for various kinds of fish such as rainbow trout, carp, eel, Ayu fish, chum salmon and red sea bream contain lipids at levels of 4~5~o, mostly derived from white fish meal used as a protein source and their contents of 093 fatty acids (mainly consisting of 20:5093 and 22:6093) are around 0.4--0.6~o of the diets. These practical diets, however, always satisfy the EFA requirement of these fishes. Watanabe & Takeuchi (1976) also examined the effect of phospholipids on growth of rainbow trout using lecithin, although there is no direct comparison with the pollock liver oil which contained only a trace amount of polar lipids. The addition of lecithin to methyl laurate at the level of 0.01 and 0.05~o did not result in any improvement in growth conditions. Stickney & Andrews (1972) also found that diets con-

T. WATANABE

of EFA efficiency. Takeuchi & Watanabe (1980) showed that C 2 2 : 2 fatty acid with one double bond at ¢o3 position, but another at or near the carboxyl end, had no EFA value for rainbow trout and chum salmon.

4,5

16

2

3

,m

EFFECT O F EFA DEFICIENCY O N S P A W N I N G

o e~

8

"= 4

I

I

2

I

i

6

I

10

i

I

14

Weeks

Fig. 1. Effect of dietary methyl esters of m3 fatty acids on growth rate of rainbow trout. Curves 1, 2, 3, 4 and 5, 4.5% methyl laurate diet containing 0.5°,0 methyl linolenate, 0.5)o methyl eicosapentaenoate, 0 5°./0methyl docosahexaenoate, 0.25~o methyl eicosapentaenoate plus 0.25~o methyl docosahexaenoate and 0.5% to3HUFA, respectively. raining 8~o beef tallow + 2~o lecithin produced lesser gains than did tallow alone and this indicates that lecithin does not enhance the utilization of saturated fats by catfish whereas enhancement has been reported in mammals (Kulikov, 1965). In later experiments, Takeuchi & Watanabe (1978) again confirmed these results by using lipids extracted from shortnecked clam (Tapes japinica) which contained about 60-70% of polar lipids. The triglyceride and polar lipid fractions of the lipids, both containing sufficient amounts of 20:5~o3 and 22:6~o3, had the same EFA value for rainbow trout. By contrast supplementation of phospholipids prepared from soybean to a diet resulted in improvement in growth of prawn (Penaeus japonica) (Teshima, 1978). Thus, not only 18:3~o3, but also the other fatty acids of m3 series, play a role as EFA for many fish species. In practical fish farming diets, however, 18:3~o3 is not very important, being used mainly for purified research diets; but ~o3 HUFA, such as 20:5~o3 and 22:6~3, are important practically as EFA. The latter fatty acids are very frequently supplemented to fish diets in the form of feed oil (pollock liver oil)just before the administration to fish. On the other hand, the methylene interrupted double bonds seem to be necessary for the appearance

The recent studies by Watanabe et al. 11978, 1979) have demonstrated that EFA deficiency greatly affected the spawning of rainbow trout and red sea bream and that EFA play as important a role in reproductive physiology as tocopherol (Watanabe et al., 1977) in fish as in higher animals (Table 1). The adult rainbow trout fed on the EFA-deficient casein diet containing methyl laurate as the sole dietary lipid for 3 months before spawning matured, but the eggs produced had a low hatching rate. The parent fish, however, unlike fingerlings were little affected by EFA deficiency in that changes of fatty acid distribution in tissue lipids were not observed in the adult fish fed the EFA-deficient diet. It is of some interest that the addition of 1"o of ethyl linoleate in place of linolenate, given to fish in error, improved egg condition to a level comparable with those obtained in the control parent fish, although addition of 18:2o96 to a diet formulation is unnecessary when 18:3to3 is included for reproduction of rainbow trout (Yu et al., 1979). The results obtained in red sea bream fed the EFA-deficient purified diet for 6 months before spawning indicated that the total egg production, proportion of eyed eggs and hatchability were significantly influenced by the EFA status in the diet and were quite low in the group given the EFA-deficient diet. These low quality eggs showed abnormality in the number of oil globules, the average being seven whereas in normal eggs it is usually one. Almost all the fish larvae obtained from the group also showed various kinds of deformations in the body. Supplementation of cuttlefish liver oil high in ¢o3 HUFA effectively prevented these pathologies. NEGATIVE EFFECT O F EFA

It has become apparent by the recent studies on the EFA requirement of rainbow trout that o93 fatty acids of linolenic family including o93 HUFA have an excellent growth-enhancing effect for rainbow trout. Especially, the EFA value of o93 HUFA is about twice as high as that of 18:3~,J3, and the requirement of rainbow trout is satisfied by the former acid at a level

Table 1. Effect of EFA deficiencyon the spawning of rainbow trout and red sea bream

Rainbow trout Control EFA-deficient Red sea bream Control EFA-deficient

Total egg production

Eyedrate (%)

Total hatch (o~,)

2375 1429

83.6 53.9

70.0 46.4

1.74 x 106 1.17 x 106

-

93.9

1.8

0.9

90.0

* Abnormality observed both in eggs and larvae.

Abnormality* (,,~,)

Lipid nutrition in fish 10 o I%w3 HUFA 8

1)',, 18:3~3

6 ,.~ ._~

4 ~ 18:3m3 2)(m3 HUFA

,.~

3%e3 HUFA

|.

i.

t

2

4

i

|

i

6

8

10

Weeks

Fig. 2. Effect of dietary levels of methyl esters of (o3 fatty acids on the growth rate of rainbow trout.

of 10~o, and by the latter acid at a level of 20~o of dietary lipids. The excess amounts of EFA, however, were found to exert ill effect on rainbow trout. The addition of 18:3t~3 or the (o3 HUFA mixture of 20:5o~3 and 22:6m3 (1:1) to diets at a level exceeding an amount 4 times higher than that required by rainbow trout resulted in poor growth and low feed conversion (Takeuchi & Watanabe, 1979) (Fig. 2). The adverse effects of excess t~3 HUFA were similar to those resulting from the use of half the quantity of 18:3o93. Yu & Sinnhuber (1976) reported that addition of 1~o 18:2to6 appeared to stimulate fish growth, but growth of rainbow trout was depressed as the dietary level of 18:2~6 was increased to 2.5 or 5.0~o. They also noted that dietary levels of (06 fatty acids higher than 1~ or an extremely high percentage of e~3 fatty acids depressed growth of coho salmon (Yu & Sinnhuber, 1979). We should be careful about the type and amount of EFA in lipids used to supplement diets, since the EFA requirement changes according to the dietary lipid levels and type of (o3 fatty acids in the lipids.

EFFECT O F DIETARY LIPIDS O N BODY COMPOSITION

The composition of body lipids is most significantly affected by dietary lipids, although addition of dietary lipids of high quality ranging from 5 to 25~ never led to ill effects in rainbow trout or carp, there was a remarkable increase of visceral fat due to the excessive energy density of the diets, however the lipid content of liver is not much affected by dietary lipid levels. This agrees with the work of Ogino et al. (1976) who found that body lipid deposition is directly related to dietary lipid levels in rainbow trout. Similar results have been seen in rainbow trout (Castledine & Buckley, 1980), plaice (Cowey et al., 1975), channel catfish (Garling & Wilson, 1976), carp (Takeuchi & Watanabe, 1979), and turbot (Bromley, 1980).

Dietary lipids lacking EFA or containing oxidation products can, however, affect body composition. Castell et al. (1972a) reported increased muscle water in rainbow trout fed the EFA-deficient diet and in addition liver respiration rate increased and blood hemoglobin content lowered. Watanabe et al. (1974a) confirmed that the whole body, muscle and whole viscera of fish fed on EFA-deficient diets were considerably higher in water content and lower in both protein and lipid contents. The content of liver lipid was also high, indicating a sign of fatty liver. It is very characteristic that the liver of salmonid fish readily reflects EFA deficiency. Chum salmon also revealed swollen pale liver with a high lipid content in EFA deficiency (Takeuchi et al., 1979c). On the other hand, the EFA-deficient diet resulted in decreased lipid content in hepatopancreas of carp (Takeuchi & Watanabe, 1977a). These changes in lipid content of fish were due to alterations in neutral lipids; the content of polar lipids in tissue remained at an almost constant level, not affected by EFA deficiency, and the level of nonpolar lipids in the liver increased by dietary non-essential fatty acids such as 12:0 and 16:0 (Watanabe & Takeuchi, 1976). Accumulation of lipids in livers of EFA-deficient animals could be traced back to impairment of lipoprotein biosynthesis (Fukuzawa et al., 1970, 1971). Dietary 18:3to3 at a level of 4 ~ resulted in fatty degeneration in the liver of red sea bream (Fujii et al., 1976; Fujii & Yone, 1976), quite different from rainbow trout in which fatty liver is prevented by addition of 18:3m3 and from turbot (Legcr et al., 1979) in which a high level of dietary 18:3m3 (3.7% of the diet) presented better growth. Excess amounts of the EFA reduced hepatosomatic index and the lipid content of rainbow trout opposite effects to those occurring in the EFAdeficient fish (Takeuchi & Watanabe, 1979). Fatty acid composition of body lipids are most clearly reflected by the dietary lipids. Fish are able to alter the dietary co6/~o3 ratio in favor of co3 fatty acid incorporation into body lipids (Sinnhubcr, 1969), but the influence of dietary lipids on the fatty acid composition of boty lipids differs between triglycerides and phospholipids. Dietary lipids affect the fatty acid composition of phospholipids to a greater degree than those in the neutral lipids. In freshwater fish dietary 18:2(o6 and 18:3o93 are elongated and desturated, and the former is converted to 20:4c~6 and 22:5co6 and the latter to 22:6o93 in phosphotipid fraction of fish, whereas in triglyceride fraction these fatty acids are deposited unaltered, increasing the concentration of 18:2(o6 and 18:3o93 (Takeuchi & Watanabe, 1977a; Farkas et al., 1980). Farkas et al. (1977) assumed that there is a constant flow of linolenate from triglycerides, via the chain elongation and desaturating sites, to the different phospholipids in carp. As long as the diet contains sufficient linolenate this fatty acid will either be deposited in triglycerides unchanged or converted to 22:6o93 and placed in phospholipids and triglycerides. The EFA status in diets is well known to have a great effect on the fatty acid composition of phospholipids in fish. A fat-free diet or an EFA-deficient diet results in elevated level of "abnormal" eicosatrienoic acid (20:3(o9) together with the elevation of monoethylenic fatty acids in phospholipids and 18:lo99 in

8

T. WATANABE

triglycerides in rainbow trout, carp, eel, chum salmon and catfish, although increased levels of 20:3o99 were not reported in plaice and turbot due to the inability of the fish to make 20:3o99 from 18:1co9 (Owen et al., 1975). Dietary 18:2o,~6 and 18:3o~3 both depress the triene levels. The appearance of 20:3o99 in phospholipids is somehow related to the disappearance of 18:3o93 and 22:6093 from the triglycerides and the accumulation of 20:3o99 starts only after the exhaustion of the linoleanate pool in the triglycerides (Farkas et al., 1977). The increase of 20:3o99 in the tissue of fish fed EFA-deficient diets agrees with the findings of other workers for the rat (Mohrhauer & Holman, 19631, chicks (Hill, 1966), and rabbits (Ahluwalia et al., 1967). For most animal studies, in which fatty acids of the linoleic type satisfy the EFA requirement, Mohrhauer & Holman (1963) have suggested that the ratio of 20:3o99/20:4o96 in the animals' lipids be taken as an index of EFA deficiency. Castell et al. (1972b) pointed out, however, that as rainbow trout require acids of the o93 type, the ratio of 20:3co9/22:6~o3 would be more appropriate for fish as an index of EFA status. Alfin-Slater & Aftergood (1968) proposed the ratio of 20:3o99/20:5o93 as an estimate of the adequacy of linolenate intake in the diet, but in the case of trout 22:6o93 appears to be the primary long chain acid of this series that accumulates in body lipids (Castel et al., 1972b). If the ratio of 20:3~o9/22:6o93 in the phospholipids is used as a criterion for evaluating EFA deficiency, all fish receiving 0.7~o or more of 1 8 : 3 o 9 3 in the diet are receiving sufficient amounts of o93 fatty acids. This judgement is based on a ratio of 0.4 or greater indicating a deficiency in mammals. When 093 HUFA such as 20:5o93 and 22:6o93 are used as EFA, the ratio of 0.4 was reached when 0.5% o93 HUFA was provided in diet (1.3~o of dietary calories), whereas the ratio became less than 0.4 in fish receiving 18:3o93 more than l~o (2.5~o of dietary calories) (Takeuchi & Watanabe, 1976b). The lipids from the fish fed 0.5~o 20:5o93 or 0.5~o 22:6o93 showed 25.3~o and 27.3% 22:6o93, respectively and the corresponding value was 32~,,, when fish were fed either an l~o 20:5~03 or l~o 22:6o93 diet, while a value of only 26.1°,'o was obtained when they were fed a diet with 3".i, 18:3o93. This also indicates therefore that both 20:5o93 and 22:6o93 are more effective as EFA than is 18:3~o3. In carp which require fatty acids of both 18:2t~96 and 18:3o93 for growth, Watanabe et al. (1975b) proposed the ratio of 20:3~o9/20:4o96 as well as the ratio of 20:3o99/22:6o93 for the EFA index in carp. When both ratios are applied for carp as indices, the former ratio becomes less than 0.4 and the latter less than 0.6 if the carp are receiving a sufficient amount of both ~o6 and o93 fatty acids. The use of these ratios obviously depends on the anabolic capacity of the animal concerned and in particular on the ability of the animal to synthesize long chain polyunsaturated fatty acids from dietary precursors at rates concomitant with physiological requirements. Data on this point are necessary before such ratios can be applied to other species newly under cultivation (Cowey, 1975). These ratios, however, are only one index for evaluation of EFA-deficient conditions of fish, and the

evaluation of EFA status should be done from physiological, histological and biochemical aspects in fish. There is one evidence that rainbow trout showed EFA-deficiency symptoms in spite of the fact that the ratio of 20:3o99/22:6o93 became less than 0.4 (Takeuchi & Watanabe, 1977b). As already metnioned, elevated dietary laurate levels increase the requirement of rainbow trout for linolenate. On the other hand, dietary laurate levels exerted no significant effects on fatty acid composition of liver lipids, while there were marked changes in fatty acid distribution attributable to the presence of increasing levels of linolenate in the diet. The linolenate-free diet produced high percentages of 16: 1, 18:1 and 20:3099, and these levels were lowered by the addition of 1~o linolenate, regardless of the amounts of laurate in the diets. Consequently, the EFA index showed low values even in the lo~growth groups receiving diets containing 1'0~;linolenate along with 9°/oor 14,%olaurate. This suggests that it is impossible to decide from EFA index alone whether or not fish are receiving a sufficient amount of EFA to maximize growth. It was also shown by Takeuchi & Watanabe (1976j that not only laurate but also palmitate was not deposited unaltered and exerted no appreciable change in fatty acid composition of rainbow trout when used as part of the lipid source with a moderate amount of o93 fatty acids. It is interesting to note that the saturated fatty acids in fish body lipid remained fairly constant at about 24~o of the total regardless of the high saturated fatty acid content in diets (Yu et at., 1977a). Stickney & Andrews (1971) reported similar results in their feeding experiment with catfish. A diet containing 10% tallow (41.% saturates) was fed to a group of catfish, and another diet containing 10°,, menhaden oil (25~ saturates) was fed to a second group. After 10 weeks at 20c~C water temperature, the saturated fatty acid content of the two groups of fish was found to be the same at about 20°,/, of the body lipids. It appears that there is also an optimum level of unsaturation if maximum growth rate is to be obtained. Excess dietary t,93 or o96 fatty acids present an unnatural or unusual condition forcing the animal to respond metabolically and to adapt to this abnormal situation (Yu & Sinnhuber, 1976). Takeuchi & Watanabe (19761 also noted that the total percentage of o93 fatty acids in polar lipids of the liver increased as the increase of dietary to3 fatty acids and seemed to reach a maximum level at around 40'0~,. A mechanism may exist in fish to regulate and maintain a proper level of body lipid saturation. It is reasonable to consider that the ratio of saturated to unsaturated fatty acids largely reflects the balance of the enzyme systems producing these fatty acids (i.e. fatty acid synthetase and various desaturases and chain elongasesl (Farkas et al., 1980). The balance of saturate to unsaturate in fish tissues is also known to be influenced by the environmental temperature (Roots, 1968; Knipprath, 1966a,b: Kemp & Smith, 1970; Leger et at., 1977: Farkas et al., 1980). ENERGY VALUE OF DIETARY LIPIDS

Lipids play an important role as a source of energy rather than as EFA in fish farm diets, especially for

Lipid nutrition in fish carnivorous fish in which the availability of carbohydrate for energy is very low. It has been considered that fish characteristically require much higher protein levels in the diet than are necessary for birds and mammals (Cowey & Sargent, 1972). This is probably due to the fact that most species of fish are carnivorous and require diets high in protein because of the low availability of complex carbohydrate as an energy source, and consequently some part of the dietary protein is consumed as energy. Addition of lipids, with EFA as an energy source, to a diet, therefore, is helpful in effective utilization of dietary protein (protein sparing effect) (Table 2). The recent results on protein requirement of fish have demonstrated that protein requirement of carp and rainbow trout were almost equal to that of swine and lower than that of broiler chickens when digestible energy other than protein is sufficient in the diet (Ogino, 1980). Namely, this is due to the fact that the feeding rate is significantly lower in fish than in mammals because of poikilothermy and that the protein requirement is changed according to feeding rate. The main protein sparing effect of dietary lipids is to replace protein which could otherwise have been catabolized and used both for energy and to synthesize lipid. The sparing of dietary protein by lipids has been extensively investigated in various species of fish (Lee & Putnam, 1973; Page & Andrews, 1973; Takeda et al., 1975; Adron et al., 1976; Takeuchi et al., 1978a,b,c, 1979a,b; Shimeno et al., 1980; Bromley, 1980). Takeuchi et al. (1978b) have demonstrated the optimum ratio of protein to lipid in diets of rainbow trout by feeding them various diets containing different amounts of lipids (5-20~o) in different protein levels (16-48~o). The optimum ratio of protein to lipid in diets of rainbow trout is 35~o to 15-20Yo, and that at these levels of lipids dietary protein can be reduced from 48Yo to 35Yo with no loss in weight gain. They also determined the optimum ratio of energy to protein in diet (DE kcal/protein ~o) by using diets with various levels of lipids (5-25Yo) at a fixed protein level of 35~o (Takeuchi et al., 1978c). With an increase in the amount of dietary lipids, both the values for PER and NPU increased, giving a maximum protein retention with best weight gain in fish fed a diet containing approx 18Yo lipid. The rate of energy retention in the fish body was 60~o, regardless of the lipid levels in the diets or the growth rate of the fish. The digestibilities of protein and lipids were not affected by the lipid content of the diets and were as high as 98~o. The experimental value of the optimum DE (kcal)/protein (~o) for the optimum growth of trout was 130 when based on the measured digestible energy, rather higher than those calculated in brook trout (Phillips et aL, 1966; Ringrose, 1971), rainbow trout (Lee & Putnam, 1973; Zeitoun et al., 1973), channel catfish {Garling & Wilson, 1976) and yellowtail (Takeda et al., 1975). Thus, protein concentration can be decreased by approx 15~o in rainbow trout diets, if high-quality lipids, capable of satisfying the EFA requirement of the fish, are added at the level of approx 18~o. It was also found to be possible to reduce the protein level of yellowtail diets from 70 to 55~o without retardation of growth if the caloric content was maintained at a high level with pollock liver oil (Takeda et al., 1975). Bro-

mely (1980) has also demonstrated that the protein sparing action of the lipid was most pronounced when turbot were fed to about three-quarters of satiation on a diet supplemented with 6~o lipid. Under these conditions 42~o of the dietary protein was converted into fish protein, compared with 32~o conversion on the basic diet which contained 0.5Yo lipid. On the other hand, omnivorous fish, such as the carp can utilize effectively both carbohydrate and lipids as dietary energy sources. The enrichment of the digestible energy content from 320~60 kcal/100 g diet by addition of lipid at levels of 5-15~o to diets resulted in no improvement in growth, feed conversion, or the value for NPU, when the dietary protein level remained around 32Yo, while in the fish fed diets containing 23~o protein values for these parameters were quite low, regardless of the DE contant in the diet (Takeuchi et al., 1979a). The optimum DE/protein for the optimum growth was 97-116 when based on the measured digestible energy. Protein sources for fish farming diets depend heavily upon fish meal, produced from demersal fish from northern waters, as a raw material. With the establishment of a 200 sea mile fishing zone, the haul of fish in the future will decrease, thus making it necessary to lower the fish meal contents of diets as far as possible, DIETARY LIPID LEVELS AND a-TOCOPHEROL REQUIREMENT

High intakes of lipids containing polyenoic fatty acids increase the animals' requirements for vitamin E, although addition of lipids to a diet is very effective in promoting utilization of dietary protein. A relationship between the requirement of animals for ~-tocopherol and the polyunsaturated fatty acid (PUFA) content of the diets has been reported by many workers, A direct relationship between the vitamin E intake and PUFA intake in man has been postulated by Horwitt (1961, 1962) who reported that elevated dietary linoleate levels increase the tocopherol requirement. Hayes & coworkers (1969) also reported in studies with dogs that although the requirement for tocopherol was directly related to PUFA consumption, this was associated with metabolism of the fat and not with an antioxidant role for the vitamin. On the other hand, Alfin-Slater et al. (1972) found no evidence of ~-tocopherol deficiency or of an increased ct-tocopherol requirement when vegetable seed oil rich in polyunsaturates were fed at a level of 30Yo of the diet without a supplementary source of vitamin E. However, few data are available on these relationships in fish. Watanabe et al. (1977) investigated in carp whether there is an increased requirement for dietary tocopherol as the unsaturation of the fat in the diet is increased. In their experiment the apparent muscular dystrophy, the so-called "Sekoke" disease characterized by a marked loss of flesh in the back which is known to be induced by the absence of ~t-tocopherol (Watanabe et al., 1970a) or oxidized lipids (Hashimoto et al., 1966; Watanabe et al., 1967, 1968, 1970), was used as an index of a-tocopherol requirement. And they demonstrated that the elevated dietary linoleate levels increase the tocopherol re-

P L P P* Ct L P L

P L

P L P L P L C

Eel

Yellowtail (I)

71:0:8 55:0:17 68:4:5 53:4:15 40:0:6 40:0:18 40:20:9

52:22:7 41:23:16

40:18:5 36:32:15 42:10:6 42:25:6 32:45:5 32:30:15 39:21:4 35:31 : 18

384 383 353 369 233 347 392

361 398

275 407 259 316 356 384 276 430

367 402

54 70 52 70 58 87 85

70 97

69 113 62 76 110 125 71 119

75 112

Calorie/protein

7.5 7.5 4.3 5.8 0.7 0.8 1.2

3.3 3.8

6.0 7.5 4.3 5.0 4.7 4.2 1.7 2.2

4.6 4.9

Per cent gain/day (%)

85 69 124 75 96 75 62

67 51

48 40 44 39 33 31 50 37

38 31

Protein required for 100 g of weight gain (%)

Cowley et al. (1975)

Shimeno et al. (1980)

-40 -22 -35

Takeda et al. (1975)

K a g o s h i m a Pref. Fish. Exp. St.+

Ohmae.~

-19

-24

26

Takeuchi et al. (1979a)

-15~ -30

-

Garling et al. (1976)

Takeuchi et al. (1978c)

Researcher

- 17

- 18

E(P) (%)

EtL~E(P)

* P. diets with high protein and low lipid content: L, diets with low protein and high lipid content: C, diets with high carbonhydrate and low lipid content. t Protein: Digestible carbohydrate: Lipid. ++Watanabe T. (1980) Lipids. In N u t r i t i o n in Fish and Diet (Edited by Ogino C.) pp. 149 186. Koseisha-Koseikaku, Tokyo.

Plaice

Yellowtail (2)

Carp (2)

Carp(l)

P: C: L% (%)

P* 49:30:5 L 36:30:15

Channel catfish

Rainbow trout

Fish

Energy content (kcal/'100 g)

Diet

Table 2. Protein sparing effect by dietary lipid in fish

Z

>. ...q

H

Lipid nutrition in fish quirement of carp, judging from the appearance of apparent muscular dystrophy. They again confirmed in carp by feeding them diets containing 15-20% pollock liver oil (Watanabe et al., 1981) the increased requirement for tocopherol. The tocopherol requirement of rainbow trout, markedly lower than that of carp (unpublished data), was also found to be affected by dietary lipid content (Watanabe et al., 1978). Feeding the tocopherol-deficient diet containing 15% methyl esters of pollock liver oil to rainbow trout of 0.9 g in body weight which had been kept on a tocopherol-free diet for 2 weeks before the initiation of feeding resulted in poor growth, low feed conversion and a high mortality along with convulsions. Addition of 5 mg of a-tocopherol to the diet effectively prevented these variables. OXIDATION OF LIPIDS IN FISH DIETS

Marine lipids high in polyunsaturated fatty acids, especially tn3 fatty acids, provide EFA resulting in good growth and feed conversion, and good energy source resulting in sparing action on dietary protein. However, these lipids are known to be quite susceptible to autoxidation when exposed to atmospheric oxygen. If adequate care is not taken in the preparation and storage of diets, the positive nutritional value of ~3 fatty acids in fish lipids becomes a negative factor for fish. The products of lipid oxidation may react with other nutrients such as protein, vitamins, etc. and reduce the dietary value of lipids or exert ill effect on fish. The effect of oxidized lipids on dietary proteins, enzymes and amino acids have been demonstrated by Roubal & Tappel (1966), Opstvedt (1974) and Ko et al. (1975). Njaa et al. (1966) found that lipid oxidation in herring meal had a greater effect on the digestibility than on the NPU value. Opstvedt (1974) also confirmed that oxidation of lipid decreases the energy value of the fish meal and probably cause a reduction in the protein quality of the meal. Oxidized lipids are also well known to be toxic for various kinds of fish. Hashimoto et al. (1966) demonstrated that the disease of carp, called "Sekoke" disease, histologically muscular dystrophy, a serious problem for carp culturists in Japan, was induced by oxidized lipids in dried silk-worm pupae used as a main ingredient of feedstuff. They found that the disease was effectively prevented by the addition of DL-0ttocopheryl acetate (50 mg/100 g diet), but not by antioxidants such as methylene blue, BHA, ethyl gallate, DPPD and ethoxyquin (Watanabe et al., 1966, 1967). Sinnhuber et al. (1968) similarly proved that addition of vitamin E could prevent the toxic or negative effects of adding 5~o highly oxidized salmon oil to the diet of rainbow trout. Fowler & Banks (1969) described symptoms occurring in chinook salmon fingerlings when rancid fish meals were fed. Recently Murai & Andrews (1974) also demonstrated muscular dystrophy incuded by oxidized menhaden oil in channel catfish. Fish fed diets containing oxidized menhaden oil without supplemental ~t-tocopherol or ethoxyquin exhibited poor growth, food conversion, and survival rates; high incidence of three distinct gross syndromes occurred-exudative diathesis, muscular dystrophy, and depigmentation. Tocopherol had a

much greater effect on all variables than ethoxyquin. The results of the preventive effect of ct-tocopherol and ethoxyquin obtained by Watanabe et al. and Murai & Andrews may conicide with those by Takeuchi (1972) who reported that ct-tocopherol may act as a peroxide decomposer and stabilizing factor of intestinal epithelium, which improves the absorption of oxidized lipids, while ethoxyquin did not improve absorption of hydroperoxide. The preventive effect of tocopherol homologues on muscular dystrophy in young carp was investigated by Aoe et al. (1972) who reported that ~-tocopherol is far more effective than fl-, ~- and 6-tocopherols, in order of decreasing potency. This may be attributable to the fact that the assimilation of non-~t-tocopherols was significantly lower than that of a-tocopherol in both carp (Higashi et al., 1972) and rainbow trout (Watanabe et al., 1978) and the interconversion between ~t-tocopherol and other tocopherols occurs little in either fish species. The low content of tissue tocopherol in the carp fed diets containing oxidized lipids suggests that muscular dystrophy induced by oxidized lipid was coupled with ct-tocopherol deficiency (Watanabe et al., 1966, 1967). It is most likely that the oxidized lipids destroyed the ~t-tocopheroi originally present. Watanabe et al. (1970a,b,c) proved that the at-tocopherol-deficient diet resulted again in apparent muscular dystrophy in carp after 90 days feeding and that the requirement of carp for ~t-tocopheroi to support the otpimal growth was about 10mg/100g of diet, at a dietary lipid level of 5~o, a value higher than that required by chinook salmon (Woodall et al., 1964) and rainbow trout (Watanabe et al., 1978). Care should be taken not to use oxidized lipids and diets should be protected to prevent further oxidiation. UTILIZATION OF HYDROGENATED FISH OIL AND ANIMAL FATS AS AN ENERGY SOURCE Hydrogenated fish oil or swine fat, lard and tallow are probably suitable as energy sources in fish diets because of their greater stability in prepared diets. In addition these lipids are widely used as energy sources in animal feeds and are easily obtained. Animal fats or hydroganted oils with high melting points are reported to be less digestible than the lipids with lower melting point that are used in formulating feeds for coldwater fish. Consequently they are seldom used in fish rations (Phillips, 1969). Recent studies on the energy requirements of fish, however, have indicated that animal fats, such as lard and tallow, or hydrogenated beef tallow, with other lipids providing the necessary level of EFA, can be used as an energy source of fish diets without any adverse effects on feed efficiency, fish growth or survival (Yu et al., 1977a,b; Takeuchi et al., 1978d, 1979d). Takeuchi et al. (1979d) determined the digestibility of beef tallow and various hydrogenated fish oils with different melting points with carp and rainbow trout and also investigated the effects of fish size and water temperature on the digestibility of these lipids (Fig. 3). The digestibility of hydrogenated fish oils was found to be affected by their melting point and increased as melting point decreased (Fig. 4). Whereas the digestibility of dietary protein was as

T. WATANABE

12

lo0[

Exp. I

F~

.~

~-Pollockliver

oil

:1~ ~

o.

S 40

k

'

to3 H U F A at 10~o of dietary lipid) is effective for n o r m a l growth of rainbow trout at each stage of their development, i.e. fingerlings, adult fish and spawning parent fish. They found that the low protein diet of high energy density is available for all stages of develo p m e n t in rainbow trout and EFA requirement does not change t h r o u g h these stages. In addition, eggs produced from the spawning adult fish fed the diet also gave good yields of eyed eggs with high hatchability comparable to those fed the control commercial diet containing 45~o protein.

Exp. II

~

i

o

~

rap53

°

20 I

10

I

50

I

100

I

150 10

50

I

100

150

REFERENCES

Body weight (gl Fig. 3. Relationship between fish size and apparent digestibility of hydrogenated fish oils with different melting points in carp.

high as 98~o without regard to that of lipids. The hydrogenated oils of m.p. 53°C was significantly low in digestibility in b o t h carp and rainbow trout, especially in the fish weighing less than 10 g. O n the other hand, the beef tallow and hydrogenated fish oil of m.p. 38°C were found to be effectively utilized by both the fish, with a digestibility of more than 700/o regardless of fish size and water temperature, indicating that these lipids are available as a dietary energy source without any adverse effects on fish, when they are used with some marine lipids to provide the necessary level of EFA. Based on the result for rainbow trout, that in terms of dietary lipid intake the requirement of EFA is a b o u t 20~o of 18:3ro3 or 10~o to3 H U F A , then dietary protein will be spared when diets contain 15~, of the dry matter as animal fats or hydrogenated fish oils with melting point less than 40°C and another 3~0 of the dry matter as high quality lipid capable of satisfying EFA requirement. Takeuchi et al. (1978, 1979, 1980) conducted a long-term feeding test (3 years) on a relatively large scale to determine whether the low protein diet (35~o) with a high energy value (18~o lipid containing beef tallow as a main energy source and

100 - R a i n b o w t r o u t

"P~,

80

Carp

~.~

"a

•

~ 60

g

"O

•¢~ 40

~ 2O I

I

I

I

I

I

30 40 50 30 40 50 60 M e l t i n g p o i n t ( ° C ) o f hydrogenated fish oil

Fig. 4. Relationship between melting points of hydrogenated fish oils and their apparent digestibility in rainbow trout and carp. The signs of P and B in the figures indicate those obtained from pollock liver oil and beef tallow. Mean _+ SD in the right figure was calculated from the values determined at different water temperatures.

ADRON J. W., BLAIR A., COWEY C. B. & SHANKS A. M. (1976) Effect of dietary level and energy source on growth, feed conversion and body composition of turbot (Scophthalamus maximus L.). Aquaculture 7, 125 132. AHLUWALIA B. G., PINCUS G. & HOLMAN R. T. (1967) Essential fatty acid deficiency and its effect upon reproductive organs of male rabbits. J. Nutr. 92, 205 214. ALFIN-SLATER R. B. & KANEDA T. (1962) Comparative bioactivity of linoleate and arachidonate in the rat. Fedn Proc. 21, 285. ALFIN-SLATER R. B. & AETEROOOD L. (1968) Essential fatty acids reinvestigated. Physiol. Rev. 48, 758-784. ALFIN-SLATER R. B., SmMMA Y., HANSEN H., WELLS P. ~¢; AFTERGOOD L. (1972) Dietary fat composition and tocopherol requirement Ili. Quantitative studies in the relationship between dietary linoleate and vitamin E. J. Am. Oil Chem. Soc. 49, 395~,02. AOE, H., ABE 1., SAITOT., FUKAWA H. & KOYAMA H. (1972) Preventive effects of tocols on muscular dystrophy of young carp. Bull. Japan. Soc. scient. Fish. 38, 845-851. ARM S., NOSE T. & HASHIMOTO Y. (1971) A purified test diet for the eel, Anguilla japonica. Bull. Freshwat. Fish. Res. Lab., Tokyo 221, 161-178. BROMLEY P. J. (1980) Effect of dietary protein, lipid and energy content on the growth of turbot (Scophthalamus maximus L.). Aquaculture 19, 359-369. CASTELL J. O., SINNHUBER R. O., WALES J. H. t~ LEE J. D.

(1972b) Essential fatty acids in the diet of rainbow trout (Salmo 9airdneri): Physiological symptoms of EFA deficiency. J. Nutr. 102, 87-92. CASTELL J. D., SlYNHUBER R. O., WALES J. H. & LEE J. D. (1972a) Essential fatty acids in the diet of rainbow trout (Salmo gairdneri): Growth, feed conversion and some gross deficiency symptoms. J. Nutr. 102, 77 86. CASTELL J. D., LEE D. J. & SINNHUBER R. O. (1972c) Essential fatty acids in the diet of rainbow trout (Salmo .qairdneri): Lipid metabolism and fatty acid composition. J. Nutr, 102, 93-100. CASTELL J. D. • COVEY J. F. {1976) Dietary lipid requirements of adult lobsters, Homarus americanus (M.E.I.J. Nutr, 106, 1159--1165. CASTELL J. D. (1978) Review of lipid requirements of finfish. EIFAC/FAO Symposium on finfish nutrition and fi'ed technolooy, Hamburg, West Germany, 20-23 June. CASTELL J. D. (1979) Review of lipid requirements of finfish. From Proc. World Syrup. on Finfish Nutrition and Fishfeed Technology, Hamburg 20- 23 June, 1978. Vol. 1. CASTLEDINE A. J. & BUCKLEYJ. T. (1980) Distribution and Mobility of 3 fatty acids in rainbow trout fed varying levels and types of dietary lipid. J. Nutr. 110, 675-685. COWEY C. B. & SARGEYTJ. R. (1972) Fish Nutrition. Mar. Biol. 10, 383 492. COWEY C. B., ADRON J. W. & BROWN D. A. (1975) Studies on the nutrition of the marine flatfish. The metabolis of glucose by plaice (Pleuronectes platessa) and the effect of dietary energy source on protein utilization in plaice. Br. J. Nutr. 33, 219-331.

Lipid nutrition in fish COWEY C. B., ADRON J. W., OWEN J. M. & ROBERTS R. J. (1976a) The effect of different dietary oils in tissue fatty acid and tissue pathology in turbot Scophthalamus maximus. Comp. Biochem. Physiol. 53B, 399-403. COWEY C. B., OWEN J. M., ADRON J. W. & MIDDLETON C. (1976b) Studies on the nutrition of marine flatfish. The effect of different dietary fatty acids on the growth and fatty acid composition of turbot (Scophthalmus maximus). Br. J. Nutr. 36, 479-486. DUPREE H. K. (1969) Influence of corn oil and beef tallow in growth of channel catfish. U.S. Fish. Wild. Set. Tech. Pap. No. 27, 13. FARKAS T. ~ CSENGER I. (1976) Biosynthesis of fatty acids by the carp, Cyprinus carpio L., in relation to environmental temperature. Lipids 11, 401-407. FARKAS T., CSENGER I., MAJOROS F. & OLA.H J. (1977) Metabolism of fatty acids in fish I. Development of essential fatty acid deficiency in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture 11, 147-157. FARKAS T., CSENGER I., MAJOROS F. ~,~ OL.~H J. (1978) Metabolism of fatty acids in fish II. Biosynthesis of fatty acids in relation to diet in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture 14, 57-65. FARKA'S T., CSENGER I., MAJOROS F. & OL,g,n J. (1980) Metabolism of fatty acids in fish III. Combined effect of environmental temperature and diet on formation and deposition of fatty acids in the carp, Cyprinus carpio Linnaeus 1758. Aquaculture 20, 29-40. FOWLER L. G. & BANKS J. L. (1969) Tests of vitamin supplements and formula changes in the abernathy salmon diet. U.S. Bur. Sport. Fish. Wild. Tech. Pap. No. 26, 3-19. FuJII i . , NAKAYAMA O. & YONE Y. (1976) Effect of o93 fatty acids on growth, feed efficiency and fatty acid composition of red sea bream (Chrysophys major). Rept. Fish. Res. Lab., Kyushu Univ. No 3, 65-86. FuJn M. & YONE Y. (1976) Studies on nutrition of red sea bream-XIII. Effect of dietary linolenic and 03 polyunsaturated fatty acids on growth and feed efficiency. Bull. Japan. Soc. scient. Fish. 42, 583-588. FUKUZAWA T., PRIVETT O. S. & TAKAHASHI Y. (1970) Effect of essential fatty acid deficiency on release of triglycerides by the perfused rat liver. J. Lipid Res. 11, 522-527. FUKUZAWA T., PRIVETT O. S. & TAKArlASH1 Y. (1971) Effect of essential fatty acid deficiency on lipid transport from liver. Lipids 6, 388-393. FURUKAWA A., TSUKAHARA H. & FUNAE K. (1966) Studies on feed for fish V. Results of the small floating net culture test to establish the artificial diet as complete yellowtail foods. Bull. Naikai re#. Fish. Res. Lab. 23, 45-56, GARLING D. U & WILSON R. P. (1976) The optimum dietary protein to energy ratio for channel catfish fingerlings. J. Nutr. 106, 1368-1375. GATESOUPE F. J., LEGER C., METAILLER R. & LUQUET P. (1977a) Alimentation lipidique du turbot (Scophthalmus maximus L.) I. Influence de la longueur de chaine des acides gras de la s6rie o93. Ann. Hydrobiol. 8, 89-97. GATESOUPE F. J., LEGER C., METAILLER R. & LUQUET P. (1977b) Alimentation lipidique du turbot (Scophthalamus maximus) II. Influence de la supplementation en m6thyliques de lfi.ads linolennue et de la competementation en acides gras de la s6rie o99 sur la croissance. Ann. Hydrobiol. 8, 247-254. HASHIMOTO Y., OKAICHI T., WATANABET. & FURUKAWA A. (1966) Muscular dystrophy of carp due to oxidized oil and the preventive effect of vitamin E. Bull. Japan. Soc. scient. Fish. 32, 64-69. HAYES K. C., NIELSEN S. W. ,~6 ROUSSEAU J. E. (1969) Vitamin E deficiency and fat stress in the dog. J. Nutr. 99, 196-209. HIGASHI H., KANEKO T., USHIYAMA M. & SUGIHASHI T. (1964) Effects of dietary lipids on fish under cultivation II. Effect of ethyl linoleate, linolenate and ethyl esters of

13

polyunsaturated fatty acids on deficiency of essential fatty acids in rainbow trout. Bull. Japan. Soc. scient. Fish. 30, 778-783. HIGASHI H., KANEKO T., ISml S. & SUGIHASHI T. (1966) Effect of ethyl linoleate, ethyl linolenate and ethyl esters of highly unsaturated fatty acids on essential fatty acid deficiency in rainbow trout, d. Vitam. 12, 74-79. HIGASHI H., TAKEDA K. • NAKAHIRA T. (1970) Studies on the roles of tocopherols in fish (I) Tocopherol content and ratio of each member of tocopherol-homologues in marine animals. Vitamins 42, 1-7. HIGASHI H., TERADA K., MORINAGA H. t~ NAKAHIRA T. (1972) Studies on roles of tocopherols in fish (II) Mutual conversion of tocopherol-homologues in carps, when ct, 7 or 3 tocopherol was given to them (Preliminary Report). Vitamins 45, 121-125. HILL E. G. (1966) Effect of dietary linoleate on chick liver fatty acids: Dietary linoleate requirement. J. Nutr. 89, 465-471. HONJO T. (1965) The effect of cod liver oil to the growth and the content of vitamin A in rainbow trout. Aquaculture 13, 15-21. HORWITT M. K., HARVEY C., CENTURY B. & WITTING U (1961) Polyunsaturated lipids and tocopherol requirements. J. Am. diet. Ass. 38, 231-235. HORWITT M. K. (1962) Interrelations between vitamin E and polyunsaturated fatty acids in adult men. Vitamins Horm. 20, 541-549. KANAZAWAA., TESHIMAS., SAKAMOTOM. & AWAL MD. A. (1980) Requirement of Tilapia zillii for essential fatty acids. Bull. Japan. Soc. scient. Fish. 46, 1353-1356. KANEKO T., TAKEUCHI M., HIGASHI H. & KIKUCHI T. (1967) Effect of dietary lipids on fish under cultivation III. Effect of methyl esters of highly unsaturated fatty acids, methyl linoleate and methyl palmitate on the fatty acid composition of rainbow trout flesh lipids. Bull. Japan. Soc. scient. Fish. 33, 47-55. KEMP P. & SMITH M. W. (1970) Effect of temperature acclimatization on the fatty acid composition of gold fish intestinal lipids. Biochem. J. 117, 9-15. KNIPPRATH W. G. & MEAD J. F. (1966a) Influence of temperature on the fatty acid pattern of mosquitofish (Gambusia affinis) and guppies (Lebistes reticulatus). Lipids i, 113-117. KNIPPRATH W. G. ,~¢ MEAD J. F. (1966b) Influence of temperature on the pattern of muscle and organ lipids of rainbow trout (Salmo gairdneri). Fish. Ind. Res. 3, 23-27. KNIPPRATH W. G. & MEAD J. F. (1968) The effect of the environmental temperature on the fatty acid composition on the in vivo incorporation of [~1-14C] acetate in gold fish (Carrasius auratus L.). Lipids 3, 121-128. Ko T. Y., YANAGIDA T. & SUGANO M. (1975) Effect of oxidized fats on the nutritive value of proteins VI. Changes in available lysine and digestibility of proteins accompanied with lipid oxidation; Experiments with a model system. Nippon Noaei Kagaku Kaishi 49, 435-437. KULIKOV V. M. (1965) The utilization of phosphatide concentrate in the animal industry. Musloboino-Jirouaga Promyshlemnost 22, 1-8. LEE D. J., ROEM J. N., Yu T. C. & SINNHUnER R. O. (1967) Effect of to3 fatty acids on the growth of rainbow trout, Salmo gairdneri. J. Nutr. 92, 93-98. LEE D. J. & PUTNAMG. B. (1973) The response of rainbow trout to varying protein/energy ratios in a test diet. J. Nutr. 103, 916-922. LEGER C., BERGOT P., LUQUET P., FLANZY J. ¢~ MEUROT J. (1977) Specific distribution of fatty acids in the triglycerides of rainbow trout adipose tissue: Influence of temperature. Lipids 12, 538-543. LEGER C., GATESOUPEF., METAILLERR., LUQUETP. ~¢, FREMONT L. (1979) Effect of dietary fatty acids differing by chain length and co series on the growth and lipid corn-

14

T. WATANABE

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