PRINCIPLES OF FISH NUTRITION

WERNER STEFFENS Head of the Department of Fish Nutrition, Institute of Inland Fisheries, Berlin and Professor for Fish Culture and Fish Nutrition, Humboldt University, Berlin Translator B. D. HEMMINGS Translation Editor Dr L. M. LAIRD Department of Zoology, University of Aberdeen ELLIS HORWOOD LIMITED Publishers Chichester Halsted Press: a division of JOHN WILEY & SONS New York Chichester Brisbane Toronto

5 Vitamins 5.1 GENERAL

Vitamins are low-molecular-weight compounds of widely-differing composition which are essential to life, but which cannot as a rule be synthesized (or are synthesized only in insufficient quantity) by the higher animals, and hence must be supplied as part of their diet. They possess very specific functions in the process of cell metabolism. The term vitamin arose historically and should be understood primarily as a collective term for a group of biologically defined compounds. Not every vitamin is an essential requirement for every species of animal, nor is it always required in the same amount. Sometimes synthesis of a particular vitamin does take place within the animal organism, chiefly as a result of the activity of microorganisms within the digestive tract. Certain vitamins may also be synthesized from precursors (provitamins). Vitamins very often exert a catalytic action as constituent parts of enzyme systems and hence they are needed in only quite small amounts, although there may be exceptions to this in the case of certain vitamin-like substances (e.g. choline). The absence of a particular vitamin leads to serious metabolic disorders which are properly referred to as avitaminoses and which are frequently fatal. Inadequate supply of vitamins results in deficiency symptoms which are frequently non-specific and are grouped together under the heading of hypovitaminoses. In cases of deficiency it is seldom possible to trace the cause to lack of a specific vitamin: Usually there is a quantitative deficiency in the supply of several vitamins which manifests itself in a more or less non-specific growth retardation and susceptibility to disease which is very difficult to diagnose accurately. Compounds which nullify or impair the activity of vitamins are termed antivitamins. The individual vitamins were first denoted merely by capital letters, but since I960 they have had internationally-agreed names (Table 119). The traditional subdivision into fat-soluble and water-soluble vitamins is still in common use. Despite

Vitamin A were the subjects of special attention. In Japan likewise intensive research of a similar nature was initiated. Today it is possible to estimate the vitamin requirements of the principal commercial species as far as concerns the essential features, and to provide adequate vitamin supplementation for compound feeds. Nevertheless, there remain many open questions and further studies are needed, if only because the whole problem is very complex, with many interacting factors which may sometimes have opposing effects (Aoc et al. 1971). Summaries concerning the vitamin requirements of fish may be found in Halver and Coates (1957), Dupree (1966). Kitamura et al. (1967a), Halver (1957a, 1972, 1980. 1982. 1985). Aral et al. (I972a). Steffens (1969c 19745), Ketola (1976a) and Poston (1986a).

5.2

VITAMIN A

Vitamin A is an alicyclic polyenoic alcohol. It is found only in the animal kingdom and especially in liver, where it is present in large amounts. The widely-occurring compound pcarotene. which is abundant in green plants and in particular in carrots, has the character of a provitamin. Depending on the animal species, various mass ratios for the p-carotenc/retinol transformation are applicable, ranging from 2 in the case of i ats to 8 for cattle. The carotenoids also exhibit additional biological functions in fish (Tacon 1981). Vitamin A is of importance for normal vision (adaptation to darkness) and in cases of deficiency, night-blindness (nyctalopia) may be produced. The protective action of retinol towards the epithelium is also very important (mucous membrane formation from mucopolvsaccharides). Where vitamin A is lacking the mucus layers dry out. The onset of xerophthalmia of the eye is also typical of this process, which accounts for the earlier designation for vitamin A of axerophthol. In circumstances of vitamin A deficiency the danger of bacterial infection and parasitic invasion is increased because of damage to the epithelium. Furthermore, retinol plays a role in the normal development of young animals in connection with bone formation and fertility. An ample supply of Vitamin A also enhances cold resistance as was shown by studies with goldfish (Chen et al. 1985). In animals, dietary fats and bile acids promote the absorption of Vitamin A and p-carotene. Storage occurs in the form of esters (acetate and palmitate) which nowadays are synthesized for use as feed additives. For rainbow trout the retinol content of the diet has a pronounced effect on the vitamin level in the fish (Higashi et al. 1962; Keiz 1965; Braekkan et al. 1969; Georgiev 1971, 1972). The same applies to brook trout and brown trout (Poston 1969b.c; Poston et al. 1966: Pickering

1978). The retinol supplied in the feed is very well absorbed. At higher temperatures elevated contents in the fish can be expected. Retinol contents in the pyloric caecae are appreciably greater than those in the liver. Vit; min AI (C2(,H29OH) is partially converted to Vitamin A2 (C20H2 7OH) probably mainly in the blind caecae. prior to transport and storage in the liver. During embryonic development the vitamin A reserves iri the egg do not undergo any noticeable changes. After hatching, however, the retinol content invariably falls (Steffens and Karst 1965). In brown trout eggs the carotenid content ranges from

Vitamins 0.99mg% to 1.63 mg%, and in rainbow trout eggs from 0.54 mg% to 1.09mg%.The losses during embryonic development were inversely proportional to the carotenoid content (Snarevic and Sachnenko 1972). It has been demonstrated that in numerous species of fish the p-carotene ingested in the diet can be converted into vitamin A. Relevant studies include those on Perca fluviatilis and other cyprinids (Morton and Creed 1939; C,7czuga and Czerpak 1976), cod (Neilands 1947)..channel catfish (Dupree 1970a) and rainbow trout (Steffens and Karst 1972). For brook trout it was shown that the conversion of p-carotene to vitamin A is temperature-dependent. As the enzyme which oxidizes p-carotene to retinol in the intestinal mucosa exhibits only very little activity at temperatures below 10C. water temperatures greater than this are essential for effective conversion in salmonids (Poston 1969c; Poston et al. 1977). In goldfish the conversion of P-carotene and lutein into vitamin A has been reported to occur at temperatures of 15C-24C. Lutein is probably converted only into vitamin A2 (Del Tito 1983). In young rainbow trout, vitamin A deficiency manifests itself in reduced appetite, poor growth (Fig. 69), lower relative liver weights, anemia, loss of body colour in the early stages, succeeded by hemorrhages in the eyes and skin as well as fin erosion, flexing of opercula and increased mortality (Kitamura a al. 1967a, b). All these effects, with the exception of the flexing of the opercules, are reversible within a few weeks of retinol addition to the diet. For brook trout numerous results confirm the absence of any effect resulting from vitamin A additions to the diet with respect to growth, feed utilization or mortality (Phillips et al. 1955d, 1964c). However, a beneficial effect on the haematocrit value was reported (Poston 1969b). Vitamin A deficiency gave rise to hemorrhages. Best performance was obtained in response to a combination of vitamins A and E. Vitamin A acetate and palmitate were equally well utilized by the salmonid organism (Poston etal. 1966). Similarly, brown trout there appeared to be no relation between growth and the level of retinol palmitate in the diet. In an experiment lasting 32 weeks, no significant growth differences were observed between groups receiving 0, 18000, or 100000IU of retinol palmitate per kg of diet, and similarly no differences in skin structure were apparent (Pickering 1978). No evident of hypervitaminosis was detectable in brook trout even at levels of 600000 IU per kg of diet. Massive doses of the order of 2 million IU/kg, however, resulted in lesions similar to those observed in higher animals, namely poor growth, impaired feed utilization, reduced haematocrit values and serious damage (erosion and necrosis) of the tail fins in 70% of the fish after 20-24 weeks. There was. however, no apparent increase in mortality. For rainbow

trout supplementation with 124000 IU vitamin A/kg dry diet did not give rise to toxic symptoms as compared with 4000 IU/kg (Hilton 1978a). Apparently there was no evidence of any correlation between the retinol supply and ascorbic acid deficiency symptoms, as postulated by Primps (1971). The maximum tolerable dose for rainbow trout was considered to be 900000 IU/kg diet, while amounts in excess of 2.7 million IU/kg are toxic (Hilton ct al. 1983). The symptoms of hypervitaminoses included growth depression, poor feed utilization and fin lesions, together with pale livers, spinal deformities and elevated mortality. As the content of vitamin A in the diet increases so does the vitamin A content in the liver, while its Fe-content decreases. Consequently a connection

Fig. 69 Growth of young rainbow trout as a function of vitamin A content in the diet (from Kitamura etal. 19676). between vitamin A and Fe-metabolism may be inferred. Honjo (1965), on the basis of retinol storage in different organs of rainbow trout receiving a diet containing cod-liver oil as a source of vitamin A, established a daily vitamin A demand for this species of the order of 1000 IU/kg live weight, which is equivalent to about 50000 IU/kg diet. Based on daily gains the requirement for juvenile fish of less than 7 g live weight amounted to over 2500 IU/kg diet, and for larger rainbow trout about 2500 IU/kg diet or less (Kitamura et al. 1967b). Nowadays most compound feeds for salmonids are supplemented with considerable quantities of vitamin A. It is of no consequence whether these take the form of liver oils or synthetic additives such as retinol acetate or palmitate. As losses of the vitamin occur during storage of the feed, the supplement should not be set at too low a level. Vitamin A addition to the diet is also indicated for cyprinids. In young carp vitamin A deficiency symptoms include anorexia, growth retardation, pale coloration, hemorrhage of skin and fins, protrusion of the operculum and exophthalmia. Appetite, growth and body colour were shown to be generally improved following the supply of retinol acetate but the other lesions were irreversible (Aoe et al. 1968). For goldfish, retinol deficiency likewise became manifest in hemorrhages

Vitamins in the eyes and other places, in exophthalmia, sloughing of scales and poor feed intake as well as elevated mortalities. A typical response was the appearance of nonspecific infection among the avitaminosis group (Jones et al. 1971). Having regard to the deficiency symptoms, and the level of retinol in the hepato pancreas, the daily demand for vitamin A has been estimated as 100-500 ID/kg bodyweight, or 4000-20000 ILJ/kg of dry diet (dry weight). The carp-rearing diet developed at the Institute for Inland Fisheries, Berlin-Friedrichshafen, contains 6000IU/kg. According to Tafro and Kiskaroly (1986) female breeding carp (spawners) require 12000-15000 IU/kg of dietary vitamin A during the period of gonadal development. The great importance attaching to the supply of vitamin A for the growth of fish was particularly evident in experiments with Heteropneustis fossilis. Fish of 20 g initial weight given a retinol-free diet exhibited only a short-lived growth period, lasting until their internal vitamin A reserves were exhausted, and after 90 days weighed only 12 g while fish which received the same diet supplemented with vitamin A attained an average weight of 36 g during the same period. In a parallel trial using smaller fish the same results in principle were observed. In this case fish of 3 g initial weight on the vitamin-A-deficient diet weighed only 1.6g after 90 days, but those receiving vitamin-A-supplemented diet reached an average weight of 5.7g (Gos-wami and Basumatari 1988). When vitamin A was incorporated into the diet of the previously-deficient group, they began to grow again after 10 days.

5.3

VITAMIN D

The group of D-vitamins is composed of those closely-related compounds possessing antirachitic activity. The compounds of the greatest physiological interest from a dietary viewpoint are vitamin D2 (ergocalciferol C2sH44 O) and vitamin D? (cholecal-ciferol C27H440). They originate in vegetable or animal tissue as products of the corresponding provitamins ergosterol and cholesterol (7-dehydrocholesterol) through the action of natural U V radiation, or are produced industrially from certain strains of yeast by irradiation (activation). The provitamins, which do not exhibit anti-rachitic activity, are of no importance for animal feeding. Vitamin D promotes the absorption of calcium (and also phosphorus) in the small intestine and has a positive effect on bone mineralization. In this context cholecalciferol acts as a precursor of the hormone 1.25-dehydroxy D which is the biologically-active form for this part of the mineral metabolic process. As the fish are able to take in considerable amounts of calcium from the surrounding water via the gills, the supply

of Vitamin D is particularly important in the case of waters poor in calcium. Natural sources of vitamin D2 include sun-dried green plants, while fish livers and tissue fat are also rich in vitamin Dv In animal nutrition vitamin D? is the preferred form. In higher animals over dosage may give rise to hypervitaminosis accompanied by serious disease symptoms. Our present state of knowledge concerning the vitamin D requirement of fish is still very incomplete. Nevertheless, the amounts required by the salmonid organism may be taken to be quite small. Even though no calciferol could be detected in their diet, juvenile rainbow trout failed to exhibit any deficiency symptoms for up to 110 days (Kitamura el al. 1967a). The only change was a slightly elevated blood count.

Vitamin D On the other hand Barnett et al. (1978) showed that vitamin D was essential for normal growth of rainbow trout For brook trout the addition of vitamin D to the diet had no effect (Phillips et al. 1955d); it could not replace cod-liver oil. Increasing the vitamin D2 content in the diet of brown trout from 6000 Ill/kg to 24000 IU/kg was also without effect (McCartney 1968). In certain circumstances overdosage can lead to symptoms of hypervitaminosis in fish. Trofimova (1962) failed to achieve any positive result from the addition of vitamin D2 in the form of an oil preparation to the diet of rainbow trout, and the fish even gained 8% less than the controls: however, other factors may have been responsible for this. Massive vitamin D2 supplementation of the diet of brook trout at a level of 3.75 million lU/kg resulted in an increase of the Ca-content in the serum and elevated haemocrit values relative to the controls. There was also evidence (although not significant) of poorer growth. No increase in mortality was observed (Poston 1969e). For juvenile rainbow trout of 8.3 g initial weight D3 supplementation of the diet with 4000, 104000 or 1 004000 IU/kg (dry diet) had no effect on gain, feed utilization or losses, and hypercalcaemia was not present. Levels of Ca, Mg, P and Na in the vertebrae and in the whole body were unaffected. However, those fish which received 1 004000 IU/kg exhibited increased levels of Ca, Mg and P in the skin. Nephrocalcinosis was not observed, so that this did not appear to be connected with Dv over dosage (Hilton and Ferguson 1982). In a 16-week trial with channel catfish of 0.5 g initial weight, vitamin D deficiency gave rise to poor growth and diminished levels of minerals, Ca and P in the organism. At 29C, 500 ID/kg dry diet of vitamin D3 in the diet was adequate for normal growth and unimpaired turnover mineral metabolism (Lovell and Li 1978). Higher dosages of up to 1 million I U/kg of vitamin D3 gave the same result and did not lead to any negative consequences. A lack of Ca and especially P in the diet aggravated the deficiency symptoms in the absence of D 3. Even in the presence of vitamin D3, P-deficiency produced an adverse effect on growth and mineral content but this did not happen in response to Ca-deficiency. According to Andrews et al. (1980) young channel catfish exhibited maximum rates of growth in response to dietary levels of 1000-4000 IU D3/kg. Growth impairment was apparent at a level of 50000IU D3/kg. The sole indications of vitamin D deficiency and hypervitaminosis were depressed rates of growth and feed utilization. No sign of altered levels of minerals in the vertebrae was apparent dependent on dietary levels of vitamin D in these experiments. Administration of

vitamin D3 was also unnecessary for the rearing of juvenile channel catfish in ponds where a certain proportion of the diet was of natural origin (Launere/o/. 1978). Daily intraperitoneal injections of 10000 IU D:7kg increased the serum calcium level in Amphipnouscuchia, which war, greater the higher the calcium content of the water in which the fish were kept (Srivastav 1983). As a rule cholecalciferol (vitamin D3) appeared to be more beneficial than erogcalciferol (vitamin D2 ) both for rainbow trout and channel catfish (McLarent et al. 1947; Andrews et al. 1986). For rainbow trout cholecalciferol is at least three times as effective as ergocalciferol in meeting the vitamin D requirement (Barnett et al. 1979). For young catfish, however, the effect of ergocalciferol at 1000 lU/kg or less was the same as that of cholecalciferol. but amounts of 2000-20000 lU/kg of D

Vitamins resulted in higher gains than equal doses of D2. A conclusive decision concerning the necessary level of Vitamin D supplementation for conventional compound feeds cannot be arrived at, as the raw material composition exerts an important influence. Where the feed contains animal oils in significant amounts, it may he dispensed with. Nevertheless calciferol (Ruhdel 1964) or vitamin-D-containing nils (Phillips et ul. 1964 g.h) are often added to pelletted diets. A vitamin D3 supplement of 20 (XK30(X) lU/kg in the diet should probably be regarded as the maximum. I ;i some cases much smaller quantities (300-500 IU/kg)or none at all may be used. Even ten times this amount (20000 ID/kg) did not have any adverse effect on rainbow trout (Kitamura et al. 1967a).

5.4

VITAMIN E

The group of very closely similar compounds having vitamin E activity are referred to by the collective term tocopherols. Among them, a-tocopherol (5.7.8-tritnethylto-col. C:uH5i,O:) has the great biological activity so that only this compound enter< into consideration from the nutritional standpoint. It is largely insensitive to heat, but is very rapidly attacked by oxidizing agents. Besides the naturally-occurring D-a-tocopherol, there is the synthesic DL-atocopherol which can be utilized by fish as a source of vitamin E, while G-, y- or 6tocopherols are only utilized to a small extent (Watanabe et al. 1981a). Or course ytocopherol exhibits a very marked degree of protective activity against fat oxidation (Takahashi etal. 1983). Retention, transport and distribution of DL-a-tocopheryl acetate by rainbow trout take place following hydrolysis in the digestive tract in a similar manner to that of D-a-tocopherol (Hung et al. 1982). Large amounts of a-tocopherol are present in green meal, vegetable oils and seedlings. Vitamin E is responsible for a number of important tasks in the control of DNA formation and in connection with respiratory chain phosphorylation, as well as in carbohydrate turnover and fat metabolism. In addition it functions as a natural antioxidant with a protective action towards lipids, a role in which it can be superseded by synthetic antioxidants. It protects the biological phospholipid-con-taining membranes, for example the plasma membranes of erythrocytes, against damage (Bell and Cowey 1985). The vitamin E requirement is greater where there is a higher level of unsaturated fatty acids in the diet and in the fish tissues. Thus a deficient supply of tocopherol leads, especially with high levels of unsaturated fatty acids, to reduced haematocrit values and increased fragility of the erythrocytes (Table 120). Oxidation of fats and prolonged storage bring about a reduction of the cc-

tocopherol content in the feed (Hung et al. 1980). Tocopheroi is also involved in the synthesis and excretion of gonadotrophic hormones via the diencephalon / hypothesis system. A close connection also exists between tocopherol and selenium. Vitamin E hinders the formation of peroxides and assists in the normal decomposition of previously-formed peroxides, through formation of hydroxy-fatty acids. In higher J animals a deficiency of vitamin E results in liver damage, muscular dystrophy and impaired reproductive performance. As a means of estimating the a-tocopherol requirement, a study of the ascorbic

Vitamin E Table 120 Effect of various a-tocopheryl acetate levels in the diet of rainbow trout on haematocrit values, as a function of the nature of the dietary fat. Duration of test four months, water temperature 7C-13C, =30 (from Boggio et al. 1985) acid-stimulated lipid per oxidation in the liver microsomes is a promising method. Low concentrations of malondialdehyde are signs of an adequate intake (Covvey et al. 1983a; Wilsons al. 1984). i/As in other animals, vitamin E is essential for the maintenance of normal life processes. Nevertheless symptoms of avitaminosis are rare because a certain amount of tocopherol is present in numerous feedstuffs. Fatty liver degeneration and anaemia were observed in rainbow trout fed rancid fat from dried silkworm larvae (Kawatsu 1964). Vitamin E supplementation prevented the development of anaemia, while the administration of numerous vitamins of the B-complex (Bj, B2, nicotinamide, pantothenic acid, B6, folic acid, B12) as well as chlorine was ineffective. However, B-vitamins given simultaneously with vitamin E enhanced its effect. When B-complex vitamins were administered the red-cell count was below 500000/mm3, but in response to DL-a-tocopherol it exceeded 106/mm3. When a diet containing 10% fatty acids derived from fish oil was fed to rainbow trout of 14 g initial weight, no differences in growth or feed utilization were apparent during 16 weeks between vitamin E contents (as DL-a-tocopheryl acetate) ranging from 20 to 100 mg/kg of the diet (Cowey et al. 1983). Low levels of vitamin supply, however, resulted in increased erythrocyte fragility (haemolysis). With increasing dietary vitamin levels the vitamin E content of most organs increased, especially in the liver and kidneys, but also in the muscle and skin (Table 121). The studies of Bell et al. (1985) showed likewise that as the dietary level of DL-a-tocopheryl acetate increased the vitamin E content of various tissues of rainbow trout also increased. The rise was highest in response to an ample supply of the

vitamin. Inthepresenceof a simultaneous deficiency of vitamin E and selenium, feed utilization was unsatisfactory, growth was poor and the fish exhibited low haematocrit values with noticeable haemolysis (Table 122). Elevated mortality rates, however, were not observed, but fluid accumulated in the abdominal cavity (exudative diathesis). Oberbach and Hartfie! (1988) reported that rainbow trout fed a diet rich in polyunsaturated fatty acids exhibited lowered haematocrit and haemoglobin levels after 18 weeks which returned to normal again during the ensuing period up to 44

Vitamin E weeks. At the close of the experiment, haemolysis was still slightly in evidence. The serum levels of aspartic-aminotransferase (glutamic-oxalacetic transaminase), crea-tinine kinase and lactic dehydrogenase were indicative of damage to the liver and muscle tissue (dystrophy) in cases of inadequate tocopherol supply (Q-20mg/kg diet). Symptoms of selenium deficiency were better alleviated by dosage of vitamin E than by selenium supplementation. With respect to comparative feeding trials of unoxidized and moderately oxidized fish oils at levels of 12% in the diet of rainbow trout fingerlings, the quality of the fat had no effect on the fish, but a deficiency of vitamin E produced similar symptoms in both groups at a temperature of 6C-12C, including growth retardation, poor feed utilization, low haematocrit value, fragility of erythrocytes, muscular lesions and elevated mortality (Cowey et al. 1984). When rainbow trout fry of 2 g initial weight received a diet containing 7.5% of highly oxidized oil (peroxide value >300 mmol/ kg) for 24 weeks, tocopherol deficiency resulted in reduced erythrocyte counts, lowered haematocrit and haemoglobin levels and increased haemolysis. In addition, haemosiderosis occurred in the spleen together with ceroid deposition in the liver. Mortality was attributable to the combined effects of liver damage and anemia. When the diet contained fresh oil at the same level such effects were not apparent even in the absence of vitamin E. Ethoxyquin had a protective action which was very much inferior to that of vitamin E (Mocciaefa/. 1984). Administration of tocopherol for a period of 30 days was successful in overcoming growth deterioration due to myopathy in the survivors (Kubota et al. 1982). Incorporation of carotenoids in muscle tissue was enhanced by an elevated tocopherol supply (500 mg/kg diet compared with 26 mg/kg for controls). Following the administration of feeds containing 50 mg canthaxanthin/kg, trout (350-630 g live weight) receiving ample supplies of vitamin E exhibited a higher level of pigment in the flesh than the controls (Pozo et al. 1988).

The difference in the carotenoid content persisted during prolonged cool storage even though both groups exhibited fading of colour as the period of storage increased. According to studies of Watanabe et al. (1987a), ct-tocopherol in the diet of rainbow trout counteracted the tendency caused by histamine-rich diets to growth retardation and damagesusceptibility of the stomach wall. Vitamin E deficiency also -, led to a noticeable decline in the immune response and to a reduction in the level of J non-specific resistance factors (Blazer and Wolke 1984). An adequate supply of vitamin E thus serves as a prophylactic against disease for fish as well as for warmblooded animals. Thus the immune response of rainbow trout towards Yersinia ruckeri was much more pronounced in fish which received atocopherol in the diet than in the corresponding vitamin-E-deficient group (Table 123). In addition, the total serum globulin level and the phagocytic index were significantly higher in response to adequate dietary vitamin E supply. Complete replacement of vitamin E by synthetic antioxidants is thus not possible for a variety of reasons. Deficiency symptoms occasioned by a lack of vitamin E have also been recorded in other almonid species. In Oncorhynchus tshawytscha the symptoms comprised poor growth, severe anemia (480000 red cells/mm3 and a hemoglobin level of 4.0 g/100 ml compared with > iO6 red cells/m3 and 8.0 g/100 ml for hemoglobin), the occurrence of numerous immature erythrocytes, adhesion of the gill membranes, epicarditis. ceroid deposition in the spleen, exophthalmia and as cites. These symp

Vitamins toms were especially pronounced if the diet contained 5% herring oil without addition of vitamin E; they became less in response to 1 % herring oil without vitamin E, and disappeared in response to a-tocopherol. For the diet containing 5% herring oil triglycerides the a-tocopherol requirement was estimated as 5-30 mg/kg dry diet (Woodall et al. 1964). For brown trout also, vitamin E deficiency resulted in poor growth and a severe reduction in the haematocrit (of about 50% after 16 weeks) and high mortality (Poston 1965). A typical manifestation was sudden death. Survivors recovered in response to adequate tocopherol supply, when the haematocrit value increased and the losses declined. For young brook trout a beneficial synergistic effect between vitamins E and A on erythropoiesis was observed (Poston 1969b). For good growth in experiments on this species elevated vitamin E and also vitamin A levels were necessary. A higher DL-a-tocopherol content on its own was not sufficient. Relations between tocopherol and selenium deficiencies became apparent from experiments with Salmo salar (Poston et al. 1978). Lack of vitamin E doubled the loss of fry in the first four weeks. The same happened in response to lack of Se or absence of Se plus vitamin in the diet. The addition of 500 IU tocopherol/kg of dry diet plus 0.1 mg Se/kg reduced the losses in less than two weeks. Salmon of 0.9 g initial weight exhibited the following signs of avitaminosis (with or without Se): extreme anaemia, pale gills, anisocytosis, poikilocytosis, elevated plasma protein levels, exudative diathesis, blistering of the skin, yellow-orange discoloration of the liver, enlargement of the gall bladder with dark green bile, muscular dystrophy, elevated fat and moisture contents in the organism, periodic feebleness and interruption of swimming activity. In order to overcome the muscular dystrophy, administration of vitamin E plus Se was required. Injury due to overdosage of vitamin E was observed in the cas of brook trout (Poston and Livingston 1971a). The addition of 5000 mg of DL-a-tocopherol/kg of diet (compared with 500 mg/kg for controls), in one experiment lasting 20 weeks with young fish at 12C, led lo a 6.5% reduction in growth, a haematocrit value depressed by about 18% on average and a low linoleic acid content in the liver. Probable side-effects consisted of an elevated fat content in the liver and a diminished relative liver weight. Mortalities did not occur.

Vitamin E In the case of carp, the most striking sign of prolonged (90 day) feeding of an a-tocopherolfree diet is the occurrence of muscular dystrophy. There is also a decrease in the levels of myosin and actomycin (Watanabe et al. 1970a,c; Aoe et al. 1972). In addition, poor growth and feed utilization, depressed function of the islets of Langerhans and the pituitary gland, and increase of serum protein contents were observed. Miyazaki and Kubota (1981b) also observed deposits of ceroids and haemosiderin. Symptoms such as muscular dystrophy and poor growth together with poor appetite and high mortality are typical characteristics of the Sekoke disease which occurs, according to studies by Hashimoto et al. (1966), in repsonse to feeding of oxidized oils and can be prevented by the addition of DL-a-tocopheryl acetate (500 mg/kg) to the diet. A close link between vitamin E and fat metabolism is suggested by the fact that in carp a lack of a-topcopherol does not only lead to elevated moisture and diminished protein levels in the musculature, but also the linoleic acid content of all tissue fat is reduced. Enhancing the proportion of unsaturated fatty acids in the form of dietary linoleic acid increases the vitamin E requirement of the carp (Waranabe et al. 1977a,b; Watanabe and Takashima 1977). According to studies by Miyazaki (1986) myopathy was Induced in carp by oxidized fatty acids and tocopherol deficiency. Administration of different forms of fat (beef tallow, fish oil, corn germ oil, linseed oil) at a dietary level of 12%, with different proportions of a-tocopheryl acetate (80 or 500 mg/kg) had only an insignificant effect on the fatty-acid composition of the fish flesh, even though a certain influence on the proportions of the more highly-unsaturated acids of the co-3 and o>-6 series could not be excluded. For large amounts of polyunsaturated acids in the diet, a level of 80 mg vitamin E/kg was not sufficient to prevent impaired growth and inferior feed conversion ratios. In the linseed-oil group the higher tocopherol level in the diet resulted in a higher fat content in the edible portion of these fishes (Runge et al. 1987,1988; Schwarz et al. 1988).
N/

Vitamin E also has an effect on the hypophysis-ovary system and hence plays an important

role in the reproductive process as it also does in higher animals. In the ovaries of sexuallymature carp the vitamin E level rises prior to the onset of the spawning period, and decreases thereafter. An elevated vitamin requirement must therefore be reckoned on during the spawning season (Mezes 1986; Tafro and Kiskaroly 1986). When female carp were

given intramuscular injections of a-tocopherol over a prolonged period (six weeks) at a level of 30 mg/week in two doses, there was a marked rise in the level of vitamin E in the ovaries accompanied by

; \a decrease in the level of malon-dialdehyde dehydrogenase (Mezes and Vadasz 1984). Tocopherol deficiency led to an inferior state of development of the ovaries. The gonadosomatic index (relationship of gonadal weight to body weight) was only 3.3% compared with 14.1% in control fish on an adequate diet. Moreover the moisture content of the ovaries from the deficient group was strongly iflcreased and the fat and protein contents correspondingly reduced. The phospholipids were of course very seriously affected (Watanabe 1985). Channel catfish, in experiments by Dupree (1968), exhibited lethargic behaviour, pale discoloration of the skin, ascites and occasionally exophthalmia in response to vitamin E deficiency. After 16 weeks the mortality rate was 100%. When feeding oxidized oil in the absence of tocopherol, the symptoms included growth retardation,

Vitamins exudative diasthesis, muscular dystrophy, loss of pigment, fatty livers, anemia, lesions of pancreatic and kidney tissue and high mortality (Murai and Andrews 1974). Channel catfish fingerlings of 70 g or 140 g ionitial weight, however, when fed for long periods on a diet which contained 1 % cod-liver oil and 4% lard together with adequate Se, exhibited no adverse effects on growth or feed utilization due to vitamin E deficiency. Less than 20 g DLa-tocopherol/kg diet resulted in the sole haematological finding of elevated haemolysis and haemosiderosis in the kidneys and pancreas. Based on the ascorbic-acid-stimulated lipid per oxidation in liver microsomes. feeds for channel catfish should contain at least 50 mg/kg of atocopherol and in the presence of high concentrations of polyunsaturated fatty acids, the level should be even higher (Wilson et al. 1984). Vitamin E deficiency symptoms are independent of stocking density and vitamin C status. In every case, however, significant interactions exist between vitamin E and selenium as for rainbow trout. (Gatlin etal. 1986b). In the case of channel catfish there may possibly be differences between different strains in respect of their susceptibility to vitamin E deficiency (Gatlin et al. 1986a). Channel catfish fillets exhibited improved frozen storage stability when the diet on which the fish were reared contained elevated levels of a-tocopherol (>200400mg DL-a-tocopheryl acetate/kg dry diet). The oxidative spoilage of fat was thus curtailed (O'Keefe and Noble 1978). Young of Oreochromis niloticiis (0.5-8 g) showed no significant deficiency ] symptoms in the absence of vitamin E supply when the diet contained only 5% fish oil (as methyl esters). Higher oil contents (10%-15%) however, in the absence of a-tocopherol for three weeks, gave rise to depressed feed intake, poor feed utilization, impaired growth and increased mortality. These symptoms could be prevented by the addition of 500 mg vitamin E/kg diet. With rising oil contents, the absence of dietary vitamin E produced an increase in moisture content, a reduction in protein and fat content and exhaustion of the vitamin E reserves in the whole fish (Satoh et al. 1987a). For practical feeding of Tilapia on low-fat diets, experience indicates that a level of 50-100 mg/kg of a-tocopherol in the diet is sufficient. Young turbot of HOg liveweight receiving a-tocopherol in the diet showed a linear increase in the a-tocopherol content of the liver. Levels in the spleen and serum showed a similar response, but the vitamin E content of muscle was hardly affected at all. In addition non-atocopherol isomers were absorbed from the diet, but the content in the fish fell rapidly after cessation of the supply and after 20-25 days they were no longer detectable. In vivo

conversion of non-a-tocopherol isomers into a-tocopherol could not be demonstrated (Feldheim 1985; Schulz 1986; Schulz and Feldheim 1986; Feldheim etal. 1987). Just as in carp and other fish species, in the ayu (Plecoglossus altivelis) vitamin E plays an important role in the reproductive processes (lakeuchi el al. 1985, cited in Watanabe 1985). Deficient tocopherol supply has the effect of inhibiting spawning in a proportion of the fish, and the survival rate of both eggs and fry is low. Very close relationships exist, as already mentioned, between the dietary fat content (and also the type of fat in the diet) and the requirement for vitamin E (Table 124). For this reason, the amount of a-tocopherol which should be added to compound feeds can vary quite widely. It is to a large degree also dependent on the natural vitamin E content of the basic constituents and the presence of other