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 p-

carotene. 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

(C20H27OH) 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,7£czuga 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 10°C. 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 15°C-24°C. 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 anti-

rachitic activity. The compounds of the greatest physiological interest from a dietary

viewpoint are vitamin D2 (ergocalciferol C2sH44O) 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 D v 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 29°C, 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-a-

tocopherol which can be utilized by fish as a source of vitamin E, while G-, y- or 6-

tocopherols are only utilized to a small extent (Watanabe et al. 1981a). Or course y-

tocopherol 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 7°C-13°C, =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 6°C-12°C, 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 damage-

susceptibility 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 a-

tocopherol 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 12°C, 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-tocopherol-

free 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 sexually-

mature 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 DL-

a-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 a-

tocopherol 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 (>200—400mg 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-a-

tocopherol 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