c

c







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
à

 
B. D. HEMMINGS
à



Dr L. M. LAIRD
Department of Zoology, University of Aberdeen


 


Publishers ‡ Chichester
Halsted Press: a division of 


New York ‡ Chichester ‡ Brisbane ‡ Toronto
£
º
 
 
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
º
 
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 
1971).
Summaries concerning the vitamin requirements of fish may be found in Halver and Coates
(19£7), Dupree (1966). Kitamura 
 (1967a), Halver (19£7a, 1972, 1980. 1982. 198£).
Aral 
(I972a). Steffens (1969c 1974£), Ketola (1976a) and Poston (1986a).

 º


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 
198£). 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 
1962; Keiz 196£; Braekkan 
1969; Georgiev 1971, 1972). The
same applies to brook trout and brown trout (Poston 1969b.c; Poston 
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 196£). In brown trout eggs the carotenid content ranges from
º
  
0.99mg% to 1.63 mg%, and in rainbow trout eggs from 0.£4 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 c 
 
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 
 1977). In goldfish the
conversion of P-carotene and lutein into vitamin A has been reported to occur at temperatures
of 1£°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

 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 
19££d,
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 
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 

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. ‰ Growth of young rainbow trout as a function of vitamin A content in the diet (from
Kitamura 
19676).
between vitamin A and Fe-metabolism may be inferred.
Honjo (196£), 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 £0000
IU/kg diet. Based on daily gains the requirement for juvenile fish of less than 7 g live weight
amounted to over 2£00 IU/kg diet, and for larger rainbow trout about 2£00 IU/kg diet or less
(Kitamura 
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 
 1968). For goldfish, retinol deficiency likewise
became manifest in hemorrhages
º
  
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 
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-£00 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 12000-1£000 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    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 £.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.

£.3 º


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 @  (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 1.2£-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 
 1967a). The
only change was a slightly elevated blood count.
º
 
On the other hand Barnett 
 (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 
19££d); 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 @ 
supplementation of the diet of brook trout at a level of 3.7£ 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.£ 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, £00
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 D3.
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

(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 £0000IU 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
¦ 
 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 
1947; Andrews 

1986). For rainbow trout cholecalciferol is at least three times as effective as ergocalciferol in
meeting the vitamin D requirement (Barnett 
 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
º
  
resulted in higher gains than equal doses of @ 
¦ 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
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-£00 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 
1967a).

£.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 (£.7.8-tritnethylto-col. C:uH£i,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 
 1981a). Or course y-
tocopherol exhibits a very marked degree of protective activity against fat oxidation
(Takahashi 
 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 
 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 198£). 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 
tocopherol content in the feed (Hung 
 1980). Tocopheroi is also involved in the
synthesis and excretion of gonadotrophic hormones via the diencephalon / hypothesis system.
¦ 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

º
 
Table 120 ² 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 
198£)
acid-stimulated lipid per oxidation in the liver microsomes is a promising method. Low
concentrations of malondialdehyde are signs of an adequate intake (Covvey 
 1983a;
Wilsons
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 £00000/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 
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 
(198£) 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

º
 
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 
1984).
When rainbow trout fry of 2 g initial weight received a diet containing 7.£% 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 
1982).
Incorporation of carotenoids in muscle tissue was enhanced by an elevated tocopherol supply
(£00 mg/kg diet compared with 26 mg/kg for controls). Following the administration of feeds
containing £0 mg canthaxanthin/kg, trout (3£0-630 g live weight) receiving ample supplies of
vitamin E exhibited a higher level of pigment in the flesh than the controls (Pozo 
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 
 (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  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  
  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  

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

 
º
 
toms were especially pronounced if the diet contained £% 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 £% herring oil triglycerides
the a-tocopherol requirement was estimated as £-30 mg/kg dry diet (Woodall 
1964). For
brown trout also, vitamin E deficiency resulted in poor growth and a severe reduction in the
haematocrit (of about £0% after 16 weeks) and high mortality (Poston 196£). 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 
 

(Poston 
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 £00 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 £000 mg of DL-a-tocopherol/kg of diet (compared with
£00 mg/kg for controls), in one experiment lasting 20 weeks with young fish at 12°C, led lo a
6.£% 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 
1970a,c; Aoe 
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 
(1966), in repsonse to
feeding of oxidized oils and can be prevented by the addition of DL-a-tocopheryl acetate (£00
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 
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 £00 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 
1987,1988; Schwarz 

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 198£).
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 £0 mg/kg of a-
tocopherol and in the presence of high concentrations of polyunsaturated fatty acids, the level
should be even higher (Wilson 
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 
 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 
1986a).
Channel catfish fillets exhibited improved frozen storage stability when the diet on which the
fish were reared contained elevated levels of a-tocopherol (>200²400mg DL-a-tocopheryl
acetate/kg dry diet). The oxidative spoilage of fat was thus curtailed (O'Keefe and Noble
1978).
Young of     (0.£-8 g) showed no significant deficiency ] symptoms in
the absence of vitamin E supply when the diet contained only £% fish oil (as methyl esters).
Higher oil contents (10%-1£%) 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 £00 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 
1987a). For practical feeding of à

 on low-fat diets, experience
indicates that a level of £0-100 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 20-2£ days they were no longer detectable.  
conversion of non-a-tocopherol isomers into a-tocopherol could not be demonstrated
(Feldheim 198£; Schulz 1986; Schulz and Feldheim 1986; Feldheim 
1987).
Just as in carp and other fish species, in the ayu c
  vitamin E plays an
important role in the reproductive processes (lakeuchi 
198£, cited in Watanabe 198£).
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