VITAMIN AND MINERAL REQUIREMENTS WITH FISH

J. KOENIG

I. INTRODUCTION

At the beginning of the century, following the discovery of vitamins, there was a time when deficiency and a specific symptom were associated. For a certain number of vitamins, those which were discovered in the beginning, one appelation was kept: Axerophtol or vitamin A, Aneurin or vitamin B₁. The specific avitaminosis-symptom is the first approach to this problem.

However, a more discrete deficiency will lead to non specific disorders: anorexia, apathy, lack of growth. But these disorders must not be neglected, as, in rearing, they are of such importance and consequences that they can lead to catastrophies in profitability and be the forerunners of more severe physiological disorders.

We know that vitamins have an active and specific participation in all the metabolisms, which allow an organism to live and build itself up, by transforming the elements, nutriments that it absorbs, this is anabolism; and by destroying and eliminating another part, this is catabolism.

Vitamins are prosthetic groups -in other words, biologically active parts enzymes. Each vitamin can enter into a more or less great number of enzymes where it will have a specific function. Some vitamins have an unique biological function as is the case of Vitamin E.

The vitamins of groups B have been studied and their roles in the metabolism are rather well known. Their constitution is very specific and any slight modification of structure can block the reaction that they are meant to catalyse. These neighbouring compounds of vitamins are antivitamins. Certain medicaments and raw fish flesh contain antivitamins.

If a great number of reactions, where the vitamins of groups B intervene, is known, there still remains a few points which are difficult to understand; in particular, for vitamin A whose exact role is still unknown, while yet very important in the synthesis of proteins.

We have seen that there are two actions limits for vitamins, the inferior Limit -expressed generally in mg per kg of ration- below which appear specific symptoms; and the minimum superior Limit for growth and normal behaviour. There is another superior level where therapeutic actions can appear within the frame ofvitaminotherapy.

By this section, minerals logically fit in along side with vitamins. Some, for example macroelements participates in the structure of the organism. Microelements on the contrary, have a catalytic role and often they join with vitamins in their actions.

We must not forget to speak also of the technological questions when vitamins are incorporated into compound food. Questions linked with their more or less great fragility. Indeed, only vitamins C causes a practical problem.

As this report concerns fish let us ask ourselves the questions: do the requirements in vitamins and minerals for fish differ from those living in an atmospheric environment? If, on general, the biological functions are same, the specific symptoms are different for fish, with regard to their anatomic and physiological particularities. Gills instead of lungs for example; which means that abnormalities of the gill organs in relations to certain deficiencies may be remarked.

II. VITAMINS

a) General functions of vitamins

1. Role played in the intermediary metablisms

Table 1 - Synopsis of the vitamins of their active forms and of their metabolic functions
W.F. KORNER and J. VOLLM (1976) - Doc.ROCHE

2. WHERE DO VITAMINS TAKE EFFECT ?

2.1. Plasmic membranes

Their role is essential in living beings: They constitute the interface between the organism and the environment, they maintain the constancy of the internal medium,they ensure the respiratory exchanges; the membranes actively defend the organism against agressions.

With fish the plasmic membranes in addition ensure:

-   the constant internal osmotic pressure,

-   the excretion of soluble substances.

The plasmic membranes consist in the double layer of phospholipids whose lipidic branches turn inwards and which have one hydrophilous molecule located on the outside of the membrane choline inositol. This double hydrophobous and hydrophilous character enables the membranes to have very particular properties: they constitute a barrier for exterior elements but can admit certain specific elements: thanks to the globular proteins found in the membrane. This is how in the gill menbaranes of the fish. So as to balance the internal osmotic pressure, organoids known as “ the sodium pump” extract Na ions from the internal medium and cast them out; this against the osmotic gradient. We can clearly see that these plasmic membranes or “intelligent” organs.

Nevertheless, the constitution and functioning of the membranes depend on a certain number of vitamins: INOSITOL and CHOLINE are constituents of phospholipids, and although structural elements of the membrane, they are considered as vitamins, as they can entail deficiencies.

The molecule of phospholipids comprehends 2 unsaturated fatty acids (UFA) which must have very specific constitutions permitting them to function correctly. These UFA are long chains of 22 to 24 carbons atoms. Some species such as the trout can use a C 18 precursor to synthetize, through elongation, the C 22 and C 24 UFA. This precursor is linolenic acid which is essential.

To do this, the trout will require catalysists supplied in the diet: Hiothine, whose importance in the gill membrene is certified by the characteristics deformation in the case of deficiency.

Panthotenic acid, a prosthetic group of the coenzyme A, is also necessary in the synthesis of UFA. Its deficiency also provokes the extremities of the filaments to thicken.

In all cells of intensive metabolism, the free radicals can take a toxic effect. The membranes then play a protector, either in capturing them or in destroying their product: peroxide.

One role played by vitamine E is the capture of the free radicals before they take effect at UFA level, in forming peroxides. A deficiency in vitamine E causes the destruction of the muscular masses: muscular dystrophy.

Selenium, a compound of Glutation-peroxides, take effect conjointly but after vitamin E, in destroying the peroxides which have formed vitamin E and selenium are thus complementary; Selenium ensure the safeguard of vitamin E.

2.2. Mucous membranes

These are fragile tissues which must stay humid continously: ocular, intestinal mucous membranes,…

VITAMIN A is necessary for the integrity of these mucous membranes. A deficiency leads to their keratinization, in other words, the formation of a corneal layer.

2.3. Erythrocytes

Their function, in the transport of oxygen, lead to intense metabolis. where sevaral vitamins intervene, more especially:

FOLIC ACID, is necessary for the formation of erythrocytes, a deficiency leads to megaloblastic anemia in salmonids, in other words cells which are abnormally enlarged with a segmented and strangled nucleus.

VITAMIN B₁₂ is employed for the formation of red corpuscles. A deficiency creates a microcytic hypochrome anemia.

2.4. Cartilagenous tissues

VITAMIN C hydroxylates lysine and leucine, a stage which is necessary for the synthesis of collagen and of cartilages. A deficiency in vitamin C leads to the formation of a cartilage of poor quality and in fish to different bone structure deformations.

Therapeutic use of Vitamin C with fish is its capacity to activate the healing of wounds by stimulating the formation of collagen.

b) PRINCIPAL SYMPTOMS OF DEFICIENCY WITH FISH

1. Non-specific symptoms

SLOW GROWTH AND A LOW CONSUMPTION INDEX. These are the first signs which indicate vitamin deficiency, the other causes being eliminated (inflection or unbalanced diet). The economic importance of these first symptoms must be emphasized.

ANOREXIA or lack of appetite, is another non specific symptom of vitamin deficiency.

EXOPHTALMIA-protruding eyes; which occur when the osmotic pressure is badly regulated diseases (especially V.H.S).

ABNORMAL COLOURING, moreover a dark colour. However, a decolourization can occur when there exists a Niacin deficiency.

DWARFISH-Remarked in salmoneids, can be related to different deficiencies Vitamin B₂, Vitamin C, Zinc.

2. Specific symptoms

The non-specific symptoms are, general, the first to appear and these are then followed by the specific symptoms. There is a gradation in the specific symptoms: these are, when remarked in the beginning stages, reversible, which means that they can regress when the vitamin lacking is administered. But after a certain time, the lesion become too important and can not be cured; the last stage is death. By this, we learn that we must act immediately when the first symptoms appear.

It is possible to detect a deficiency in its early stages, in other words before the specific symptoms appear, by applying the appropriate biological tests. For example, the consumption of raw fish containing thiaminase causes a nervous disease in trout caused by the destruction of vitamine B₁. The quantity of transcetolases in the blood (an enzyme whose prostetic group is vitamin B₁) permits to detect the start of the deficiency before the specific symptoms appear. Biochemical tests are widely employed in the studies of vitamins.

The following tables give the symptoms of deficiencies per vitamin and for the principal types of fish reared. We shall describe the most characteristic symptoms one by one.

Table 1: Major signs of vitamin deficiencies in fishes
(Hugh A.POSTON, 1985-Doc. ROCHE)

Vitamin A

Deficiency in vitamin A provokes characteristic lesions of the eye in salmoneids. As there exist several types of ocular lesions, linked with different deficiencies, we shall with this point alone.

Vitamin E

A deficiency is often provoked by the use of rancid oil in which toxic peroxides are found.

Lack of vitamin E leads to muscular distrophy which is the destructuration of the muscular masses, with a small enlargement, we can remark the alteration of the fibers and the proliferation of the conjunctive tissue.

The use of an antioxydant (Ethoxiquin) can prevent the formation of peroxide but can not replace vitamin E.

Vitamin B₁

In trout, the deficiency provokes the dizziness diseases (loss of balance) seen in Danemark, when the basic food employed was raw fish. The distribution of 10 g/m³ of vitamin B¹ in the water or 12 mg/kg of food will eliminate these symptoms.

Vitamin B₂

Deficiency in vitamin B₂ provokes the cataract in salmoneids. We shall examine the characteristics in the part dedicated to ocular disorders.

Vitamin B₆

Pyridoxal-phosphate being necessary for the synthesis of several neuro-transmitters, the most typical symptoms of its deficiency will be nervous disorders: epileptic movements, hyperirritability, abnormal swimming or spinning, the rapid apparition of “rigor mortis”. Among the characteristic symptoms, it has been remarked that the trout is unable to snap up its food as it is not capable of sensing distance.

Panthotenic acid

With trout and channel catfish, the lack of panthotenic acid provokes the swelling of the branchial scales of the filament extremities, known as clubbed gills.

Biotine

Lack of biotine also provokes the hyperplasy of the extremity of the branchial scales in trout but not in channel catfish. Dermatites called the “blue slime patch”where the skin breaks off in fragments has been confirmed when biotin is lacking.

Niacin

The most charateristic symptom of a deficiency in niacin in trout and channel catfish is the discolouring of the skin under the influence of the sun. Exposure to light (or UV rays) causes skin and fin lesions in carp also.

Folic acid

Folic acid being necessary for the formation of trod corpuscles, its deficiency leads to anemia characterized by the destruction of erythrocytes and the presence of senile pre-erythrocytes (megaloblastic).

Vitamin B₁₂

Vitamin B₁₂ has much the same function as folic acid is necessary for the formation of red corpuscles. Deficiency leads to the reduction and fragmentation of red corpuscles. Vitamin B₁₂ and folic acid used to be given in the form of fresh animal products in fishculture, and was considered, before industrial vitamins were made available, to be irreplacable by dry products.

Vitamin C

This vitamins intervenes in a large number of enzymatic reactions and in the most varied cases: synthesis of collagen, cartilages, synthesis of adrenalin from where it has an antistress effect, reproduction system, etc…with salmoneids. The most common and most spectacular manifestations are the bone deformations of the spine (scoliosis, lordosis, dwarfism, gibbosity) along with the shortening of the operculums.

Channel cat-fish show the same deficiencies as trout.

With eel, deficiency in vitamin C provokes haemorrhagies in the head region.

Cholin

It has a structural function in entering into the phospholipids, which form an integral part of the membrane. Nevertheless, cholin acts in the same way as does a vitamin; a deficiency leads to the liver turning yellow due to the infiltration of lipids.

Inositol

Like cholin, inositol enters into the composition of phospholipids. A deficiency does not lead to a real characteristic symptom, if not the scaling of the fins in carp.

c) SECURING THE REQUIREMENT

1. Upkeep requirements

The upkeep requirements have been defined, in starting with a synthetic diet lacking in the vitamin where deficiency is be studied. These are artificial conditions which make that in practice, a specific deficiency is very rarely observed. In varying the vitamin dose rates in ascending order starting at zero, we shall remark: A total deficiency provokes the stoppage of growth, specific symptoms and death. With a feeble dose, mortality is no longer remarked; if the dose is increased, the specific symptoms then disappear; finally, with the correct dose, growth becomes normal.

Certain biochemical parameters can be following on parallel, along with the transcetolase activity of red corpuscles for vitamin B 1. These parameters permit the detection of a deficiency from the beginning, before the specific symptoms which can cause confusion, such as the deformation of the gill filaments which are often difficult to define. It may indeed concern a deficiency in biotin, in panthotenic acid, in vitamin C, but also a toxicosis or a pathological state.

Certain vitamins interact with other elements, the requirements in vitamin E are proportional to the rate of unsaturated fatty acid. In addition, the needs in unsaturated fatty acids rise in Winter for salmoneids, when the temperature of the water drops. Thus a certain amount of vitamin E which is suitable in Summer may not be so in Winter.

Thus, we must be very prudent when using the tables of requirements and consider them more so as adjustable work hypothesis depending on the formulation and environmental conditions of the rearing.

Table 2 - Minimum requirements in vitamins per fish ¹
(cf, NCR, 1981; NCR, 1983) - POSTON, 1985

1 - Listed amounts do not allow for losses during processing and a storage. Levels needed may vary different environment condition.
2 - indicates requirement not tested or not known
3 - indicates dietary needs but in undefined quantities.

Table 2 - Minimum dietary vitamin requirements for fishes (continued)

2. Therapeutic requirements

It has been remarked that certain vitamin, employed in greater amounts than in upkeep requirements, can have therapeutic affects. The therapeutic doses are usually 10 to 100 times more than the doses.

-   As examples of therapeutic employments, we can mention:

-   Vitamin C which is employed to speed up the healing of wounds.

Cholin which is commonly employed to reabsorb the Lipidic excess of the liver caused by overfeeding. But the most interesting cases of investigation is the immuno-stimulating effect of certain vitamins against bacterial agressions. We shall see this further down.

3. Special requirements

We call special requirements the supplementary requirements resulting from the use of certain nutriments or medicaments.

-   The presence of a thiaminase, which destroys vitamin B¹, in the flesh of the fish has been pointed out. Certain vegetables (cabbage), empolium, have the same effect.

-   The use of sulfaguanidin reinforce the symptoms of deficiency in Vitamin K.

-   “High energy” food rich in UFA require the supplement of vitamin E.

4. Toxicity

Certain vitamins employed in excess can be toxic. Nevertheless, the toxicity threshold is generally extremely high, so that this sort of accident, except when a grave error is committed, is not to be feared.

An historical example is that of these polar explorers who had to eat bears'liver, so as to survive, and which is very rich in vitamin A. This caused a bone necrosis, which is characteristic of hypervitaminosis A.

A hypervitaminosis was provoked in trout in distributing 2.2 million of I.U/kilo of ratio, which is 1,000 times the upkeep dose. This hypervitaminosis is manifested by a reduction in growth, anemia, and a necrosis of the caudal fin.

30,000 I.U of vitamin D₃ (normal dose x 50) provokes a significant decrease in growth in the channel catfish.

d. VITAMINS AND THEIR MAIN FUNCTIONS

1. Vitamins and reproduction

LIGNIERE pointed out in 1949, of trout fry, that a deficiency in vitamin E, provoked an atrophy of the ovarian chain and testicles; a supplement of vitamin E ensured normal sized organs.

With the Coho salmon, the breeders lacking vitamin C produce fry with a vitamin C deficiency, showing characteristic deformations. Another batch of breeders with a normal level of vitamin C produced fry exempt of deformation.

This shows the importance of food well provided in vitamin E and C for breeders, so as obtain healthy offspring.

The case of caroteneids used in fishculture for pigmentation (canthaxanthin and soon astaxanthin) has not yet been defined. The report between the pigmentation of the eggs and their viability has been studied: There is a critical level of carotenoid (1–3 ug/g-egg) above which it is less than 50 %. This data is empirically confirmed by the aquaculturists who prefer coloured eggs to white eggs. Carotenoids act on the respiratory functions and as provitamin A.

2. Vitamin and defenses of the organism

Vitamin C plays and important part in the metabolism of tyrosin, especially in the synthesis of adrenalinand of noradrenalin-antistress hormones. These hormones, called “of urgence”or catecholamines, permit the organism to face up to an agression in metabolizing an energetic cardiac and circulatory potential.

The catecholamines are synthetized from tyrosin in four stages, 3 of which mobilize vitamin C:

We can use vitamin C for fish each time a decrease in performance after treatment is remarked or after a stressing operation such as sorting out. Distribute 60 g per kg of live weight for 3 consecutive days. The method is the use of pellets having an oily covering.

Vitamins and immunity

During the past ten years, a number of studies revealed that certain vitamins have an immunostimulating effect. These effects were principally studied concerning vitamin C and more recently vitamin E.

Vitamin C

The resistance of Channel catfish to infection by means of an enterobacteria EDWARDSIELLA TARDA was studied. A decrease in mortality was remarked when these fish received 150 mg of vitamin C/kg of food. The test was carried out at 2 temperatures: 23°C and 33°C. At 33°C, vitamin C had less effect than at 23°C. This confirms the importance of the temperature in the mechanism of resistance to agression.

This test was followed by a more specialized experiment where a gradient of vit. C, of 0 to 3 000 mg/kg of food was given and different immunitary parameters were measured. There was total mortality at 0 and zero at 3 000. The production of antibodies was at its maximum at 3 000 mg along with the hemolytic activity of the complement. We can remark here that the immuno-stimulating effect is obtained with doses of 30 times more than upkeep doses.

Vitamin E

Recent studies, where the immuno-stimulating effect of vitamin E on calves was studied, proved that the distribution of vitamin E before and after infection, had a positive effect on the production of agglutinative antibodies and increased the % of success of the vaccination of calf infected by Brucella abortus.

With fish, there are few studies available, however a stimulating effect on the immunitary responses of trout was discovered with the use of vitamin E, after a period of 17 weeks of deficiency, no growth problem occurred. The Authors concluded that the deficiency in vitamin E would be responsible for certain immunodepressive states. This method of prevention against disease by reinforcing the natural defenses seems very promising, knowing the disappointing results obtained with the use of antibiotics, because of their ability of adaptation of infectious agents.

It was believed that this peculiar quality of vitamins could also apply for defense against viral diseases, as it was proven that vitamin C stimulated the production of interferon, a non specific immunitary response.

Practical tests carried out on these lines in the aim of fighting against the V.H.S of trout, proved disappointing, as the distribution of vitamin C increased the mortality. It was concluded that, in experimental conditions, the virus taking advantage of the cellular metabolism also profited from the stimulation of this by the vitamin. As remarked, this method is not simple and can exert an inverse effect of that required thus the necessity to ensure precise research for application.

The immuno-stimulating effects with pyridoxin and pantothenic acid with Channel catfish has been pointed out.

3. Vitamins and the nervous system

Nervous symptoms are often the first to show up deficiencies. The nervous tissues have indeed an intense metabolism, which explains why they are the first to be affected.

VITAMIN B₁ - Its deficiency in trout, leads to hyperirritability, loss of balance (giddeness). Several biochemical relations have been found between Thiamin and the nervous system; Thiamin also participates at the synthesis of acetylcholin, a mediator at synopses level.

PYRIDOXIN - Pyridoxal phosphate is essential for the synthesis of neuro-endocrine substances such as serotonin from tryptophan. For this reason, deficiency in vitamin B₆ is manifested by nervous disorders.

e) PARTICULAR PROBLEMS

1. Vitamins, amino-acids, oligo-elements in ocular diseases

Ocular disorders in fish are often of nutritional origin. However certain eye troubles can be of other origin: physical shock, anorexia, parasites, gassy oversaturation. When the ocular pathology is of nutritional origin, the lesions are generally bilateral.

VITAMIN B₂. Deficiency in riboflavin was the first to be described for ocular symptoms. This is a lesion which commences in the cristalline cortex by a cataract which progressively spreads and becomes opaque, then the cornea becomes affected, finally the ocular globe is liquefied. With the use of a synthetic vitamin, this type of problem has practically disappeared.

AMINO-ACIDS - This concerns another type of cataract completely different from the former one and linked with the degradation of the quality of the diet ingredients, following the rarefaction of the fish meal from Peru in 1973. The use of vegetable protein in excess led to a deficiency in methionin which caused a different type of cataract than that caused by a deficiency in vitamin B₂; The cristalline was affected, but the cornea remained undamaged. Then, liquefaction and necrosis occured, but the nucleus (Centre of the cristalline) was retained by the peripherical fibrous tissus. The addition of methionin to the diet prevented the development of the disorder.

ZINC - In 1973, the manufacturers also used white fish meal, coming from filleted fish meant for human consumption, a meal which was prepared on board. This meal, although relatively poor in proteins was of excellent quality but was excessively rich in mineral matter. The use of this type of meal caused a cataract which started in the perinuclear region in trout fry of less than 1 g and in the cortex region in older fry (more than 4 g). The evolution of the cataract continued with the liquefaction of the cristalline. It has be enpointed out that this cataract was caused by a deficiency in Zinc, although the meal contained 70 – 80 mg. The deficiency is, in fact, induced by a excess of calcium which blocks the Zn available. In adding either Zn (200 mg/kg of food, sulphate) or a chelating agent, this symptom disappears.

The deficiency was first defined in the U.S.A in 1978; the correction was applied to food for trout in France in the eigties. Since then, type of cataract no longer causes any problem.

VITAMIN A - Deficiency in Vitamin A is the only on which implicates exophthalmia with the depalcement of the cornea. The cristalline become opaque and the retina degenerates. This deficiency was produced at laboratory level, but in practice, it is not a treat to fishculture. The generalized use of vitamin premix makes that the apparition of this cataract by means of avitaminosis is very unlikly to occur.

The same does not apply for deficiencies in amino-acids nor minerals, as already seen, they can be the consequence of formula modifications.

In a less spectacular way, bad sight in fish in rearing will lead to a bad perception of the food, which is also detrimental from an economic viewpoint. The fish must be carefully surveyed for this.

2. Problems caused by carotenoids

Carotenoids are employed for two reasons with fish.

-   Vitamin A so as the cover the physiological requirements.

-   Canthaxanthin and more recently astaxanthin for the pigmentation of salmon. Vitamin A, canthaxanthin and astaxanthin belong to the same biochemical family of carotenoids. The two latter molecules possess chetone functions.

At a certain period, the French Authorities were all against the use of canthaxanthin, which was pejoratively qualified as a colouring. So as the avoid confusion with the natural product, the commercial name“salmon trout”was prohibited the fish breeders then choose the name“pink trout”. This name was then prohibited in the eighties and the only authorized one is “trout with natural reinforced colouring”, a complicated and vaguely pejorative expression.

This side issue in the reglementation calls for some precision: Although manufactured by synthesis, canthaxanthin is prevalent in nature, especially in the skirret or chanterelle mushroom from where it takes its name, as for astaxanthin it colours crustacea.

Canthaxanthin is widely employed and gives a pinky orange colour, Astanxanthin, which will soon be put on the market, gives a much similar colour but a little deeper in colour which can be obtained quickly. In addition, and this is its strong point, it is the same pigment which gives natural salmon pink.

Canthaxanthin and astaxanthin are not only simple pigments but also provitamins A.

Thus, there is no justification for unfavourable prejudice against these synthetic products which exist in nature and which merit better than a dissuasive commercial denomination from the authorities.

These substances are employed to colour not only the flesh but also the eggs, which benefit from a noted preference by the fishculturers. We have seen that this preference is justified, by their performances, although the reasons for these good performances have yet to be defined.

f) SOURCES OF VITAMINS

1. Natural and synthetic vitamins

The ingredients used in the formulas contains natural vitamins, however the amounts employed can vary and are not very reliable. It is advisable to cover the needs, by adding vitamins to the premix, However, we must take into account the liposoluble vitamins contained in oil.

Premix contains not only vitamins, but also a neutral support (gluten or wheat middlings) so that its rate of incorporation in the food will represent 1%. It would be indeed impossible to incorporated in a homogenous way the vitamins whose incorporation rates are from 10 – 5 to 10 – 7. industries furnish such premix to food manufactures.

Certain rules must be abided by so as to obtain a good concentration of vitamins. The first and most important is not to put a vitamin premix in direct contact with an oligo-elements premix, as the latter is a powerful destructful agent to vitamins. These two products will be added separately when the food is being manufactured.

Another important precaution to taken is the separate addition of cholin (chlorine) which is a hydroscopic liquid which can also affect vitamins.

2. Technological problems arising by the incorporation of vitamins

Apart from vitamin C which will study alone, the other preparations of vitamins are stable.

Vitamins are more or less fragile organic molecules which are prone to two types of agressions:

1. During manufacture, the pressing process which agglomerates meal produces and the obligation of having to employ a die after humidification and heating. The process of extrusion causes yet more harm to the vitamins.

2. When stocked, the vitamins contained in the food mixture are in contact with more or less agressive elements: oligo-elements, peroxides. This, in hot and humid conditions can accelerate the process of degradation.

With the exception of vitamin c, solutions have been found in all the other cases, which permits their concentration in the food, for at least 6 months. Certain vitamins are presented in an oily solution form (A and E): If employed in this form, they will be quickly destroyed. The coating technique permits good conservation.

Certain vitamins have less resistance during the manufacture of the food:

VitaminB₂ - 25 % can be destroyed during manufacture.

Panthotenic acid - This is an unstable and viscous solution. It is used in the solid form of calcium panthotenate, which has an activity of 46 %.

Nicotinic acid - 20 %can be lost during extrusion.

Folic acid - 10%can be lost during the industrial processing.

Vitamin C - The vitamin C, furnished by industries, is, up to present, fragile. Losses of 88 % after 8 Weeks of storage in food stocking rooms having a temperature of 21° C have been recorded: but only losses of 10 % are recorded if the stocking room is kept at 5° C. The losses during manufacture can vary from 20 % to 60 %, depending on the precess of manufacture employed.

Deficiency in vitamin C, in salmoniculture, is a common thing and is probably at the origin of the body deformations remarked. Controls carried out by us often show rates of less than 100 mg/Kg.

For these reasons, it is advisable:

- To add a much larger amount of vitamin C than demanded: 500 mg/Kg.

- To conserve the food for not more than one month in a place which has a temperature of not more than 20°C. The use of a cold storage room is advisable in the Mediterranean climate.

- To add vitamin C after the pressing or extrusion by means of an suspension coating. However, there is still hope of seeing it commercialized in a more stable form. Ascorbic acid sulphate would be the stocking form in the organism of vitamin C and ascorbic acid the active form. The organism mobilizes the active form from a pool of sulphate.

The sulphate form is much more stable and could be produced industrially. However, this view point is not the unanimous opinion of the specialists and tests are at present being carried out.

III - MINERALS

Two types of minerals which are strictly indispensable for living beings can be remarked.

1. MACRO-ELEMENTS which are the architectural elements of the organism. They are found in concentrations of 10–2 to 10–4: p, Ca, Mg, Na, K, s.

2. MICRO-ELEMENTS or OLIGO-ELEMENTS which intervene as enzyme molecule compounds and as biocatalyzers, their role can be compared to that of vitamins, with which they sometimes interfere. Their concentrations vary for 10–4 to 10–8: Fe, Cu, Mn, Zn, Co, I, Se.

The border between these two categories is not well defined, as certain elements are both structural and catalytic.

An essential difference must be remarked from a nutritional view point between animals with pulmonary respiration and those breathing through the gills. While with mammals and birds, the mineral supply is uniquely obtained through food, with fish the major part of minerals can be absorbed under soluble form through the gills and skin. For this reason, We must relativize the nutrition and requirements for minerals and taken into account their tenor in the water.

Numerous experiments show that when fish are deprived of calcium in their food, they will normally have a provision in their organism: This element being directly absorbed from the water through the gills and skin.

The use of Ca⁴⁵ with Carassius aurata has permitted to remark that 90 % of Ca absorbed by the organism can come from the water.

a) Calcium and phosphore

The most apparent role played by these two elements being the constitution of bone tissue, it is normal that they be associated.

With mammals, 99 % of Ca and 86 % of P are contained in the bones and teeth. With fish, an important part of these two elements is contained in the scales and skin: around 40 %in trout. The relation Ca/P in the whole organism of fish varies from 1.5 to 2.1.

Ca, apart from its structural function it also ensures: the coagulation of blood, muscular contraction, nerve transmissions, and osmoregulation.

Phosphore is necessary for a great number of the essential metabolic functions. It consists of adenosin triphosphate (A.T.P), phospholipids, DNA and RNA. P plays a part in the energetic transformations, the control of permeability of the membranes.

The symptoms of deficiency in Ca are difficult to detect in fish due to the feeble diet requirement rate demanded. In using a yeast basis in the diet, especially lacking Ca, the input was 0.1 %. A loss of appetite, feeble growth and a poor consumption index was then remarked in trout and carp. In practice and with the employment of a classical diet consisting of fish meal basis, it is very improbable to find a deficiency in Ca.

As for phosphore, the water on general does not contain a high rate of this element, except in the case of polluted water where P constitutes an eutrophic element. In marine waters, the rates are very low: 0.1 mg/l.

As there exists a tendency for economic reasons, to use more vegetable products instead of animal products, there often appears a deficiency in P, as this is then less assimilable.

The symptoms of deficiency in P have been studied for the channel catfish and Cyprinus carpio. The signs generally remarked are poor growth, low consumption index and bone deficiencies, We remark in carp a frontal swelling and an increase in muscular fat.

The input of P for fish is that recommended in assimilable form. The normal food at disposal supplies remarkable quantities but often not very digestible.

In animal meal, P is found in the form of tricalcic phosphate of which the the carp assimilates 13 % and the trout 64 %.

In vegetable products, P is essentially found in the form of phytate, which is not very well assimilated (except for peneid shrimp). In the soya bean, the digestibility of P varies between 29 %and 54 %.

The requirements in assimilable Ca varies according to the richness of the water, which, in any case, are very low: 0.10 to0.25 of the ration.

The assimilable P must be supplied in amounts of 0.45 % of the ration for channel catfish and 0.90 %for Tilapia nautica.

As there is very little information available and that phosphate is very important, it seems advisable to supply 0.80 % of P to the diet of fish, under the form of monosodic or monocalcic phosphate.

b. Magnesium

The role played by Mg is not well known but very probably of great importance. Deficiencies in Mg have been provoked in channel catfish, carp, red sea-bream, rainbow trout. The symptoms are not very characteristic: poor growth, anorexia, apathy, muscular relaxation, mortality. Convulsions have been remarked in carp.

Apart from the absolute requirements in Mg, this most be supplied in proportion with Ca and P. According to the water and species, the food supplies must contain from 0.02 to 0.07 %of the Mg ration.

NEPHROCALCINOSIS is a disease which is characterized by white renal deposits of co₃ca, which can reach the size of a walnut in trout and greatly decrease production. The apparition of this disease depends principally on the rate of carbonic gas found in the water. However, this disease occurs more often and as a greater effect in waters lacking Mg.

In the studies which were carried out on the transfer of fish to seawater, Mg plays as important physiological role, when trout were submitted to stress, with a rise in the temperature and the salinity. This stress leads to a characterized syndrome which is followed by death. At the same time a high increase in magnesiemia (up to 300 %) was remarked, as if the membranes couldn't stop the entrance of ions. The hypothesis of blocking the tranmission of the nervous influx by Mg was proposed.

c. Sodium, Potassium, Chlore

These elements are important on account of their participation in the essential physiological processus: osmoregulation, basic-acid balance, chlorhydric stomacal secretion, maintenance of the membrane, potential and transmission of the nervous influx.

Na, K, Cl are found in fresh water and in sea water in more or less great quantities, for this reason, it has not been possible to characterize a dificiency in any one of these elements.

Fish can tolerate excesses of NA Cl in the diet of up to 12% without unfavourable effects occurring: probably, because it is easy to excrete the excess through the gills and skin.

d. Sulphur

This element is found in certain essential amino-acids: METIONIN, CYSTIN, … The requirements in S will be covered when those of sulphured amino-acid are.

e. Iron

The deficiency in iron causes a microcytic hypochromic anemia in trout and carp, … The requirements will be covered for trout with 150 mg/kg in iron.

Growth is not affected by a deficiency in Iron.

Ferruginous water can be toxic, ferric hydroxide forms a deposit on the gills. In such a situation, it is advisable to filter the water over pebbles before using it in fishculture, the ferric hydroxide will settle and the water will be fit for use in fishculture.

Dietetic from: Citrate, chlorine.

f. Iodine

Deficiency in iodine causes goitre, a swelling of the thyroid gland, corresponding to a hyperplasy of this tissue.

The requirement in I for fish are around 1 to 5 mg per kilo of ration.

Deficiency in I can occur in mountainous regions and with certain food: meaty food. In these conditions and in the absence of specific syndroms of deficiency, the distribution of a supplement of iodized protein led to an increase in growth of 10 % with rainbow trout.

With the use of marine products in the rations, the danger of a deficiency in I is feeble. However, as fish meal is scarce, it seems advisable to add potassium iodide.

g. Copper

Copper is employed to for red corpuscles. The requirements in copper are around 1 mg/kg of ration for carp, trout and Channel catfish.

Excess copper (16 – 32 mg/kg) leads to poor growth and slight anemia in Channel catfish. The common employment of sanitary baths with So₄ Cu in Salmon culture, gives reason to belive that there are probably more dangers of toxicity than of deficiency.

Vitamin C has a detoxifier effect concerning Copper and other different heavy metals.

h. Manganese

With carp and trout, tests have showen that the deficiency in Mn occur with 4 mg/kg and causes feeble growth, withered fins and dwarfism. A supplement of 12 mg will suffice so as to ensure normal growth and development. The use of white fish meal will provoke a deficiency in Mn and cause cataracts to appear.

i. Zinc

Zinc is an important biochemical element which enters into the composition of different enzyms (cocarboxylase) and proteins (eye).

Its deficiency in trout causes reduced growth, cataracts, dwarfism, wasting away of the fins, and mortality.

As stated before, concerning ocular troubles, deficiency in Zn has occurred in fishculture, when white fish meal was employed. The symptoms can be prevented by adding 200 mg/kg ration of zinc sulphate.

The use of white fish meal also causes, according to studies carried out in japan, a deficiency in Manganese and Vitamin B², and in this case, it is advisable in supplement in consequence.

With carp Channel catfish, the deficiency in Zn causes feeble growth and mortality, while no cataracts or dwarfish are remarked. The addition of 15 to 30 mg of Zn/kg ration prevents these symptoms.

j. Selenium

Selenium is the key element of glutathione-peroxidase which destroys toxic peroxides coming especially from rancid oil. The symptoms deficiency are similar to those when vitamin E is lacking, the principal one being muscular dystrophy.

The requirements in Se are feeble, around 0. 15 to 0. 40 mg/kg for salmoneids. At rates of 13 mg, it become toxic.

k. Cobalt

This found in the molecule of vitamin B¹² and does seem to have a special fraction, when the requirements in vitamin B¹² are ensured.

IV. CONCLUSION

Without vitamins, certain precautions concerning their conservation are necessary: In particular, never place in direct contact vitamins, oligo-elements, cholin chloride, but incorporate them into the other ingredients contained in the food.

The food must be conserved in good conditions: away from humidity and in a temperature of not more then 20°C. In these conditions, it is advisable to renew fresh food every month. In Mediterranean climates, it is preferable to stock it in a cold room.

Vitamin C will continue to create problem until a stable form can be commercialized. In these conditions, we must remember that a delay in growth and body deformations may caused by a insufficient amount of Vitamin C resulting from the bad conditions of conservation.

Experience has proven that important modifications in the formulation can give rise to certain deficiencies. It is normal to conjecture that the tendency to decrease animal products in favour of vegetable products will lead to the reconsideration of vitamin and especially mineral requirements.

Finally, vitaminotherapy, especially for specific and non specific immunostimulation, permits access to a research method which is only beginning to be explored and which is full of promise.

CARENCE EN ACIDE PANTOTHENIQUE

CARENCE EN VITAMINE C

DYSTROPHIE MUSCULAIRE
(saumon atlantique)

TROUBLES OCULAIRES

MOLECULE DE PHOSPHOLIPIDE