ASCORBIC ACID IN AQUA NUTRITION

Dr.M.MURUGANANDAM

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ASCORBIC ACID IN

AQUA NUTRITION

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First Edition 2013

ISBN-978-9982-22-301-0

Author

Dr.M.Muruganandam. Email vaccine.m@gmail.com Publisher Einsteein Bio-Engineering Research Foundation, South India.

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Dedicated to

Rev.Fr.V.S.Manickam S.J

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Preface

This is a Monograph for Ascorbic acid in Aqua

Nutrition. Ascorbic acid has various functions and

physiological roles in animals. It’s stability in feed is very

low. So Scientists now find out alternative way to

develop for increased stability in Aqua feed, these were

detaily discussed in this book. Other important

Functions of Ascorbic acid such as Stress Management,

Role in reproduction, disease resistant were also

discussed briefly. During the preparation of monograph

many students help me and I took information from

many authors references, most of them, I mentioned in

bibliography. I thank very much to all of them.

M.Muruganandam.

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Contents

1. Introduction

2. Common Functions

3. Stability in Feed

4. Deficiency Signs

5. Stress Management

6. Gametogenesis

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1. Introduction

Discovery

The Hexuronic acid discovered in

1928 by Albert Szent – Gyorgy which was

later found to be identical with the anti

scorbutic component and subsequently

named “Ascorbic acid. In 1937, Albert

Szent – Gyorgy was awarded the Nobel

prize in medicine for the discovery of

ascorbic acid (vitamin C). Mc. Laren et

al., observed that the arrest of growth and

pathologies in rainbow trout fed a diet

devoid of ascorbic acid. After that,

kitamura et al., first clearly demonstrating

the requirement for ascorbic acid in fish

rainbow trout.

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Vitamin C can occur in three

forms. One is a reduced form or L –

ascorbic acid, second one is Oxidized

form or de hydro ascorbic acid, and third

one is Intermediate form or free radical,

unstable and reactive.

The active form is a white,

odorless, acid – taste powder. Ascorbic

acid is well water soluble, only a few

alcohols soluble and insoluble in fat

solvents such as ether and chloroform. In

aqueous solution it is prone to oxidation in

the presence of metallic catalysts, alkali,

light and oxygen, the molecule is

destroyed at 1900 C.

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Biosynthesis

Vitamin C is an essential

nutrient in teleost fishes due to the

missing enzymatic activity of L-

gulonolactone oxidase needed for ascorbic

acid biosynthesis. In other systematic

groups of osteichthys, chondrostein in

particular, the study of gulonolactone

oxidase activity indicated kidney tissue is

where ascorbic acid is synthesized in

sturgeon (Acipenser fulvercens) and

paddle fish (Polyodon spathula).This

discovery suggest that other systematic

units among lower vertebrates (Pisces and

cyclostomata) maintain an active pathway

of ascorbic acid synthesis and teleost are

unique in having lost this ability. It has

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been suggested that shrimps have a

limited ability to synthesize vitamin C

although endogenous vitamin C synthesis

less not seem to meet the requirement in

young crustaceans.

Digestion process

In practical fishmeal based diets,

ascorbic acid is oxidized to the dehydro

form as such as absorbed by mucosal cell.

This oxidation at the intestinal level would

considerably diminish the role of vitamin

C as an antioxidant. The ability to reduce

de hydro ascorbic acid back to ascorbic

acid in the intestinal tissue of rainbow

trout has been demonstrated and this

conversion is apparently a part of the

transport system. There are various

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vitamin C derivatives and different forms

are used as vitamin C source in aqua

nutrition. Ascorbic acid is less stable

compare to these various forms. The

hydrolysis, absorption, digestion of

ascorbic acid and its derivatives are vary

based on stability, digestion and Bio

availability the vitamin C source is

selected for aqua feed.

Interactions with other compounds

Vitamin C and E play important

role in numerous biological conditions,

such as ageing, cataracts, DNA Damage,

diabetes, neurodegenerative diseases,

cardiovascular diseases and cancers.

.Interaction between vitamin C and E

affects their tissue concentration, Growth,

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lipid oxidation and deficiency symptoms

in yellow perch (perca flaversiens) and

other fishes. The interaction between two

vitamins occur at the membrane – cytosol

interface and vitamin E functions as

reducing agent of the membrane bound

oxidized vitamin E.

Vitamin C also modulates the

intestinal absorption copper, decreasing

copper absorption and subsequent toxicity

by keeping copper in the reduced cuprous

state. The interaction between iron and

vitamin C has been shown to occur both at

the intestine level and invivo. In warm

blooded animals, vitamin C is known to

prevent the formation insoluble and un

absorbable iron compounds and to reduce

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ferric to ferrous iron, which is essential for

iron uptake into the mucosal cells

Ascorbic acid is also required for the

release of iron from transferrin and ferritin

and its subsequent in corporation into

hemoglobin or into other essential forms.

Kitamura et al., referred to a study

on Guinea pigs when the requirement of

ascorbic acid was increased during

tryptophan deficiency, which could

explain the development of scoliosis in

tryptophan deficient rainbow trout. The

interaction between ascorbic acid and

Folate may be related to the role of

ascorbic acid as a reducing agent and

antioxidant, protecting reduced forms of

folates in metabolism. Graded dietary

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ascorbic acid (0 , 20 , 200 mg. AA Kg- 11

AAmp) modulated the response of

channel cat fish to dietary Folate and vice

versa as illustrated by red blood cell

pathologies in a study by Duncan and

Lovell . Flavonoids provide antioxidant

protection and ascorbic acid many affect

these properties. Dabrowski et al.,

suggested that the better retention

observed for ascorbic acid derived from

dry feeds may be related to interactions of

Flavonoids in Artemia with ascorbic acid

utilization in fish larvae.

Ascorbic acid analysis

There are numerous methods for

ascorbic acid analysis. The important

methods are volumetric, calorimetric and

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chromatograph analysis. The simple and

easy methods are volumetric and

calorimetric methods. The calorimetric

method is easy, rapid and a large number

of samples can be analyzed in a short

time. This is otherwise called DNPH

method which was described by Omaye et

al. In this method, samples were prepared

by homogenizing it in 5ml of i.e. cold 5%

trichloro acetic acid (TCA). The

homogenates were centrifuged for 20

minutes at 3000 rpm then 0.5ml of

supernatant was mixed with 0.1 ml of

reaction solution (0.4g thiourea, 0.05g CU

So 4 5H 2 O and 3.0g 2,4- dinitrophenyl

hydrasine (DNPH) dissolved in 9 N H 2 So

4 and brought to 100ml) and was incubated

for a period of 3 hours at 37o C . Then

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0.75ml ice cold 65% H2 SO 4 was added

and mixed well. The mixtures were

allowed to stand at room temperature for

30 minutes and its absorbance was re

recorded at 520mm.

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2. Common Functions

Vitamin C is an essential nutrient

for growth, reproduction and stress

reduction. It is mainly involved in many

metabolic reactions. If there is no

appropriate amount in feed, it will lead to

develop disease condition mainly scurvy.

If supposes add requirement level in feed,

the disease conditions will disappear. It

acts as very good antioxidant and essential

for wounded tissue repair. Vitamin C is

also very good Immunostimulant. During

vaccine development, it also used as

adjuvant. It has some synergetic reaction

with some other nutrients. It is also very

much essential for skeletal morphological

development. Because it has main role in

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collagen synthesis. So vitamin C

deficiency influences the formation of

skeletally deformed young ones. During

feed formulation, there is wide chance for

loss and leaching in processing, storage

and introduce in water. These were detaily

discussed in next chapter.

Growth

Vitamin C is one of the important

minor nutrients for growth. Its

requirement varies for larvae, grower and

brooders. But more amount require during

stress condition, stress induce weight loss

during culture. Its requirement also varies

species to species. During larval stage,

larvae feed live organisms which have

more amount of Vitamin C. after shifting

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to artificial feed; we must monitor the

requirement level for improving the

maximum growth. Studies indicated that

the vitamin requirement decreases during

age. Halver stated that, primary growth

involves formation of membranes, cellular

and intra cellular components. Vitamin C

is required for these hydroxylation

reactions.

Dietary requirements appear to be

between 30mg to 100mg ascorbic acid per

kg dry diet ingredients. This level of

intake will satisfy growth requirements of

most fishes but will not ensure an

adequate supply for any stress

encountered in the aquarium environment

The rainbow trout may have the highest

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growth requirement at about 100mg

ascorbic acid per kg diet since other fish

have been reported to grow adequately

when the dietary levels were 30mg / Kg

and catfish have been reported to need

only 10mg /Kg. diet. The rainbow trout

reports were for fish fed at very rapid

growth rakes in 150 C water may have

increased the demand for more vitamin C

for more tissue synthesis. Growth

requirements are there fore probably

related to not only species reared but rate

of tissue synthesis. The source of the

vitamin is also an important consideration

on measuring growth requirements.

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Immunostimulant

Vitamin C acts as

Immunostimulant. Friedmann et al stated

that Immunostimulant stimulate the

macrophage immune-force of the fish to

eliminate unwanted pathogens in their

blood stream. Furthermore, it also relieves

allergies that may occur due to depressed

functionality of the immune system by

supplementing the fish’s diet with these

immunostimulants. The use of

immunostimulants for preventions of

diseases in fish is considered an important

attractive and promising field.

Elevated doses of dietary

vitamin C have been shown to enhance

several non – specific responses such as

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macrophage activation, lymphocyte

proliferation, natural cytotoxicity,

complement activity. Waagbo et al also

reported that elevated antibody levels after

vaccination have been observed in fish fed

high level of vitamin C . Most of the trials

on the modulating effect of vitamin C on

antibody production after vaccination

have shown an improvement of the

response.

Dietary vitamin C enhances

phagocytosis and it influence on

macrophages activities. The increasing in

plasma lysozyme with increasing level of

vitamin C was reported by waagbo et al.,

Many researchers observed that an

increase in complement activity in fish fed

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vitamin C at long term feeding, and also

increases blood peripheral lymphocyte

level. Amnah A.H.Rayes reported that

phagocytic activity and phagocytic index

of O.niloticus fed on diet contain vitamin

C and S. cerevisiae were significantly

elevated than the control group, also

lysozyme concentration serum albumin,

and globulin and A/G ratio.

Antioxidant

The role of ascorbic acid as an

antioxidant evolved in vertebrates over

400 x 10 6 years ago independently of the

rise of oxygen atmospheric pressure total

present level. The ascorbate requirement

is differ in fishes living in water of various

oxygen tensions. Derivatives of ascorbic

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acid with increased stability against

oxygen, especially sulfated esters, were

found in cysts of brine shrimp frequently

exposed to air during prolonged periods.

During embryonic development and after

hatching, naupli of Artemia mobilize

ascorbyl esters into free ascorbic acid. The

presence of ascorbyl sulfate sulfohy

drolase has been documented invitro but

in hardly explains the hydrolyzing

capacity observed invivo.

The antioxidant defense system

provides protection against potentially

harmful ROS (reactive oxygen species)

produced constantly during aerobic cell

respiration. In fish, as in mammals,

endogenous antioxidants such as GSH

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(Glutathione) and the antioxidant enzymes

CAT (cataloes) and Sod (superoxide

dismutase) as well as dietary antioxidants

such as vitamin C and Vitamin E protect

cells against oxidative damage. Vitamin C

synergistically with vitamin E, selenium,

zinc and copper in cell membrane

protection. Essential trace metals such as

copper (Cu), manganese (Mn) and iron

(Fe) are incorporated as functional redox

centers of antioxidant enzymes. However,

excessive amounts of some trace metals

may also serve as pro-oxidants.

Furthermore, the Solomon body contains

ample amount of n-3 PUFA that are

highly susceptible to oxidation. There

fore, to achieve a balance between the

amount of Ros produced in the body and

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the levels of antioxidant to protect against

them, it is important to establish well-

balanced diets with an optimal

composition of pro- and antioxidants.

Vitamin C involves in inactivating

harmful free radicals produced through

normal cellular activity. The antioxidant

function of vitamin C could in part, at

least, enhance immunity by maintaining

the functional and structural integrity of

important immune cells.

Metabolism

Vitamin C is the major intracellular

reducing agent in fish tissue. It is also

used to maintain a metallic ion in a

metallo enzyme system in the active

reduced state for electron transfer.. Tucker

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and Halver reported that vitamin C

involved in many hydroxylation reactions

in the body. The conversion of pro

collagen into collagen tissue by

hydroxylation of proline and lysine

residues Which is a basic growth process

in the formation of cellular membranes

Most fish have low level of gulonolactone

dehydrogenize and therefore require

ascorbic acid in the diet. It is involved in

collagen and cartilage formation in bone.

It is involved in mixed function oxidize

activity, in detoxification systems, in

hydroxylation of steroids and hormone

functions. It is involved in the reduction of

iron to enhance absorption. It is also used

to synthesis of tissue and during

metabolism of numerous essential

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anabolic components as well as

detoxification of xenobiotics, toxicants

Steroids and pollutants.

Wounded Tissue repair

Higher amount of vitamin C in diet

is necessary for wounded tissue repair in

fishes. Halver et al detaily reported that,

when Coho salmon or rainbow trout were

fed diets with different levels of vitamin C

content. Fish fed diet treatments of 100mg

c/kg diet repair wounds slowly where as

those fed diets with vitamin C at 200, 400

or 800mg/kg repaired both muscle and

skin tissue more rapidly directly

dependent upon the vitamin C in takes up

8 to 10 times the growth requirement. Fish

fed 50 or 100 mg/ kg diet has 3 week post

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incision wounds with poorly reunited

epidermis with the dense dermal collagen

layer hypo plastic fish fed 200 mg vitamin

c / kg diet showed fine collagen fibers and

immature fibroblasts with regenerating

muscle fibers in the musculature Coho

salmon showed more rapid repair that the

rainbow trout fed the same level of

vitamin C indicating an apparent lower

requirement for tissue repair fish fed diet

with 400mg or100mg vitamin c/ kg diet

had very rapid wound repair and after

3weeks on these wound repair diets , the

incision was well repaired and collagen re

connected with scabs regenerated and

muscular are near by normal in tissue

histology. The rates of wound repair

suggested that 4-8 times the growth

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requirement would be necessary to

promote the most rapid wound stress

repair.

Gametogenesis and

Embryo development

Vitamin C is essential for

biosynthesis of hormones is important for

sexual maturation and prevents or reduce

the oxidation of biomolecule. The seminal

plasma has high amount of vitamin C

compared to blood plasma. If there is

lesser amount of vitamin C in seminal

plasma leads to formation of abnormal

embryos. The fertilization rate – also

depends the amount of vitamin C present

in seminal plasma the increasing survival

rate of embryo and hatchability depends

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on vitamin C present in brooders diet.

Bloom and Dabrowski reported that the

both fecundity and embryo survival also

increases with an increasing supply of

vitamin C through diet. Vitamin C is

necessary for successful breeding in fishes

it mainly involves in sperm quality, ovary

maturation, embryo development, survival

rate, etc. Many researches proved that

vitamin C is very much essential for

normal development and survival of

gametes, embryo and larvae. Vitamin C

role in reproduction was detailed

discussed in last chapter.

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3. Stability in Feed

Stability of ascorbic acid is

much more problems in aquaculture feeds.

Several attempts have been made to

improve the stability of ascorbic acid.

Different forms are used such as protected

forms (Coated forms) and derivatives

(different chemical forms). The protected

forms are obtained which coating vitamin

C granules with fatty acid, cellulose or

silicones which are impermeable to

oxygen and metabolic catalysts. These

coated forms help to increase stability of

vitamin C in Aqua feed and reduce the

leaching. The stability levels differ in

various coated forms. The alternative

sources of ascorbic acid derivatives also

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have been proposed as solution to the

problem of instability of vitamin C in

aquaculture feeds. Several of these forms

are tested for stability in main types of

feeds. The main stability forms are

sulfates and phosphates of ascorbic acid.

They are more resistant to heat, light and

oxidation and less water soluble.

Important Factors

The stability of vitamin C depends

upon temperature, oxidation, and

environment, presence of metabolic

catalysts and time of processing and

storage. Heiteid et al demonstrated that the

effect of light on destruction of vitamin c

in milk stored in ruby, amber and clear

bottles. They found that lower losses of

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vitamin c occurred in milk kept in clear

bottle. Similarly in the current

experiments losses in clear polythene bags

were higher than in black bags at the same

temperature. Wanninger also reported that

the temperature, O2, PH & light are

important factors which are stimulate the

rate of destruction of vitamin c. Amerio

and Benvenuti, also stated the vitamin c is

extremely prone to oxidation and during

feed processing, heat and moisture can

induce a rapid decay of it.

Processing Loss

The different pelleting procedures

have great influence on stability of

vitamin c. During feed processing, water is

injected, steam and extrusion led to losses

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of vitamin c content up to 90%. However

that for commercial diets the above

reported losses could be reduced. Wilson

et al , reported that severe losses during

extrusion of pellets for channel cat fish

Ictulurus punctatus and up to 50% of

original content. Eva et al also reported

that the L. ascorbic acid in feed was lost

during processing and storage at room

temperature. However Hilton et al also

found that after processing and 6 weeks of

storage at room temperature, all of the

ascorbic acid was lost in feeds

supplemented with 20 – 128 AA/kg. So

that the feed processing is one of the

major causes for losses of vitamin C. If

use good processing technique, we can

reduce in the losses level of vitamin C.

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The relevant of dietary ascorbic

acid was affected by storage conditions,

exposure to light and length of storage.

Temperature of storage also of affected

the stability of ascorbic acid. Sandnes and

Utne reported that fish feeds stored at

room temperature (200 c) lost nearly all of

their vitamin C after 16 weeks of storage

compared to a loss of 70% after 24 weeks

of storage at 40c. Soliman et al stated that

after 12 weeks storage at 40c Na –

ascorbate was still present as 13.9% of the

original content and ascorbic acid as

17.8% the freezing storage greatly reduce

ascorbic acid losses, while at room

temperature, the ascorbic content of feed

was reduced from 400mg/kg to loss than

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100mg/kg Within 5 weeks (Pelleted feeds)

or within 8 weeks (extruded feeds)

Field Loss

In culture filed, Losses begin during

the immersion of feed in water. Soliman et

al also reported that a high correction

between losses of vitamin c from the feed

due to leaching during immersion time.

Yamamoto stated that vitamin c losses up

to 99% during the 5 minutes of immersion

into water. The losses up to 10% of

original contents of vitamin c after 10

seconds immersion water was reported by

Hilton et al.

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Recommend for Rectification

Yamamoto reported that with five

minutes of immersion, more or loss all the

vitamin c was leached in feed. So that the

important is to reduce the time of

immersion as far as possible by the use of

improved feeding husbandry i.e. through

the use of automatic feeders or by

increasing feeding frequently and the

usages of palatable feeds. Another way is

use of coated from vitamin c or derivative

forms of vitamin c also help to reduce

leaching. Otherwise, increasing level of

dietary vitamin c resulted in significantly

increased retentions of vitamin c after

processing and storage even through the

losses, in absolute terms, were higher in

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high vitamin c in Feeds. So that the more

rate supplementation levels prevent

deficiency signs in fishes. So if follow

these management approaches, it will help

to reduce the major level of vitamin c

losses in feed.

Stability of Coated Forms

The coated forms help to increase

stability in vitamin c in Aqua feed and

reduce the leaching. The stability levels

differ in various coated forms. In this

chapter, various coated forms and their

stability in aqua feed were discussed.

Polymer coated

Ascorbic acid (PCAA)

The synthetic polymer coated

ascorbic acid (PCAA) is more stable then

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crystalline vitamin c (Ascorbic acid) and

other coated forms also tested in Ethyl

cellulose coated ascorbic acid and

glycerin coated ascorbic acid. The

researcher also reported that the PCAA

has superior stability and at the same time

full bio availability. The ascorbic acid loss

during pelleting was 29% for crystalline

ascorbic acid and 19% for PCAA. After 6

weeks at room temperature 73% of PCAA

was still retained in feed. Based on this

reports PCAA is one of the good sources

for increase stability of vitamin c in feed.

Ethyl cellulose coated

Ascorbic acid (ECAA)

Ethyl cellulose coated ascorbic acid

(ECAA) exhibited higher stability when

stored in a freezer (-200 c) and refrigerator

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(50 C) than when stored at room

temperature. All ECAA in feed was lost

after 6 weeks of storage at room

temperature where as the loss was not

quite as serious in commercially produced

feed, in which 25% was left after 5 weeks

at room temperatures. Sandnes and Utne

reported that commercial trout feed

contained only 17% of ECAA after 4

weeks at 200 c. In a Pelleted catfish feed,

the half times for uncoated ascorbic acid

and ECAA were approximately 2.3

months and 2.6 months respectively.

Glycerides coated

Ascorbic acid (GCAA)

Good vitamin activity for ascorbic

acid coated with a mixture of mono – di

and tri-glycerides in tilapias feed. The

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residual amount of vitamin c in the feed

after 12 weeks storage 40c was 45.5%

compared with 17.8% in the control. The

GCAA should a higher stability than

crystalline ascorbic acid. The leaching of

dietary ascorbic acid increased with

increasing immersion time and water

temperature. But GCAA has hydrophobic

coating it increase its stability. The

stability of GCAA under different storage

conditions was in decreasing order

respectively, freezer (-200 c) refrigerator

(5.8 0 c), room temperature in black bags

(22 – 240 c) and room temperature in clear

bags (22 – 240 c).

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Stability of coated forms

Soliman et al reported that

normally, 70% of the ascorbic acid

content in feed was decreased. The coated

form of vitamin c has lesser stability

during processing and storage in trout and

catfish feed. Losses after production were

in the range of 49 – 5%, after 1 month of

storage retention could not be improved

by varying extraction conditions.

Robinson found that 50% loss after feed

manufacture of catfish feeds when fat

coated of Ethyl cellulose coated forms of

vitamin c were used. The losses are found

during storage varied from 38 to 100%

after 1 month and depended on the

moisture content of feed.

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Ascorbic acid Derivatives

The protected forms of ascorbic

acid have been indentified in fish tissues

and have been manufactured industrially.

First L – ascorbyl -2- sulfate was

indentified. Then ascorbyl -2- di-

phosphate and ascorbyl -2- tri-phosphate

also identified from fish tissue they have

the very good stability because in their

structure, at the c-position of lactone ring

have been used to increase it resistance to

oxidation The requirement levels of each

derivatives vary because their hydrolysis,

absorption, digestion and bioavailability

levels also vary. The other forms such as

ascorbyl palmitate, ascorbyl glycerides

etc. are also used as ascorbic acid sources

in feeds. During the stability forms

45

selection for feed formulation another one

important aspects is cost effect of

derivatives. We always give importance

select suitable source which is lease cost

with best quality. The important

derivatives are discussed one by one.

Ascorbic phosphates

In this form phosphate combines

with ascorbic acid to form stabilized

ascorbic acid these compound hydrolysis

in the digestive tract of the fish releasing

the ascorbic, which is then absorbed into

the blood Fish allows efficient absorption

and storage of ascorbic acid in liver.

Mainly two derivatives are used in

commercial feeds, one is ascorbyl-2-

46

mono phosphate and another one is

ascorbyl -2- polyphosphate These two

forms are found to be quite stable that is

retentions were higher than 90% in the

stored extrudes, feeds and above 85% in

the stored semi – moist feed. Both forms

are stable to oxidation and have relatively

long self – life.

Ascorbic -2- sulfate

Ascorbic -2- sulfate is very stable

vitamin c – source, resisting oxidation,

since the labile -2- position ascorbic acid

is now stabilized with an electron dense

sulfate unit. This compound has been

found in the tissue of fish, rat and man.

Enzymes for its synthesis have been

reported. One of the important functions of

47

ascorbyl -2- sulfate is storage form in fish.

They also reported that fish salmons fed

ascorbyl -2- sulfate showed fixation into

collagen and support cartilage with the

same distribution as labeled ascorbic acid,

indicating the fish has ability for

absorption and use of the material in their

tissue. Halver et al. ., reported that one

long term feeding study with ascorbyl -2-

sulfate and rainbow trout in seawater for

one year symptoms when this compound

was used as vitamin c sources. Ascorbyl -

2- sulfohydrolase present in salmon tissue,

converts the ascorbyl -2- sulfate into

ascorbic acid and this enzymes may be the

modulator of cellular levels of ascorbic

acid in fish. However Dabrowski and kock

stated that the stable ascorbyl -2- sulfate is

48

poorly absorbed by fish intestine and

results in lower tissue accumulation

relative to native ascorbic acid.

Ascorbic palmitate

The utilization of ascorbyl

palmitate could require an indicate

enzymes, lacking in the first state of life

of fish. In fact, during the cited trial fish

fed ascorbyl – palmitate exhibited lesser

growth only during the first 8 weeks. If

compared with control group Albreksen et

al reported that ascorbyl palmitate

administrated with rainbow trout finger

lings (600 mg / kg AA) showed ant

scorbutic activity to a lesser extent that

ascorbic acid.

49

Ascorbic -2- glycoside

Ascorbic -2- glycoside is a recent

addition to the vitamin c source in the

market. It is stable and appears to be well

digested and absorbed to supply the

vitamin c needs of fish. More works needs

to be done to assess its efficacy as vitamin

c source for resistance to stress and

disease.

Bio availability

The concentration of free ascorbic

acid in the liver was used as an index of

the ascorbic acid status of the fish. The

biological activity of ascorbyl phosphate

is superior to the dietary ascorbic acid.

Buddington et al reported that the affinity

of carriers was higher for the mono

50

phosphate than native ascorbic acid.

Tucker and Halver stated that rainbow

trout stored ascorbic acid in body in

significantly lower concentration of

ascorbate in liver, kidney and intestine of

rainbow trout and common carp. In the

activity of ascorbyl -2- sulfate was four

times less than ascorbic acid. Dabrowski

and kock demonstrated that ascorbate -2-

sulfate shows a low bioavailability for

rainbow trout to be good. In catfish,

conclusion, for reduce the losses, we will

add more amount of vitamin c in feed is

good. If we add protected or coated form

or whatever it may be we add more

quantity leads to chance of reaching more

amount in our target fish, and another

thing is If we give importance to

51

processing methods and storage

temperature which are also help to reduce

the leaching or denature of vitamin c in

feed.

The coated vitamin c increases

stability during processing storage and

also immersion time in water, the stability

depends on types of coating what we used.

The studies showed that among all the

coating PCAA has more stability and

other coated forms are also good with

lesser stability.

Vitamin c derivatives help to

increase stability of vitamin c in feed

compare to all derivatives phosphate are

suitable for feed. The sulfate also suitable

but it has how bioavailability. But

52

generally bioavailability of all the

derivates is good. Further research is need

to other derivatives for their usages in fish

and feed.

53

4. Deficiency signs

Vitamin C is essential for normal

growth immunity and reproduction in

fishes and shell fishes. It has a major role

in enzyme activity. The minimum dietary

requirement of vitamin C in most fish

species has been estimated in the range of

10 to 60 mg/kg dry diet, below this level

of supplementation, fish shows deficiency

symptoms. In this article, vitamin c

deficiency disease and requirement levels

were discussed.

Impaired Collagen synthesis

The collagen is a vital component

for the formation of exo-skeleton of

prawns, bone cartilage and connective

tissue of fish. Vitamin C is necessary for

54

the synthesis of collagen and it is an

important for formation of hydroxyl

proline and hydroxyl lysine. The

minimum dietary vitamin c requirement to

maintain normal collagen formation in the

tissue of fish is essential to 5-10 mg/

10gm diet. Halver was using C14 radio

labeled ascorbic acid and identified the

skin, caudal fin, snout cartilage head and

jaw, gill support cartilage and bones as

Collagen forming area in the fish. The

impaired Collagen formation affect the

bones and other structural tissue giving

tissues to the classical vitamin C

deficiency syndrome like, black lesions

below exoskeleton chronic soft shelling,

opaque whitish muscle, big head with

flapped gill cover, incomplete molting and

55

increased rates of mortality. Meanwhile

those symptoms are quite common among

farm-reared animals especially when

forming is practiced at high density.

Table: 1 General scurvy Signs (sources-Halver 1995 b)

o Anorexia, Weakness, Aching Joints

o Hair Loss

o Dry Skin ,Loose Teeth,

o Bleeding Gums,

o Paranoia / Depression,

o General impaired collagen formation.

56

Table: 2 Vitamin C requirements for young

growing fish (sources-Halver 1995a)

Fish

No Scurvy

(mg / kg)

Max.

Growth

(mg / kg)

Max.

Health

(mg / kg)

Rainbow

trout

Coho

Salmon

Channel

Catfish

Common

Carp

50

30

20

10

100

50

30

30

300-600

200-500

100-300

100-300

57

Decalcification

Dabrowski et al reported that

inadequate intake of dietary ascorbic acid

results in decalcification, which leads to

twisted gill filament in rainbow trout.

Cleft branchiostegal membrane, an

opercular abnormality commonly

observed among milk fish fry and

juveniles and he also reported that, the

decalcification process in seen, when the

content of vitamin C in diet is below

30mg / kg. The Vitamin C deficiency

affects the development of gill support

structures the branchiostegal rays and

opercular deformity in milkfish, distortion

of gill filament cartilages and snout

opercular were observed in scorbutic fish.

58

Skeleton morphology

Wimberger stated that, the absolute

amount of vitamin C in the diets or an

interaction of vitamin C with some other

unidentified factors, that the presence or

absence of vitamin C in the diet affected

body shape, jaw and skull morphology.

The vitamin C deficiency has profound

morphological consequences and it has

been qualitatively documented in Coho

salmon, rainbow trout, channel catfish,

yellow tail, Tune, guppies and carp. The

vitamin C deficiency leads to scoliosis and

lordois of the spine (abnormal lateral or

dorsiventral curvature) Some times

actually resulting in broken backs.

Wimberger reported that the measurement

59

of the skull of vitamin C deficient fish

shoved that their skeleton was relatively

shorter the fish fed with vitamin C

sufficient diet. The shortened heads, blunt

snouts were observed in vitamin C

deficient fish. Wimberger also reported

that the fish fed vitamin C deficient diet

differed in caudal peduncle

measurements, shorter snout and oral jaw

measurement than fish fed the vitamin C

sufficient diet.

Minimum requirement level

The body pool of ascorbic acid is

maintained minimum critical level and it

is influenced by level of ascorbic acid in

diet. The dietary requirement appears

between 50-100 mg/kg ascorbic acids.

60

This level of intake will satisfy growth

requirements of the most fishes. The

rainbow trout may have the highest

growth requirement at about 100mg

ascorbic acid per kg diet the other fish

have been reported only 10 mg/kg diet.

The values if 10 -30 mg vitamins C/kg

recommended for normal growth and

Collogen formation in most of the fishes.

But In the European cat fish Silurus glanis

has diet supplementation with 50mg/kg

vitamin C showed significant positive

effect on growth performance. For shrimp

species up to 10,000 mg / kg ascorbic acid

requirement was reported. Generally

larvae of fish get vitamins from live

plankton feeds.

61

In conclusion, Vitamin C deficiency

leads to impaired Collogen synthesis,

decalcification, changes in body

morphology and increasing rate of

mortality. So the evolution of optimum

level of vitamin C for all cultivable fish

species are needs for attain maximum

growth and avoid above said vitamin C

deficiency diseases.

62

5. Stress management

Stress may be defined any

conditions which adversely affect life or

productivity in production system and the

maximum express of stress is the death

from fright or stock even though no

physical injury is involved . Vitamin c

plays a protective role against different

stress present in farming conditions.

Vitamin C also involve in detoxification,

disease resistance, etc. in this article,

mainly discussed about vitamin C role in

stress management in fishes.

Stress mechanism and Indicator

The stress responses are classified

into primary, secondary and tertiary

components. The primary effect involves

63

increased activity of the hypothalamic

pituitary internal axis, resulting in

increased levels of cartecholamine and

gluco corticoid hormones which induce a

wide variety of secondary effects

metabolic hematological, hydro mineral

and structural changes. The secondary

changes ultimately result in tertiary effects

i.e. those which are manifest as gross

changes in physiogical performance at the

whole organism level.

The tertiary effect of the

aquaculture industry is the decrease in

disease resistance of stock generally

accepted as being attributable to the

immunosuppressive effect of elevated

level of cortisol .increased levels of

64

circulating cortisol affect the teleost

immune system at many levels The

plasma cortisol concentration is a good

indicator of stress. Often plasma cortisol

levels rise at the beginning of a chromic

stress situation Donaldson also reported

that environmental stresses increased the

synthesis of corticosteroids by the anterior

kidney of the fish; therefore, variation in

haematic cortisol levels could be a good

indicator of stress.

Expression

During stress condition, vitamin C

level decreases in body storage and

finally, vitamin C deficiency symptoms

are expressed in many cases. Mayer et al

reported that the laboratory studies which

65

showed that chronic exposure to

taxaphene increased the vitamin C

requirement of channel cat fish and

express deficiency signs such as scoliosis,

which could be reversed by

supplementing the diet with vitamin C. in

addition; studies with other pollutants can

alter the vitamin C status of fish tissues.

The symptoms of vitamin C deficiency

such as scoliosis, eye lens damage fin

degeneration and epidermal damage have

been found in fish exposed to oil.

Suggesting that oil exposure may deplete

teleost vitamin C reserves. Further more

fish held at high stocking density stresses

leads to lower final weight than fish held

at lower stocking density.

66

Yasunori Ishibashi et al also

reported that the fish which were exposed

to the stress and fed on a diet containing

no vitamin C supplement, vitamin C

deficiency signs such as reduced growth

and high mortality were manifested early

in comparison with the vitamin C

supplementary group. Also the condition

factors, haemotocrit values and

heptosomatic indices tended to the

reduced by exposure to the stress. Thomas

et al also stated that the adverse

environmental stimuli associated with a

loss of gill vitamin C reserves. The

vitamin C has been implicated in the

control of the iron or osmoregulatory

functions of gill tissue, possibly by

inhibition on Na +K+ ATPase. It follows

67

proper management practice and handling,

we will reduce certain amount of farm

made stresses.

Stress reducing process

VitaminC breaks on steriodogenesis

through peroxidation of unsaturated lipids

there by preventing their conversion into

cholesters which are important

components of cortisol. The important

stress response and indicator is an

increasing level of plasma corticosteroids.

So vitamin C breaks these reactions. Early

experiments with salmanids appear to

confirm that steriodogenesis was inhibited

by high level of vitamin C in the

interregnal tissue. In higher animals, the

concurrent administration of vitamin C

68

can greatly decrease the extent of

cytogenetic damages induced by the

dietary concentration of aflotoxin. Similar

protective effect of vitamin C has been

also observed in invitro systems.

Stress management

Different strategies have been

proposed as being potentially beneficial

for reducing the physiological effects of

stress in farmed fish; one of the important

ways is feeding elevated levels of vitamin

C. It increases growth and immune

responses. It also reduces the influence on

various environmental stresses. During

more addition of vitamin C in diet, leads

to no harmful because the excess amount

of vitamin C eliminated by excretion.

69

Proper maintenance and good

management of the system will also

helpful to reduce the stress.

Detoxification

The effects of dietary vitamin C on

the resistance of fish to environmental

pollution have been established vitamin c

is reported to have antitoxic properties and

it also cure the cancerous neurons. The

exposure of fish to environmental stress or

pollutions causes ascorbate depletion from

body tissues. In that time, ascorbate

content of anterior kidney of rainbow trout

drops, while haematic cortisol increases.

Mazik et al. reported that ascorbate

depleted channel catfish appeared more

sensitive to environmental stress such as

70

elevated ammonia and reduced oxygen

levels in ponds. The stress induces

fluctuations of vitamin C content in each

organ in fish depending on the species and

on the strength of the stress. The decline

of vitamin C may reflect an increased

vitamin C requirement for detoxification.

Vitamin C is important in maintaining the

redox state of the cell and may act directly

to reduce the superoxide anion radical

generated during exposure to xenobiotics.

Halver clearly stated that the role of

vitamin C in detoxification. The vitamin C

is involved in detoxification process in

liver and kidney tissue, mixed function

oxidizes are involved in catabolism of

xenobiotics, drug, toxicants, steroids and

71

nutrient metabolites. Most of these

detoxification processes involves

hydroxylation reaction. Vitamin C

deficiency reduces cytochrome

proactively. An increased tolerance of fish

with high stores of ascorbate to

proactively. An increased tolerance of fish

with high stores of ascorbate to

environment pollutants has been reported.

Disease resistance

Bacterial activity and leucocytes

respiratory burst activity were both found

to be significantly reduced by stress.

Production of specific antibody following

immunization with Aeromonas

salmonicida was found to be significantly

reduced greater levels of specific antibody

72

in fish fed the vitamin C diet. The

supplementation of vitamin C enhances

antibody production against Edwarsiella

ictaluri in channel catfish and against

vibrio anguillarum in rainbow trout and

also enhances phagocytic activity and

serum lysozyme levels in turbot. Vitamin

C has been suggesting as having a positive

role in the amelioration of stress.

The serum lysozyme activity was

elevated high stocking density except in

fish fed the vitamin C supplemented diets.

This elevation of lysozyme activity could

be due to moderate stress and

supplementation of vitamins in the

particular factor. Complement activity

also seems to be related to dietary vitamin

73

C levels in some species such as channel

catfish, Atlantic salmon and rainbow

trout.

The dietary vitamin C for Japanese

eel Anguilla japonica at the dose

762mg/kg diet for three weeks showed

significantly higher lysozyme activity of

mucus and serum compared to control

group was reported. The post dietary

administration of vitamin C for Japanese

sea bass Lateolabrax japonicas at

489mg/kg diet for 8 weeks there was

significant increase of lysozyme activity

in serum with the increase of dietary

ascorbic acid was reported. Feeding of

turbot with vitamin C leads to enhanced

phagocytic activity at dose of 800mg/kg

74

whole phagocytic activity serum lysozyme

were markedly increased at dose of

2000mg/kg diet. Feeding of rainbow trout

with vitamin C supplemented diets caused

a significant increase in lysozyme activity

at dose of 1000 ppm were also reported.

In conclusion, vitamin C has

protective role against environmental

stresses. Vitamin C reduce the

immunosuperssion which is caused by

stress and it act as Immunostimulant in

fishes. So higher levels of vitamin C in

diet is required under stress condition.

Vitamin C also reduces effect of

corticosteroid hormone on target cells and

involve in detoxification process.

Moreover all stresses can reduce the

75

vitamin C status in fish and finally express

as disease condition. If like to overcome

the stress conditions, it needs elevated

levels of vitamin C in diet. This is one of

the important ways for stress

management.

76

6. Gametogenesis

The role of vitamin c in

reproduction has been derived from its

reducing properties and defined in terms

of multiple biological actions such as

involved biosynthesis of steroids and

peptide hormones and prevention of

reduction of the oxidation of bio

molecules. In this chapter, mainly discuss

the role of vitamin c in seminal plasma

and sperm quality, because the fish

seminal plasma has higher concentration

of vitamin c compared to vitamin c level

in blood plasma and the low level of

vitamin c in seminal plasma of rainbow

trout was associated with high percentage

of abnormal embryos in off spring”.

77

Sperm quality

Many substances can function as

antioxidants in seminal plasma and other

extracellular body finds and vitamin c is

probably one of the most important

relatively abundant compounds. Cieresko

and Dabrowski reported that there are

much high concentration of vitamin c in

fish seminal plasma (30 – 60ppm) than in

blood plasma (1-10ppm). This suggest

that vitamin c may be even more

important as an antioxidant in male

reproductive tract that in blood plasma.

Vitamin c in fish seminal plasma appeared

to be well protected against oxidation by

seminal plasma proteins. The peroxidation

damage to spermatozoa can be reduced by

78

vitamin c during the reproductive season,

which may affect sperm fertilizing ability

and they also reported that ascorbyl

monophosphate supplemented have, more

abundant polyunsaturated fatty acids,

because of the lower level of peroxidation.

The antioxidant activity of ascorbic acid

towards lipid peroxidation was also

demonstrated in bovine seminal plasma.

The vitamin c deficiency leads to reduced

sperm concentration motility, fertility,

protein concentration and antiproteinase

activity of seminal plasma of rainbow

trout. There is growing evidence that

vitamin c may have a role in cell

proliferation and gene expression so

vitamin c important to maintain sperm

quality.

79

Deformed embryos

The low fertilization success of

spermatozoa from fish without vitamin c

in seminal plasma was reported by

Dabrowski and Cieresko. The low

paternal vitamin c status may cause

mutations to sperm and birth defects. The

sperm motility and concentration were

impaired by vitamin c deficiency and it

also reduced the ability of sperm to

fertilizer eggs. Dabrowski and Cieresko

was also reported the supplementation of

the diet of three years old male rainbow

trout with a high dose of vitamin c

increased its concentration with in the

reproductive tract and decreased the

percentage of abnormal embryos in

80

progeny. The low level vitamin c in

seminal plasma of rainbow trout was

associated with a high percentage of

abnormal embryos in the off spring.

Among abnormal embryos,34.8%were

haploids and aneuploids, as revealed by

flowcytometric measurement of DNA

content. The UV irradiated sperm gave

rise to progeny with abnormalities similar

to those resulting from sperm with low

level of antioxidants in seminal plasma”

and they also reported that the low

antioxidant levels have been implicated in

damage to sperm DNA. So it leads to birth

defects.

81

Seminal plasma vitamin C

The rainbow trout fed a diet with no

ascorbyl mono phosphate. Supplemented

had the lowest vitamin c concentration in

seminal plasma and the highest

malondialdehyde production in sperm.

While the vitamin C decreases towards the

end of the reproductive season, the

malondialdehyde production tended to

increase.

These result demonstrated that

dietary ascorbyl monophosphate

supplements could increase seminal

plasma vitamin c concentration and

suggest that peroxide damage to

spermatozoa can be reduced during the

reproduction season. The vitamin c levels

82

spermatozoa can be reduced during the

reproduction season. The vitamin c levels

in seminal plasma are related directly to

dietary concentration of vitamin c.

Protection of spermatozoa

In rainbow trout spermatogenesis is

completed about two months before

spawning started and spermatozoa are

stored in lumen and then are released to

sperm ducts. Spermatozoa are stored in

sperm ducts throughout the reproductive

season, which can last six months, during

the long period of spermatozoa storage,

vitamin c in seminal could be a good

candidate to product spermatozoa from

attack by reactive oxygen generated by a

83

population of degraded sperm and other

sources such as in filtrating phagocytes .

In conclusion, there is a very

limited report in vitamin c role in male

reproductive system of fish. The vitamin c

deficiency leads to reduced sperm

motility, fertility and increased percentage

of abnormal embroys. So vitamin c is very

much essential for male fish brood stock

nutrition.

Egg maturation

The brood stock nutrition is

important part for successful breeding in

fishes. Deficiency of vitamin c can

directly influence on gamete quality of

male and female fishes. Hilton et al also

reported that vitamin c may have a critical

84

function in fish reproduction and the

feeding of an ascorbic acid free diet to

brood stock could impair reproductive

performance. In this chapters mainly

discussed about the role of vitamin c in

biosynthesis of hormones, egg quality and

maturation.

Biosynthesis of sex steroids

Vitamin c plays part in the

biosynthesis of gonadal steroid hormones,

is important for sexual maturation. Tolbert

et al indicated that the effect of vitamin c

in the regulation of steroid genesis in

gonads, which corresponded with high

level of vitamin c found in the endocrine

tissues. Levine and morita also concluded

that the high levels of vitamin c in the

85

ovary and adrenal gland is a reflection of

their endocrine functions, in which

vitamin c may act as a regulator or co-

factor in the biosynthesis of steroid in the

follicle and adrenal cells. Waagbo et al

also stated that in the rainbow trout, level

of circulating 17-B estrodiol increased

significantly when the fish was fed

adequate amount of vitamin c thus

indication a higher deposition rate of

vetellogenin. It is synthesized liver under

the control of estrogenic hormones. Thus

the supplementation of vitamin c involves

in the biosynthesis of sex hormones and

vetellogenin.

86

Ovary maturation

Ovary has the highest content

of vitamin c among the fish tissues and

their vitamin c content changes during the

gonad maturation period. It has been

reported for cod (Gadus morhua), and for

carp (Carassisus carassus). Ishibeshi et al

also reported that the Japanese parrot fish

under experimental conditions, vitamin c

promotes maturation of the ovary.

Sandnes and Breakkan indicated that in

the wild Atlantic cod ovarian vitamin c

content which increased during the early

ovarian growth stage and decreased at the

last stage before spawning. Several studies

in silver breams, rainbow trout, crucian

carp, wild cod, wild trout and artic charr

87

reported rise of vitamin c concentration in

oocytes and depletion of body reserves of

this vitamin during gonadal

recrudescence. Seymour reported that the

ovarian vitamin c level in maturing crucial

carp decreased as result of stimulation by

carp pituitary extracts. Akiyama et al.

Showed that al vitamin c in the ovaries of

Japanese sardine was provided by the

dietary vitamin c although it was

decreased when was injecting with

luteinizing hormone releasing hormone.

So they clearly indicated that during

spawning stage, the vitamin c levels

decreased. However during maturation

stage, it was increased.

88

Embryo development

The mature salmonids, deposition

of vitamin c in the gonads highly exceed

deposition that other organs, which may

indicate the importance of vitamin c

during egg development. This is further

supported by Sato et al who found that

during embryonic in rainbow trout, the

vitamin c concentration in the egg

decreased by 75% of the initial value over

a period of 35 days. The female tilapia fed

a scorbutic diet produced embryos with

lower hatchability and juveniles with

severe spinal deformities. Low

hatchability of rainbow trout eggs from

brooders fed diets low in vitamin c has

been reported. They also reported in on

89

increased number of females with high

egg survival rates at the higher dietary

ascorbic acid intake. Blom and Dabrowski

reported that the both fecundity and

embryo survival increases with an

increasing supply of vitamin c through

diet. The reduced mortalities of rainbow

trout embryos were earlier reported by

kamarov and kenyazewa at 700mg

vitamin c/kg diet. Soliman et al found that

a supplementation of 1250mg vitamin

c/kg feed significantly increased egg

hatchability in tilapia Oreo chromic

Mossambicus. Sandnes et al also indicated

that vitamin c in the brood stock diets is

essential for improvement of the egg

quality such as hatchability in rainbow

trout. Soliman et at, showed that some

90

portion of brood stock dietary vitamin c

transfer to newly hatched fry via and it

needs to normal growth and development

of egg larvae in tilapia.

Status in egg

The growth of oocytes, embryonic

development and early performance of fry

depends upon the supply of nutrients

during vitellogenesis therefore

sequestration and packaging of substances

from the maternal blood stream into the

yolk will probably increase the maternal

demand for vitamin c and require

entranced vitamin c levels in brood stock

diets. Soliman et al found no vitamin c in

eggs and juveniles obtained from tilapia

female fed a diet devoid of vitamin c for

91

21 weeks. A diet devoid of vitamin c in

fish eggs has depends on the vitamin c in

diet.

In conclusion, vitamin c is

necessary for successful breeding in fishes

and it mainly involved in ovary

maturation, spawning embryo

development hatching, larval survival etc.

so the evaluation of vitamin c requirement

for brood stock is an important step for

achieving better seed production of all

cultivable fishes.

92

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About the Author

Dr.M.Muruganandam is a

Scientist in Einsteein Bio-Engineering

Research Foundation. He is an Editor of

African journal of Biotechnology and

International journal of Medicine and

Biomedical Research. He is also

Reviewer and Editorial board member in

Various National and International

journals.

ISBN-978-9982-22-301-0