ASPECTS OF ASCORBIC ACID

489 Pages·2013·23.27 MB·English

7tD
ASPECTS OF ASCORBIC ACID (VIT~I!N C) NUTRITION IN
OREOCHROMIS NILOTICUS AND O. MOSSAMBICUS
by
ADEL KHAMIS SOLIMAN, B.Sc., M.Sc.
Thesis submitted to the University of Stirling for the degree of
Doctor of Philosophy
Institute of Aquaculture
University of Stirling
Stirling
Scotland, U.K.
October, 1985
AFFECTIONATELY DEDICATED TO
MY
PARENTS, SISTERS & BROTHERS
/
(i)
ACKNOWLEDGEMENTS
The author is grateful to the Ministry of Higher Education of the
Arab Republic of Egypt for sponsoring his research programme and
appreciates the support of the staff of the Egyptian Education
Bureau in London. The encouragement of staff of the Faculty of
Agriculture, University of Alexandria (Egypt), particularly Professor
Khaled El-Shazly, Hussein M. El-Ibiary and Mohammed Kosba, is appre-
ciated.
I am deeply grateful to my principal supervisor, Dr. Kim Jauncey, for
his excellent supervision, the provision of laboratory facilities,
valuable suggestions throughout the course of study, revision of the
treatise and continuous encouragement.
I would also like to acknowledge the support and assistance of
Professor Ronald J. Roberts, Director of the Institute of Aquaculture,
especially with respect to histopathological examination of a multi-
tude of samples and for his valuable suggestions and continuous
encouragement.
In addition, I would like to express my appreciation for the invalu-
able assitance of the academic, technical and secretarial staff of
the Institute, especially Mrs Elizabeth Stenhouse (Senior technician)
for her patience and understanding in obtaining laboratory supplies;
Ian MacRae (Departmental superintendent) for laboratory safety
(ii)
regulation; Ian MacGowan (Senior technician); William Hamilton
and Keith Ranson for assistance with aquarium facilities; Paul
Thomson and Mary Alexander for valuable suggestions in cutting and
staining techniques; Laura Cumming, Sheila MacEwan, Moira Stewart
and Beatrice Dale for secretarial assistance.
Special thanks must go to Mr Ronald Stewart of the University's
Audio Visual Aids department for his help in taking and printing
of the histological photographs used in this thesis, and the Library
staff, particularly Mrs Nora Glasgow of the Inter Library Loan
Division.
I am deeply indebted to Mrs Joanna Mackay for her patience and
precision in typing the manuscript.
Heartfelt gratitude goes to my parents, sisters and brothers for
their patience and encouragement.
Lastly I would like to ask my lord to bless the
efforts and the fruits of this work.
(iii)
ABSTRACT
.
Various aspects of the ascorbic acid (vitamin C) nutrition of
Oreochromis niloticus and O. mossambicus are considered in this
treatise.
The activity of L-gulono-Y-lactone oxidase was assessed in liver and
kidney of 14 teleosts of 3 genera, histochemically (qualitatively)
and biochemically (quantitatively). Activity of this enzyme was only
detected in liver and kidney of common carp, Cyprinus carpio, and
kidney of O. spilurus and O. aureus. No activity was detected in the
species considered herein.
The quantitative dietary ascorbic acid requirements of juvenile O.
niloticus and O. mossambicus were determined by feeding diets con-
taining graded levels of the vitamin (O~400mg/lOOg) and were based
on growth response, food utilization, gross body composition data,
tissue and biochemical changes and ability to prevent signs of
ascorbic acid deficiency. The recommended level of supplementation
of dietary ascorbic acid is l2Smg/lOOg and the net requirement
42mg/lOOg diet.
£.
Long-term ascorbic acid deprivation in niloticus and O. mossambicus
resulted in poor performance in terms of growth, food utilization and
survival. Other parameters evaluated included hepatosomatic index,
liver and muscle glycogen content, blood parameters, tissue ascorbate
concentrations, collagen contents, hydroxyproline and proline contents,
and serum transaminase and cholesterol levels. Signs of ascorbic acid
(iv)
deficiency were severe and included haemorrhage~ opercular deformity,
tail erosion, exophthalmia, cataract and spinal deformity (lordosis
and scoliosis). Histologically scorbutic fish showed evidence of
generalized bone changes associated with excessive production of
chondrocytes and failur~ of ossification of growing bone areas. Eye
lesions were associated with scleral collapse and also observed was
hyperplasia of gill secondary lamellae epithelial cells and pronoun-
ced steatitis.
Tissue ascorbate concentrations were correlated with dietary ascorbic
acid levels and both species exhibited highest concentrations in the
ovary, brain and testis, followed by heart, liver, gut, gills, eyes
and the lowest levels in muscle and gall-bladde~. The physiological
role for ascorbic acid in each tissue is discussed,
Eight week growth studies were conducted to evaluate the utilization
of L-ascorbic acid (AA), the sodium salt of L-ascorbic acid (NaAA),
glyceride coated L-ascorbic acid (GCAA), the barium salt of L-ascorbic
acid 2-sulphate (AA2S) and ascorbyl palmitate CAP) in diets for O.
niloticus and O. mossambicus, All five forms were added to the basal
diet, containing no ascorbic acid CAAF), on an equimolar basis to
supply 12Smg ascorbic acid/IOOg diet. All forms performed well in
terms of growth, food utilization, and prevented signs of deficiency,
Retention of ascorbic acid in diets after processing was increased by
increasing dieta~y ascorbic acid level. AA2S and GCAA were more
stable than AA and NaAA during processing and storage. The stability
of AA, NaAA, GCAA and AA2S under different storage conditions was in
Cv)
descending order as follows: Freezer (_20°C), Fridge (S-BoC), room
temperature in black bags (22-24°C) and room temperature in clear
bags (22-24°C). Leaching of dietary ascorbic acid increased with
increasing immersion time and water temperature. Stability and
price of each form evaluated suggested that GCAA is to be prefer-
red for use in fish feeds.
The antioxidant effects of ascorbic acid were investigated. The
results showed that ascorbic acid was not as effective as butylated
hydroxy toluene (BHT) in preventing in vitro oxidation.
An experiment was conducted to compare the performance of a com-
mercial trout diet with the same diet supplemented to a level of
l2Smg ascorbic acid/lOOg diet (Diet 2) when fed to O. niloticus.
Fish fed the supplemented diet performed significantly better in
terms of growth and food utilization concomittant with significantly
increased tissue ascorbate concentrations.
Sex differences in relation to dietary ascorbic acid nutrition were
investigated. Females of both species exhibited significantly higher
gonado~ and hepatosomatic indices than males. Females in both species
eXhibited higher total ascorbate concentrations in gonad, gills,
spleen, brain and blood than males whereas the reverse was true for
the eyes. Dehydroascorbic acid (DHAA) levels were very low in
tissues of both species.
Ascorbic acid depleted O. niloticus fingerlings were f,edon each of
three diets providing nil, adequate Cl2Smg ascorbic acid/lOOg diet),
(vi)
and luxus (400mg/lOOg diet) of the vitamin after small surgical
incisions had been made in dorsolateral musclature. Fish from each
group were sampled regularly over 16 days and histological evalua-
tion of the lesion area carried out as well as measurement of the
tissue ascorbate levels. Epithelial elements of the healing process
developed irrespective of the vitamin level but although fibroblast
activity was marked in all three groups, collagenisation was very
much slower in the deficient group, and in these the lesion was not
mature, even at the termination of the experiment.
The role of ascorbic acid in reproduction of tilapias was investi-
gated in three experiments. The first showed that ascorbic acid
supplementation of broodstock feed improved both hatchability and
fry condition. In the second experiment fry produced from fish fed
an unsupplemented diet and subsequently fed the same diet performed
poorly in respect of growth and food utilization. Fry produced from
broodstock fed the supplemented diet and subsequently fed the un-
Supplemented diet performed better than the previous group. This
indicates transfer of ascorbic acid from the ovary to the eggs
thence to the fry providing some protection against ascorbic acid
deficiency during the early stages of life.
(vii)
CONTENTS
ACKNOWLEDGEMENTS (i)
ABSTRACT (iii)
LIST OF TABLES (xv)
LIST OF FIGURES (xx)
LIST OF PLATES (xxii)
CHAPTER 1 : INTRODUCTION
SECTION 1.1 PREFACE 1
SECTION 1.2 ASCORBIC ACID (VITAMIN C)
1.2.1 History of Scurvy 5
1.2.2 Isolation of L-ascorbic Acid 7
1.2.3 Physical and Chemical Properties of 9
L-ascorbic Acid
1.2.4 The Biosynthesis of L-ascorbic Acid in 11
Animals
1.2.5 Metabolism of L-ascorbic Acid 20
1.2.5.1 Absorption 20
1.2.5.2 Transport 20
1.2.5.3 Distribution 22
1.2.5.4 Half-life of L-ascorbic acid 30
1.2.5.5 Catabolism of L-ascorbic acid 31
1.2.5.6 Excretion of chemically intact ascorbic, 32
acid
1.2.6 Ascorbic Acid Forms 34
1.2.7 Physiological Roles of Ascorbic Acid 39
1.2.7.1 The role of ascorbic acid in prevention 39
of anaemia
1.2.7.2 The role of ascorbic acid in biosynthesis 41
of cOllagen and wound healing
1.2.7.3 Role of L-ascorbic acid in protein 46
metabolism
1.2.7.4 Interrelationships of ascorbic acid with 50
other nutrients
(viii)
Page
1.2.7.5 The indirect biological role of 58
ascorbic acid on cyclic nucleotide
mediated hormonal activities
1.2.7.6 L-ascorbic acid and immune response 60
1.2.7.7 Role of ascorbic acid in cancer 63
1.2.8 The Experimental Methodology Employed in 65
Ascorbic Acid Studies
1.2.9 Aim of Research 71
CHAPTER 2 GENERAL MATERIALS AND METHODS
SECTION 2.1 : THE EXPERIMENTAL SYSTEMS 74
2.1.1 Recirculation system 1 74
2.1.2 Recirculation system 2 . 74
2.1.3 Recirculation system 3 77
2.1.4 Hatchery system 77
SECTION 2.2 FISHES 82
SECTION 2.3 DIETS 82
2.3.1 Diet Formulation 82
2.3.2 Diet Preparation 82
SECTION 2.4 : CHEMICAL AND BIOCHEMICAL ANALYSIS 87
2.4.1 Oil Extraction and Oil Oxidation Parameters 87
2.4.1.1 Oil extraction 87
2.4.1.2 Oil oxidation parameters 87
2.4.2 Methods of Proximate Analysis 88
2.4.3 Haematocrit and Haemoglobin Determination 89
2.4.4 Total Ascorbic Acid, Dehydro-L-ascorbic Acid 90
and L-Ascorbic Acid Determination
2.4.4.1 Total ascorbate determination 90
2.4.4.2 Dehydro L-ascorbic acid determination 91
2.4.4.3 L-ascorbic acid determination 91
2.4.5 Ascorbic Acid 2-su1phate Determination 91
2.4.6 Hepatosomatic Index and Muscle and Liver 91
Glycogen Determination