Optimun phospholipids and antioxidant levels to develop novel microdiets for gilthead seabream ...

277 Pages·2013·31.44 MB·English

PhD Thesis"
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Optimum phospholipids and
antioxidant levels to develop !
novel microdiets for gilthead !
seabream larvae "
Reda Saleh Mohamed Ibrahim"
Las Palmas of Gran Canaria, "
Spain 2013"
University of Las Palmas of Gran Canaria
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Anexo II

UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA

Departamento: Instituto Universitario de Sanidad Animal y Seguridad
Alimentaria
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Programa de Doctorado: Acuicultura: Producción Controlada de Animales
Acuáticos
Título de la Tesis
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Optimum phospholipids and antioxidant
levels to develop novel microdiets for
gilthead seabream larvae!
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Tesis Doctoral presentada por D. Reda Saleh Mohamed Ibrahim
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Dirigida por La Doctora María Soledad Izquierdo López y
El Doctor Francisco Javier Moyano López

La Directora El Director El Doctorando

!"#$%#!&’()*#*!!!!!!!!!!!!!!!!!!! Francisco Javier Reda Saleh
+,-./)$*’!012),!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Moyano López Mohamed Ibrahim

Las Palmas de Gran Canaria, a 01 de Febrero de 2013
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Optimum phospholipids and antioxidant
levels to develop novel microdiets for
gilthead seabream larvae

Reda Saleh Mohamed Ibrahim

Doctorado en Acuicultura: Producción Controlada de Animales
Acuáticos
Grupo de Investigación en Acuicultura (GIA)
Instituto Universitario de Sanidad Animal y Seguridad
Alimentaria (IUSA)

Thesis for the degree of Doctor of Phylosophy
University of Las Palmas de Gran Canaria
2013

Directors:
Prof. María Soledad Izquierdo and Prof. Francisco Javier Moyano
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List of Contents
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Page Nº
Summary ………………………………….……………………………………... I

List of Abbreviations. …………………………………………………………... III

List of Tables ……………………………………………………………………. V

List of Figures ………………………………….……………………………….. VIII

Acknowledgements …………………………..…………………………………. XVIII

Chapter 1: Introduction ………………………………………….…………….. 1
1.1 Aquaculture ………………………………….…………………………... 1
1.2 Marine fish larvae and microdiets utilization .………………………… 3
1.3 Importance of phospholipids in marine fish larvae nutrition ………… 6
1.3.1 Phospholipids as source of essential fatty acids………………… 7
1.3.2 Phospholipid requirements ……………………………………… 8
1.3.3 Phospholipid digestion, absorption and transport …………….. 10
1.3.3.1 Phospholipid digestion…………………………………. 10
1.3.3.2 Phospholipid absorption and transport ………………. 13
1.3.4 Phospholipid classes………………………………….…………… 18
1.3.5 Effect of phospholipids on skeletal development ………………. 20
1.4 Oxidative stress: Antioxidant defense mechanism ………………………. 24
1.4.1 Enzymatic defenses ………………………………….…………… 24
1.4.2 Non enzymatic defenses ……………………………….…………. 26
1.4.2.1 Vitamin E (!-tocopherol) ……………………………… 27
1.4.2.2 Selenium ………………………………….…………….. 29
1.5 Objectives ……………….……………..……………….…………………… 30

Chapter 2: Materials and Methods ……………………………………………. 32
2.1 Larvae ……………………………………………………………………... 32
2.1.1 Gilthead sea bream larvae .…………………………………. …….. 32
2.2 Experimental conditions ………………………………………….………. 32
2.3 Diets and feeding ……………………………………………….………... 32
2.3.1 Rotifers ……………………………………………………….…….. 32
2.3.2 Microdiets ………………………………………….………………. 33
2.3.2.1 Microdiets formulation.………………………………….. 33
2.3.2.2 Microdiets preparation…………………………………... 33
2.3.3 Feeding………………………………….…………………………... 35
2.4 Sampling.…………………………………. ……………………………... 35
2.4.1 Biological parameters………………………………….…………... 35
2.4.2 Proximate analysis………………………………….……………… 35
2.4.3 Digestive enzymes………………………………….……………… 35
2.4.4 Histological studies………………………………….……………… 35
2.4.5 Molecular biology………………………………….………………. 36
2.4.6 Activity test and survival………………………………….……….. 36
2.4.7 Growth ……………………………………………………………... 36
2.5 Biochemical analysis…………………………………………………….. 36
2.5.1 Proximate analysis ………………………………………………… 36
2.5.1.1 Moisture ………………………………….………………. 36
2.5.1.2 Ash ………………………………….…………………….. 37
2.5.1.3 Proteins ………………………………….……………….. 37
2.5.1.4 Total lipids ………………………………….……………. 37
2.5.1.4.1 Lipid Classes …………………………………… 38
2.5.1.4.2 Fatty acid methyl esters preparation and
quantification ……………………………………………….. 38
2.5.2 Digestive enzymes activity…………………………………………. 39
2.6 Measurement of thiobarbituric reactive substances (TBARS) ………. 40
2.7 Selenium determination ………………………………….……………... 40
2.8 Whole mount staining for skeleton studies…………………………….. 41
2.9 Molecular biology ………………………………….…………………….. 42
2.10 Statistical analysis ………………………………….…………………... 44

Chapter 3: Optimum krill phospholipids content in microdiets for gilthead
seabream (Sparus aurata) larvae………………………………….……………. 45

Chapter 4: Optimum soybean lecithin content in microdiets for gilthead
seabream (Sparus aurata) larvae………………………………….……………. 68

Chapter 5: Effect of krill phospholipids vs soybean lecithin in microdiets for
gilthead seabream (Sparus aurata) larvae on molecular markers of
antioxidative metabolism and bone development……………………………... 91

Chapter 6: Biomarkers of bone development and oxidative stress in gilthead
seabream larvae fed microdiets with several levels of polar lipids and !–
tocopherol ………………………………….…………………………………..... 123

Chapter 7: Selenium levels in early weaning diets for gilthead sea bream
larvae. ………………………………………………….………………………… 150

Chapter 8: Conclusions ………………………………………………………… 174

Chapter 9: Resumen en español………………………………….…………….. 175

References …………………………………………….…………………………. 228

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Summary
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Phospholipids (PL) are particularly important in fish larvae production for their
essential function as necessary components for cellular bio-membranes and organelles
formation, as well as for being an endogenous energy source during early
development. Besides, during early development, PL seems to induce digestive
system maturation, may promote digestive enzymes activities, and consequently play
an important role on larval digestive physiology and the metabolic pathways of the
assimilated nutrients. However, despite the many studies available denoting the
importance of dietary PL, few of them have intended to determine quantitative PL
requirements testing diets with at least five different levels of this nutrient. Thus, the
aim of the present thesis was to determine the optimum requirements of krill PL
(KPL) and soybean lecithin (SBL) for gilthead seabream (Sparus aurata) larvae, and
its influence on larval production performance and digestive enzymes activity
(Chapter 3 & 4). Another aim was to compare the effectiveness of dietary KPL and
soybean lecithin on the seabream larval rearing performance, oxidative status,
vertebral mineralization, skeletal anomalies and bone formation related genes
expression (Chapter 5). However, dietary PL have high levels of polyunsaturated
fatty acids which are molecules with a great susceptibility to peroxidation resulting in
production of harmful peroxides that affect their biological and physiological
functions, so it was important that investigate the combined effect of graded levels of
antioxidant nutrients such as !-tocopherol with dietary KPL and SBL, and the effect
of graded levels of Se derived yeast, on the seabream larval rearing performance,
oxidative status, vertebral mineralization, skeletal anomalies and bone formation
related genes expression (Chapter 6 & 7). In the light of these experiments, the results
have shown that dietary KPL are an excellent source of lipids and the optimum
inclusion levels of this ingredient in microdiets to completely substitute live preys at
larval age of 16 dph were found to be 12% KPL, providing about 10% total PL, and
8% SBL, providing about 8.8% total PL. These levels significantly improved
digestive enzymes activities, utilization and deposition of dietary essential fatty acids
and larval growth, as a consequence of a better digestion, absorption, transport and
deposition of dietary nutrients. However, despite increased on dietary SBL up to 9%
total PL improved larval survival, stress resistance, growth and skeletal development,
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dietary KPL was more effective in promoting all these parameters where their higher
content in phosphatidylcholine and n-3 HUFA, particularly, DHA, not only promotes
digestion, transport and deposition of dietary lipids, but also contributes to reduce
skeleton anomalies by up-regulating of bone formation related genes, inducing early
mineralization and resistance of vertebral bodies to reduce anomalies such as lordosis
and kyphosis. Also, inclusion of SBL markedly increased the proxidation risk as
denoted by the high larval malondialdehyde (MDA) content, as well as a high
expression of catalase (CAT), superoxide dismutase, (SOD) and glutathione
peroxidase (GPX) genes.
The inclusion of 3000 mg !-tocopherol kg-1 diet promoted the expression of genes
related to bone formation and mineralization and improved larval survival and
growth, what could be related to the better utilization of dietary lipids and to the
reduction of toxic free radicals as indicated by the low larval MDA content and the
low antioxidant enzyme genes expression (AOE). These results denoted the high
efficiency of -tocopherol as an antioxidant factor, and its positive effect on genes
expression of bone formation and mineralization.
Finally, the inclusion of Se derived yeast at 11.65 µg Se/mg diet improved larval
survival and stress resistance what could be related in one hand to the reduction of
toxic free radicals as indicated by the low levels of MDA and AOE content, which
demonstrate an adaptive response in attempting to neutralize the generated reactive
oxygen species (ROS) suggesting better oxidative status, and in another hand to the
improved utilization of dietary lipids. Moreover, the Bone Morphogenetic Protein-4,
alkaline phosphatase, osteocalcin, osteonectin, osteopontin, and matrix gla protein
genes expression in larval tissues were positively correlated to the dietary selenium
increased or larval Se tissue content increased, in relation to an adequate skeletal
development.

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