Cholesterol is found in all animal tissues and is an important component of biological cell
membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal
and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients
by plant proteins and vegetable oils tends to reduce dietary cholesterol levels

Aragão, C.

Cancela, Leonor

Colen, Rita

Dias, J.

Engrola, S.

Gavaia, Paulo J.

Laizé, Vincent

Rodrigues, V.

Viegas, Michael

Universidade do Algarve

FEEDING OUR FOOD
XV International Symposium on Fish Nutrition and Feeding
0ROGH1RUZD\²-XQH
ORGANIZERS
Book of Abstracts
www.isfnf.org
O69 
 
Effect of dietary vitamin K supplementation on larval proteome and bone marker gene 
expression in Senegalese sole  
 
Nadège Richard*, Ignacio Fernández, Luis E.C. Conceição, Paulo J. Gavaia 
 
CCMAR, Centro de Ciências do Mar do Algarve, Universidade do Algave, Campus de 
Gambelas, 8005-139 Faro, Portugal. 
Email: nrichard@ualg.pt 
 
Several studies have identified rearing environment, nutrition and genetics as causative 
factors of skeletal deformities (Boglione et al., 2003; Cahu et al., 2003). Within nutrition, 
vitamin K (VK) is a liposoluble vitamin classically involved in blood coagulation, and until 
recently largely disregarded in aquaculture. This vitamin is known to play an important role in 
bone metabolism, acting as a coenzyme for VK-dependant gamma carboxylation of Gla 
proteins as Osteocalcin and Matrix Gla Protein, and regulating Msx gene expression, through 
the recently identified VK activation of Pregnane X receptor (PXR). Since the mechanisms 
underlying the association of VK with the changes of skeletal microanatomy are still not fully 
understood (Hamre et al., 2010), the aim of the present study was to investigate the effects of 
dietary VK supplementation on skeleton deformities, larval proteome and the expression of 
selected genes involved in bone metabolism in Senegalese sole larvae.  
 
Quadruplicate groups of Senegalese sole larvae were fed from first-feeding onwards with 
rotifers and Artemia enriched with different levels of VK1 (0 to 250 mg of VK1/kg of Selco). 
Larvae were sampled at 40 DAH. Mineral content, larval growth and incidence in skeletal 
deformities were measured in order to evaluate the effects of increasing dietary VK levels in 
larval performance. Gene expression of VK related pathway was analysed by quantitative 
PCR. In addition, a comparative proteome analysis was performed by two-dimensional 
electrophoresis on entire larvae after removing the head and the gut. Larval skeleton quality 
analyses revealed that dietary VK supplementation in sole larvae leads to an improvement in 
larval quality, decreasing the incidence of skeletal deformities, as well as increasing mineral 
content. Skeletal quality has been correlated with gene expression levels of selected genes, 
which showed an altered gene expression under dietary VK supplementation. Regarding 
proteome analysis, among protein spots detected as differentially expressed between the two 
experimental groups, some were identified as being involved in clotting process, energy 
metabolism, protein folding, cellular contractile system and cytoskeleton proteins. 
Interestingly, type VI collagen, which is known to have a role in skeletal metabolism (Hall 
2005) was also identified. The potential benefits of increasing dietary VK levels in Senegalese 
sole diets during larval development will be discussed. 
 
References 
Boglione, C., Costa, C., Di Dato, P., Ferzini, G., Scardi, M., Cataudella, S. (2003) Aquaculture 227:373-394 
Cahu, C., Zambonino Infante, J., Takeuchi, T. (2003).Aquaculture 227:245-258 
Hall, B.K. (2005) Bones and cartilage; Elsewier academic press, California USA. 760 pp. 
Hamre, K., Krossøy, C., Lock, E.-J., Moren, M. (2010) Aquaculture Research 41:745-750 
 
Acknowledgements  
NR and IF acknowledge financial support from Fundação para a Ciência e Tecnologia, Portugal, through grants 
SFRH/BDP/65578/2009 and SFRH/BDP/82049/2011 respectively. This work was partly funded by project 
PDTC-MAR-105152-2008 (SPECIAL_K) from the Portuguese Foundation of Science and Technology. 
P24 
 
Effect of variable levels of dietary cholesterol and plant sterols on the growth 
performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles  
 
Jorge Dias, Rita Colen, Vera Rodrigues, Cláudia Aragão, Sofia Engrola, Michael Viegas, 
Vincent Laizé, Paulo Gavaia, Leonor Cancela 
 
CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. 
E-mail: jorgedias@ualg.pt 
 
Cholesterol is found in all animal tissues and is an important component of biological cell 
membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal 
and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients 
by plant proteins and vegetable oils tends to reduce dietary cholesterol levels. Additionally, 
the utilization of vegetable oils will increase dietary supply of phytosterols, which are known 
to compete with cholesterol for binding sites thereby lowering the intestinal cholesterol 
absorption and serum LDL-cholesterol levels. In rodent models, elevated levels of plasma 
LDL cholesterol is regarded as an osteopenic factor. On the other hand, plant sterols (beta-
sitosterol, campesterol, stigmasterol, and brassicasterol) either through its direct action on 
cholesterol absorption or in association to its potent anti-inflammatory effect have been 
shown to effectively reduce bone losses. Knowledge on the effect of such dietary variables in 
fish bone is limited.     
 
A trial was undertaken to evaluate the effect of variable dietary cholesterol:phytosterols (C:P) 
ratios on the growth performance and bone metabolism of gilthead seabream juveniles. Five 
isoproteic (crude protein, 55% DM) and isolipidic (18% DM) diets were formulated with 
purified ingredients to fulfill the known nutritional requirements of juvenile seabream. By 
using concentrated sources of cholesterol and phytosterols, the dietary C:P ratios tested were 
2.2:0 (diet CHOL 2.2), 0.7:0 (diet CHOL 0.7), 0.7:2 (diet PHYTO 2), 0.7:4 (diet PHYTO 4), 
0:4 (diet CHOL 0). Each diet was tested in duplicate groups of 30 seabream (IBW: 6.7 0.8 g) 
stocked in 100 L circular tanks, supplied with gravel-filtered aerated seawater (temperature: 
24±1°C). Fish were fed to apparent satiety, by hand, three times a day, during 56 days. 
 
At the end of the trial, fish had a 4-fold increase of their initial weight. Dietary changes on the 
cholesterol and phytosterols levels had little effect on weight gain and specific growth rate. 
However, for a constant dietary cholesterol level of 0.7%, the increase of phytosterols 
significantly reduced feed efficiency and protein efficiency ratio. Analysis of hepatic lipid 
metabolic enzymes and plasma LDL-cholesterol levels is currently underway. Additionally, 
lipid composition, phosphorus content, activities of alkaline phosphatase (ALP) and tartrate-
resistant acid phosphatase (TRAP) will be assess in vertebral bone homogenates. 
 
Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of 
Portugal (Project PTDC/MAR/70855/2006). 
 
P131 
 
Effect of variable levels of dietary cholesterol and plant sterols on the growth 
performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles  
 
Jorge Dias, Rita Colen, Vera Rodrigues, Cláudia Aragão, Sofia Engrola, Michael Viegas, 
Vincent Laizé, Paulo Gavaia, Leonor Cancela 
 
CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. 
E-mail: jorgedias@ualg.pt 
 
Cholesterol is found in all animal tissues and is an important component of biological cell 
membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal 
and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients 
by plant proteins and vegetable oils tends to reduce dietary cholesterol levels. Additionally, 
the utilization of vegetable oils will increase dietary supply of phytosterols, which are known 
to compete with cholesterol for binding sites thereby lowering the intestinal cholesterol 
absorption and serum LDL-cholesterol levels. In rodent models, elevated levels of plasma 
LDL cholesterol is regarded as an osteopenic factor. On the other hand, plant sterols (beta-
sitosterol, campesterol, stigmasterol, and brassicasterol) either through its direct action on 
cholesterol absorption or in association to its potent anti-inflammatory effect have been 
shown to effectively reduce bone losses. Knowledge on the effect of such dietary variables in 
fish bone is limited.     
 
A trial was undertaken to evaluate the effect of variable dietary cholesterol:phytosterols (C:P) 
ratios on the growth performance and bone metabolism of gilthead seabream juveniles. Five 
isoproteic (crude protein, 55% DM) and isolipidic (18% DM) diets were formulated with 
purified ingredients to fulfill the known nutritional requirements of juvenile seabream. By 
using concentrated sources of cholesterol and phytosterols, the dietary C:P ratios tested were 
2.2:0 (diet CHOL 2.2), 0.7:0 (diet CHOL 0.7), 0.7:2 (diet PHYTO 2), 0.7:4 (diet PHYTO 4), 
0:4 (diet CHOL 0). Each diet was tested in duplicate groups of 30 seabream (IBW: 6.7 0.8 g) 
stocked in 100 L circular tanks, supplied with gravel-filtered aerated seawater (temperature: 
24±1°C). Fish were fed to apparent satiety, by hand, three times a day, during 56 days. 
 
At the end of the trial, fish had a 4-fold increase of their initial weight. Dietary changes on the 
cholesterol and phytosterols levels had little effect on weight gain and specific growth rate. 
However, for a constant dietary cholesterol level of 0.7%, the increase of phytosterols 
significantly reduced feed efficiency and protein efficiency ratio. Analysis of hepatic lipid 
metabolic enzymes and plasma LDL-cholesterol levels is currently underway. Additionally, 
lipid composition, phosphorus content, activities of alkaline phosphatase (ALP) and tartrate-
resistant acid phosphatase (TRAP) will be assess in vertebral bone homogenates. 
 
Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of 
Portugal (Project PTDC/MAR/70855/2006). 
 
P220 
 
Tolerance of zebrafish embryos to supra-physiologic yolk levels of glucose  
 
Filipa Rocha1*, Jorge Dias1, Sofia Engrola1, Paulo Gavaia1, Maria Teresa Dinis1, Stéphane 
Panserat2 
 
1 CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. 
2 INRA UR 1067 NuMeA (Nutrition, Métabolisme, Aquaculture), Pôle d'Hydrobiologie, St-
Pée-sur-Nivelle, France. 
E-mail: fsrocha@ualg.pt 
 
Once spawned and fertilized the oviparous fish eggs operate as closed systems; only 
respiratory gases, heat and negligible amounts of solutes and water are exchanged freely, as a 
result of an extremely low permeability of the egg surface membranes. Currently, the 
opportunities to exert a nutritional stimulus during a stage of high metabolic plasticity, such 
as fish embryogenesis, are restricted to maternal transfer and the onset of exogenous feeding.  
 
Batches of fertilized zebrafish eggs were used to assess the utilization pattern of energy-
yielding nutrients during embryogenesis and first feeding phases. Changes on total N, free 
amino acids, free fatty acids and glucose are observed throughout the various developmental 
stages. This information on nutrient usage was cross-referenced with available gene 
expression data (ZFIN database) during embryogenesis. This info clear shows that major 
metabolic pathways of nutritional relevance seem to be already active at the blastula stage. 
Analysis of gene expression (RT-PCR) data at 6 dpf, a critical period of transition between 
endogenous and exogenous feeding will be presented.   
 
Moreover, knowledge about the effects of a nutritional supplementation strategy during fish 
embryogenesis is extremely scarce. Therefore, a set of preliminary experiments was 
undertaken to assess the technical and biological feasibility of fortifying zebrafish eggs with 
glucose by a microinjection approach. The yolk of zebrafish viable eggs, at 30% epiboly stage 
(5 hpf), was enriched with various glucose solutions (0.22, 1.41 and 2.17M) by means of 
microinjection. Additional treatments comprised non injected eggs and saline solution 
injected eggs. Following microinjection, eggs and yolk-feeding larvae were incubated and 
reared under identical conditions and following standardized protocols. 
 
0
200
400
600
800
1000
1200
1400
1600
1800
Non
Inj.
Saline GLU
0.22M
GLU
1.41M
GLU
2.17M
Experimental treatments
G
lu
co
se
 c
o
n
ce
n
tr
at
io
n
 (
u
M
)
 
In comparison to non-injected eggs, we could achieve a 4, 
15 and 43-fold increase of the glucose concentration of 
eggs, showing that microinjection is an effective way to 
fortify yolk reserves in zebrafish eggs. 
 
At 6 dpf, survival was high (>92%) for eggs injected with 
saline and 0.22M glucose, and slightly lower (>76%) for 
those injected with the highest glucose concentration .  
 
These developments are the preliminary steps to a better insight on the nutritional regulation 
of key biological and metabolic processes occurring in early developmental phases of fish. 
 
Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of 
Portugal (Project PTDC/CVT/102481/2008). Filipa Rocha is recipient of FCT Grant 
SFRH/BD/74921/2010. 


Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles

Sapientia

FEEDING OUR FOODXV International Symposium on Fish Nutrition and Feeding0ROGH1RUZD\²-XQHORGANIZERSBook of Abstractswww.isfnf.orgO69  Effect of dietary vitamin K supplementation on larval proteome and bone marker gene expression in Senegalese sole   Nadège Richard*, Ignacio Fernández, Luis E.C. Conceição, Paulo J. Gavaia  CCMAR, Centro de Ciências do Mar do Algarve, Universidade do Algave, Campus de Gambelas, 8005-139 Faro, Portugal. Email: nrichard@ualg.pt  Several studies have identified rearing environment, nutrition and genetics as causative factors of skeletal deformities (Boglione et al., 2003; Cahu et al., 2003). Within nutrition, vitamin K (VK) is a liposoluble vitamin classically involved in blood coagulation, and until recently largely disregarded in aquaculture. This vitamin is known to play an important role in bone metabolism, acting as a coenzyme for VK-dependant gamma carboxylation of Gla proteins as Osteocalcin and Matrix Gla Protein, and regulating Msx gene expression, through the recently identified VK activation of Pregnane X receptor (PXR). Since the mechanisms underlying the association of VK with the changes of skeletal microanatomy are still not fully understood (Hamre et al., 2010), the aim of the present study was to investigate the effects of dietary VK supplementation on skeleton deformities, larval proteome and the expression of selected genes involved in bone metabolism in Senegalese sole larvae.   Quadruplicate groups of Senegalese sole larvae were fed from first-feeding onwards with rotifers and Artemia enriched with different levels of VK1 (0 to 250 mg of VK1/kg of Selco). Larvae were sampled at 40 DAH. Mineral content, larval growth and incidence in skeletal deformities were measured in order to evaluate the effects of increasing dietary VK levels in larval performance. Gene expression of VK related pathway was analysed by quantitative PCR. In addition, a comparative proteome analysis was performed by two-dimensional electrophoresis on entire larvae after removing the head and the gut. Larval skeleton quality analyses revealed that dietary VK supplementation in sole larvae leads to an improvement in larval quality, decreasing the incidence of skeletal deformities, as well as increasing mineral content. Skeletal quality has been correlated with gene expression levels of selected genes, which showed an altered gene expression under dietary VK supplementation. Regarding proteome analysis, among protein spots detected as differentially expressed between the two experimental groups, some were identified as being involved in clotting process, energy metabolism, protein folding, cellular contractile system and cytoskeleton proteins. Interestingly, type VI collagen, which is known to have a role in skeletal metabolism (Hall 2005) was also identified. The potential benefits of increasing dietary VK levels in Senegalese sole diets during larval development will be discussed.  References Boglione, C., Costa, C., Di Dato, P., Ferzini, G., Scardi, M., Cataudella, S. (2003) Aquaculture 227:373-394 Cahu, C., Zambonino Infante, J., Takeuchi, T. (2003).Aquaculture 227:245-258 Hall, B.K. (2005) Bones and cartilage; Elsewier academic press, California USA. 760 pp. Hamre, K., Krossøy, C., Lock, E.-J., Moren, M. (2010) Aquaculture Research 41:745-750  Acknowledgements  NR and IF acknowledge financial support from Fundação para a Ciência e Tecnologia, Portugal, through grants SFRH/BDP/65578/2009 and SFRH/BDP/82049/2011 respectively. This work was partly funded by project PDTC-MAR-105152-2008 (SPECIAL_K) from the Portuguese Foundation of Science and Technology. P24  Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles   Jorge Dias, Rita Colen, Vera Rodrigues, Cláudia Aragão, Sofia Engrola, Michael Viegas, Vincent Laizé, Paulo Gavaia, Leonor Cancela  CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. E-mail: jorgedias@ualg.pt  Cholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels. Additionally, the utilization of vegetable oils will increase dietary supply of phytosterols, which are known to compete with cholesterol for binding sites thereby lowering the intestinal cholesterol absorption and serum LDL-cholesterol levels. In rodent models, elevated levels of plasma LDL cholesterol is regarded as an osteopenic factor. On the other hand, plant sterols (beta-sitosterol, campesterol, stigmasterol, and brassicasterol) either through its direct action on cholesterol absorption or in association to its potent anti-inflammatory effect have been shown to effectively reduce bone losses. Knowledge on the effect of such dietary variables in fish bone is limited.      A trial was undertaken to evaluate the effect of variable dietary cholesterol:phytosterols (C:P) ratios on the growth performance and bone metabolism of gilthead seabream juveniles. Five isoproteic (crude protein, 55% DM) and isolipidic (18% DM) diets were formulated with purified ingredients to fulfill the known nutritional requirements of juvenile seabream. By using concentrated sources of cholesterol and phytosterols, the dietary C:P ratios tested were 2.2:0 (diet CHOL 2.2), 0.7:0 (diet CHOL 0.7), 0.7:2 (diet PHYTO 2), 0.7:4 (diet PHYTO 4), 0:4 (diet CHOL 0). Each diet was tested in duplicate groups of 30 seabream (IBW: 6.7 0.8 g) stocked in 100 L circular tanks, supplied with gravel-filtered aerated seawater (temperature: 24±1°C). Fish were fed to apparent satiety, by hand, three times a day, during 56 days.  At the end of the trial, fish had a 4-fold increase of their initial weight. Dietary changes on the cholesterol and phytosterols levels had little effect on weight gain and specific growth rate. However, for a constant dietary cholesterol level of 0.7%, the increase of phytosterols significantly reduced feed efficiency and protein efficiency ratio. Analysis of hepatic lipid metabolic enzymes and plasma LDL-cholesterol levels is currently underway. Additionally, lipid composition, phosphorus content, activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) will be assess in vertebral bone homogenates.  Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of Portugal (Project PTDC/MAR/70855/2006).  P131  Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles   Jorge Dias, Rita Colen, Vera Rodrigues, Cláudia Aragão, Sofia Engrola, Michael Viegas, Vincent Laizé, Paulo Gavaia, Leonor Cancela  CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. E-mail: jorgedias@ualg.pt  Cholesterol is found in all animal tissues and is an important component of biological cell membranes with functions such as precursor to bile acids, hormones and vitamins. Fish meal and fish oil are cholesterol-rich ingredients. Replacement of these marine-derived ingredients by plant proteins and vegetable oils tends to reduce dietary cholesterol levels. Additionally, the utilization of vegetable oils will increase dietary supply of phytosterols, which are known to compete with cholesterol for binding sites thereby lowering the intestinal cholesterol absorption and serum LDL-cholesterol levels. In rodent models, elevated levels of plasma LDL cholesterol is regarded as an osteopenic factor. On the other hand, plant sterols (beta-sitosterol, campesterol, stigmasterol, and brassicasterol) either through its direct action on cholesterol absorption or in association to its potent anti-inflammatory effect have been shown to effectively reduce bone losses. Knowledge on the effect of such dietary variables in fish bone is limited.      A trial was undertaken to evaluate the effect of variable dietary cholesterol:phytosterols (C:P) ratios on the growth performance and bone metabolism of gilthead seabream juveniles. Five isoproteic (crude protein, 55% DM) and isolipidic (18% DM) diets were formulated with purified ingredients to fulfill the known nutritional requirements of juvenile seabream. By using concentrated sources of cholesterol and phytosterols, the dietary C:P ratios tested were 2.2:0 (diet CHOL 2.2), 0.7:0 (diet CHOL 0.7), 0.7:2 (diet PHYTO 2), 0.7:4 (diet PHYTO 4), 0:4 (diet CHOL 0). Each diet was tested in duplicate groups of 30 seabream (IBW: 6.7 0.8 g) stocked in 100 L circular tanks, supplied with gravel-filtered aerated seawater (temperature: 24±1°C). Fish were fed to apparent satiety, by hand, three times a day, during 56 days.  At the end of the trial, fish had a 4-fold increase of their initial weight. Dietary changes on the cholesterol and phytosterols levels had little effect on weight gain and specific growth rate. However, for a constant dietary cholesterol level of 0.7%, the increase of phytosterols significantly reduced feed efficiency and protein efficiency ratio. Analysis of hepatic lipid metabolic enzymes and plasma LDL-cholesterol levels is currently underway. Additionally, lipid composition, phosphorus content, activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) will be assess in vertebral bone homogenates.  Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of Portugal (Project PTDC/MAR/70855/2006).  P220  Tolerance of zebrafish embryos to supra-physiologic yolk levels of glucose   Filipa Rocha1*, Jorge Dias1, Sofia Engrola1, Paulo Gavaia1, Maria Teresa Dinis1, Stéphane Panserat2  1 CIMAR/CCMAR, University of Algarve, Campus Gambelas, Faro, Portugal. 2 INRA UR 1067 NuMeA (Nutrition, Métabolisme, Aquaculture), Pôle d'Hydrobiologie, St-Pée-sur-Nivelle, France. E-mail: fsrocha@ualg.pt  Once spawned and fertilized the oviparous fish eggs operate as closed systems; only respiratory gases, heat and negligible amounts of solutes and water are exchanged freely, as a result of an extremely low permeability of the egg surface membranes. Currently, the opportunities to exert a nutritional stimulus during a stage of high metabolic plasticity, such as fish embryogenesis, are restricted to maternal transfer and the onset of exogenous feeding.   Batches of fertilized zebrafish eggs were used to assess the utilization pattern of energy-yielding nutrients during embryogenesis and first feeding phases. Changes on total N, free amino acids, free fatty acids and glucose are observed throughout the various developmental stages. This information on nutrient usage was cross-referenced with available gene expression data (ZFIN database) during embryogenesis. This info clear shows that major metabolic pathways of nutritional relevance seem to be already active at the blastula stage. Analysis of gene expression (RT-PCR) data at 6 dpf, a critical period of transition between endogenous and exogenous feeding will be presented.    Moreover, knowledge about the effects of a nutritional supplementation strategy during fish embryogenesis is extremely scarce. Therefore, a set of preliminary experiments was undertaken to assess the technical and biological feasibility of fortifying zebrafish eggs with glucose by a microinjection approach. The yolk of zebrafish viable eggs, at 30% epiboly stage (5 hpf), was enriched with various glucose solutions (0.22, 1.41 and 2.17M) by means of microinjection. Additional treatments comprised non injected eggs and saline solution injected eggs. Following microinjection, eggs and yolk-feeding larvae were incubated and reared under identical conditions and following standardized protocols.  020040060080010001200140016001800NonInj.Saline GLU0.22MGLU1.41MGLU2.17MExperimental treatmentsGlucose concentration (uM) In comparison to non-injected eggs, we could achieve a 4, 15 and 43-fold increase of the glucose concentration of eggs, showing that microinjection is an effective way to fortify yolk reserves in zebrafish eggs.  At 6 dpf, survival was high (>92%) for eggs injected with saline and 0.22M glucose, and slightly lower (>76%) for those injected with the highest glucose concentration .   These developments are the preliminary steps to a better insight on the nutritional regulation of key biological and metabolic processes occurring in early developmental phases of fish.  Acknowledgements: this research was supported by Fundação para a Ciência e Tecnologia of Portugal (Project PTDC/CVT/102481/2008). Filipa Rocha is recipient of FCT Grant SFRH/BD/74921/2010. 

https://sapientia.ualg.pt/bitstream/10400.1/4418/3/Effect%20of%20variable%20levels%20of%20dietary%20cholestrol%20and%20plant%20sterols%20on%20the%20growth%20performance%20and%20bone%20metabolism%20in%20gilthead%20seabrem%20juveniles.pdf

Effect of variable levels of dietary cholesterol and plant sterols on the growth performance and bone metabolism in gilthead seabream (Sparus aurata) juveniles

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