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Universidade Estadual do Cear UECE

Laboratrio de Carcinicultura LACAR

Morphological and histological studies on the embryonic


development of the freshwater prawn, Macrobrachium rosenbergii
(Crustacea, Decapoda)

HABASHY, M. M.; SHARSHAR, K. M. and HASSAN, M.M.S.


The Journal of Basic & Applied Zoology

Moiss Fernandes Martins


Fortaleza, 2015

Introduction

Introduction

Materials
and methods

Objective

Results and
discussion

Conclusion

Survival rate for eggs

Crustaceans:

Reproductive success
GIESEAND PEARSE, 1974; CHARNIAUX-COTTON et al., 1992

Characterization of stages

Meroblastic development

Morphological changes

PERKINS, 1972; HELLUY and BELTZ, 1991; NAZARI et al., 2000; MULLER et al., 2003
http://bit.ly/78HTgg4

Transparent chorion
Analysis of embryogenic development
Morphology and histological
External observation of structures
M. rosembergii eggs

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Introduction

Objective

Palaemonetes
argentines

Materials
and methods

Palaemon
pandaliformis

Results and
discussion

Macrobrachium
olfersi

Conclusion

Palaemonetes
potiuna

10

13

14

21

days

days

days

days

PERIODS OF INCUBATION

MULLER et al., 1999, 2003, 2004; CLARKE et al., 1990

Crab, Erimacrus Isenbeck, 360 days


Lobster, Homarus americano, 180 days
Crayfish, Pacifastacus araucania, 120 days
PERIODS OF INCUBATION

HELLUY and BELTZ, 1991; NAGAO et al., 1999; RUDOLPH and ROJAS, 2003

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion
http://bit.ly/UIg44p1

M. Rosenbergii (Palaemonidae)
Tropical and subtropical regions
Brackish water
Commercial value
SADEK and MOREAU, 1996

M. rosembergii

Incubation
http://bit.ly/34jbsdi3

Females carry their eggs under the abdomen

Protein
Yolks
Lipid
Gravid female M. rosembergii

LUO et al., 2004; PETERSEN and ANGER, 1997; CHEN et al., 1998;
WEHRTMANN and KATTNER, 1998; GIMENEZ and ANGER, 2001; YAO et al., 2006

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Objective

Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

i) Describe the embryonic developmental stages of M. rosenbergii during cultivation


in the laboratory
ii) Investigate the histological and biochemical composition of the eggs during
embryonic development of the specie

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Materials
and methods

Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Sampling
Adult prawns were obtained from Mariout Fish Farming Company
Alexandria, Egypt (May 2009)
Transported to Fish Research Station belonging to the National Institute of
Oceanography and Fisheries, at El-Khayria City, Egypt
http://bit.ly/Jjf5199g

Fiber glass tank

Diameter 1.25 m;
filled with aerated tap water (1 meter);
polyvinyl chloride tubes (PVC);
sex ratio 1:3 (male:females);
fed every day with commercial shrimp pellet;
water parameters were measured weekly
(Temperature, pH, DO and conductivity DO).

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Introduction

Materials
and methods

Objective

Results and
discussion

Conclusion

Gravid females
40 L
28 C 0.5
Continuous aeration

Second sample

First sample
3 females
Newly eggs
(orange eggs)
Weighed

Fixed in
10% formalin
Morphological and
histological studies

Deep freezer (25 C)


Biochemical analysis

*Length and weight


DEEP YELLOW

PALE BROWN

DEEP BROWN

DEVELOPMENTAL STAGES

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Histological study
i. Dehydrated in ethyl alcohol (series)
ii. cleared in terpineol
iii. embedded in paraffin

Eggs

Coloring
(1) hematoxylin and eosin
qualitative changes;
(2) sudan black
quantify lipid
(3) periodic acid-schiff stain (PAS)
quantify carbohydrates of eggs.
Analysis
Examined and captured by light microscope

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Introduction

Materials
and methods

Objective

Results and
discussion

Conclusion

Proximate composition

Orange eggs

100

Deep yellow

100

Pale brown

100

Deep brown

100

http://bit.ly/Jjfpl66k

Protein, carbohydrates and total lipids quantification

Saline solution
(1.2% NaCl)

Spectrophotometer

*(Bradford, Anthrone and Bligh and Dyer methods, respectively)


**(595 nm - 620 nm - 560 nm, respectively)

VAN HANDEL, 1965

Water content
weight
of samples

weight
of egg

Water content
LAFMM, 1979

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Statistical analysis
Only proximate composition of eggs
ANOVA (Statistic - Version 4.5)
Duncan test (P < 0.05)

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Results and
discussion

Introduction

Materials
and methods

Objective

Results and
discussion

Conclusion

Egg coloration and morphology during embryonic development

At the laboratory
conditions

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EMBRYONIC DEVELOPMENT

days
Table 1. Measurement of eggs diameters (width and narrow side) during different
embryonic development of M. rosenbergii (X SD, n=6).

Embryonic stages

Wide side (m)

Narrow side (m)

Orange eggs

681.749.60

563.376.10

Yellow eggs

69663.50

65842.07

Pale brown eggs

73546.30

68055.14

Deep brown eggs

797.531.60

71027.75

+
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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Figure 1. Photographs showing


berried females of M. rosenbergii:
(A) bright orange eggs,
(B) pale yellow eggs,
(C) dark yellow eggs and
(D) brown eggs before hatching.

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Introduction

Objective

18 days-old
214
andand
4 days-old

Materials
and methods

Results and
discussion

6 days-old20 days-old

Conclusion

8 and 10 days-old

Figure 2. Bright-field micrographs of developing embryos of M. rosenbergii, (A) 2 days-old embryo showing a small
translucent region (c) at one pole and the yolky portion (Y), (B) side view of 4 days-old embryo, (C) 6 days-old embryo
showing transparent trunk (T), (D) 8 days-old embryo with developing 2 eyes spots (e) in the yolky mass (Y), (E) 10
days-old embryo showing enlarged oval-shaped eyes (e), (F) 14 days-old embryo showing large oval-shaped eyes (e)
surrounded by striation, (G) 18 days-old embryo with prominent dark relatively rounded eyes (e), the heart region (h)
behind the yolky portion (Y) and the transparent trunk (t) curved forward at the ventral side, (H) top view of 20 daysold embryo showing a pair of rounded eyes (e), abdomen which curved forward, (Ab), segmented abdomen (Seg) and
heart region (h), (I) ventral view of the 20 days-old embryo, (J) newly hatched larvae showing the eyes (e), the antenna
(a) and the trunk (t).

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Histological studies during embryonic development

Figure 3. Camera Lucida drawings of the embryonic development of M. rosenbergii.

telson and rudiments of uroped in 22 days-old

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Introduction

Objective

Materials
and methods

primary and secondary egg membranes PGCs

Results and
discussion

Conclusion

organ analog (OA)

Figure 4. Histological
structures
of eggs
ofmuscle
M. rosenbergii
theand
embryonic
development.
heart,
eyesinternal
and
tailorgan,
in 14 during
days-old
eyestalk,
elongated
body
presence
of yolk(A,
in B)
20Fertilized
days-oldegg
eye
analog
CEA
stage, showing first egg membrane (MB), Yolk (Y) and vetelline membrane (Ve). Hx and PAS stains, respectively.
(C) 6 days old embryo, two stands of PGCs (arrow at the dorso-medial region behind the yolk (Y), PAS stain. (D, E) 8
days-old embryo showing organ analoge (OA). PAS and Hx stains. (F) 10 days-old embryo showing eye analog
(CEA). Sudanblack stain. (G, H) 14 days-old embryo showing the heart (H), eyes (E) and the tail muscle (T). Hx and
PAS stains, respectively. (I, J) 20 day-old embryo showing the formation of eyestalk (ES), internal organ (IO),
elongated body (EB) and presence of yolk (Y).

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Biochemical composition during embryonic development

Table 2 Proximate composition of eggs (g/egg) of M. rosenbergii during the embryonic development
(X= SD, n= 6).

Embryonic stages

Protein

Lipid

Carbohydrate Moisture (%)

Orange eggs

26.5 1.47B 18.3 0.95A 3.4 0.153A

49.23 0.757D

Yellow eggs

26.7 1.56B

17.4 0.60A 2.3 0.20B

55.90 3.01C

Pale brown eggs

27.2 1.22B

16 0.31B

2.1 0.32B

68.53 1.65B

Deep brown eggs

30.9 1.14A 9.7 0.47C

1.9 0.15B

76.23 2.64A

Means bearing the same superscript in the same column are not significantly different (P<0.05).

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Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

Discussion

Hatch
out:separated
20 days
Higher
lipid
andatprotein
long larval
PGCs
are
of28
theCsomatic
cellsstages
early
in the embryonic stage
Biochemical
changes
changesto those of
PGCs
are easily
discernible,
similar
Eggs
size
stage
ofreflect
development
in morphogenesis
other
vertebrates and invertebrates

Egg morphology of M. rosembergii

Biochemical composition

FUJIMOTO et al., 1976, 1977; GIMSBURG, 1997; GINSBURG et al., 1990

Egg
weight increases
during
theenergy
embryonic
Carbohydrate
and lipid
main
source;
Granulated
cytoplasm
Quails
development
Protein
structural substance

Histological studies

PGCs
similarity in mechanism of the
migration
End of the yolk
beginning of physiological
water
functions and
thecontent
heart began to beat

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Conclusion

Introduction

Objective

Materials
and methods

Results and
discussion

Conclusion

It was concluded that, variations in the biochemical compositions of eggs reflected


changes in their morphogenesis during the embryonic development.
The findings of this study contribute to a better knowledge of this species, due to
their important participation in food chains.

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Thank you for your kind attention!