Manual for the Determination

of Egg Fertility in Penaeus monodon

Michael R. Hall, Neil Young,

and Matt Kenway

Penaeus monodon breeding stock

Photo: Matt Kenway, AIMS

PMB No 3, Townsville Mail Centre PO Box 222, Deakin West

Qld 4810, Australia ACT 2600, Australia

CONTENTS

Introduction

Section I - Breeding biology of Penaeus monodon

Preface
Ovarian development 1
Ovarian development 2
Ovarian development 3
Ovarian development 4
Ovarian development 5
Mating
Spawning
References

Section II - Development of the egg: Post-fertilisation to hatching

Preface
Video 1 - Low resolution QuickTime movie (770Kb)
Video 2 - Medium resolution AVI movie (2.78Mb)
 Time after spawning 0-30 minutes
Time after spawning 1 hour
Time after spawning 1 hour 30 minutes
Time after spawning 2 hours
Time after spawning 3-4 hours
Time after spawning 5-6 hours
Time after spawning 7-8 hours
Time after spawning 9-10 hours
Time after spawning 11-12 hours
Time after spawning 13 hours

Section III - Distinguishing fertilised and unfertilised eggs

Preface
Differentiating 1 Differentiating Between Fertilised and Unfertilised Eggs
Differentiating 2 Fertilised Eggs
Differentiating 3 Unfertilised Eggs
Difficulties 1 Mixture of Fertilised and Unfertilised Eggs
Difficulties 2 Egg Membranes
Difficulties 3 Developmental Abnormalities 1
Difficulties 4 Developmental Abnormalities 2
Difficulties 5 Developmental Abnormalities 3
 INTRODUCTION

Prawn farmers rely on wild stocks of Penaeus monodon for the production of larvae. Hatcheries
preferentially source females that have a nearly fully developed ovary, ie a gravid female, which are in
an immediate pre-spawning state to meet required production schedules. However, fluctuations in the
availability of wild broodstock, on a day to day basis as well as a seasonal one, coupled with variable
spawning performance, make hatchery operations the weakest link in the production cycle. Often post-
larvae from hatcheries cannot be reliably supplied to farms in the numbers required for grow-out
production schedules. For this reason some hatcheries stockpile wild female broodstock with
undeveloped ovaries, known as ‘blanks’, which are induced to spawn at a later date, as insurance to
match larval production with grow-out cycles. The supply of wild broodstock to satisfactorily meet the
need of the prawn farming sector is a precarious state and is set to deteriorate further if, as is predicted,
Australian production expands. Even if additional trawling grounds can be found from which to source
the extra wild broodstock required, the continued reliance on natural populations will ensure that the
capability for hatcheries to meet production schedules will remain in a fragile state.

Epizootics during the grow-out period have had a profound impact on farm production. Recent evidence
indicates that wild broodstock are carriers of potential pathogenic organisms, such as viruses, and that
the incidence of infection varies seasonally. At various times of the year over 95% of wild broodstock
trawled from Australian waters can harbour viruses that may be pathogenic (Walker et al. 1998). The
vertical transmission of these viruses from mother to larvae is a continuous threat to production. In
Australia several groups of researchers are developing diagnostic tools to assist in the identification of
pathogenic organisms within broodstock. Success overseas in the production of specific pathogen free
(SPF) broodstock has resulted in managerial regimes that have curtailed the frequency of epizootics.
For example, in Taiwan the screening of wild broodstock and the production of captive reared
broodstock, which are certified SPF with regards to White spot syndrome virus (WSSV), has resulted in
grow-out cycles free of WSSV epizootics (Lo et al. 1998). Although the use of SPF broodstock does not
result in disease resistant, or even disease tolerant stock, it is proving to be one effective managerial
control measure which minimises the likelihood of epizootics due to an identifiable pathogenic organism
which is responsible for disease and production losses. The variability in the supply of wild broodstock
and the potential to introduce disease onto the farm is placing considerable interest on the production of
closed-life cycle broodstock from farm stock.

Although closing the life cycle of P. monodon will add to production costs, the industry would have
complete control over the captive reared broodstock and they would have a known life health history.
The use of captive reared broodstock may be the most ecological and economical approach to ensure a
sustainable industry. However, before the transition will occur the industry requires evidence that the
spawning performance of captive reared broodstock is comparable to that of wild broodstock.

Commercial hatcheries in Australia report periods when wild spawner performance is unexpectedly
poor. Inferior performance has been observed in association with conditions such as:

● Seasonal variability – broodstock caught on near-shore spawning grounds in north Queensland

in August, which are thought to be the first broodstock to develop mature eggs after a dormant
period during winter, often perform poorly in hatcheries.
● Location of trawling – broodstock captured from off-shore grounds are sometimes reported as
 inferior to those from near-shore grounds.
● Multiple spawnings – hatch rates can decrease with progressive spawnings after eyestalk
ablation.
● Resident time in hatchery – if females molt in the hatchery before spawning hatch rates can be
poor.
● Pond reared broodstock – performance of pond reared broodstock have been reported to be
inferior to that of wild broodstock.
● Partial spawnings – females may only ovulate part of the egg mass, resulting in poor egg output
per spawning
● Stress spawnings – females may spawn without normal spawning behaviour, such that the egg
mass is shed in one spot as a gelatinous pile, and these generally fail to hatch.

Most hatcheries use hatch rates as a key performance indicator of broodstock. Hatch rate, however, is
influenced by several factors. For example, male performance is dependent on sperm quantity per
spermatophore and fertilisation capability of the sperm, ie ‘sperm quality’. Similarly, female performance
is dependent on such parameters as total eggs spawned per spawning, egg quality, and hatchability.
During egg formation in the female all of the nutritional and other requirements for successful hatching
are ‘packaged’ in the egg at this time. In peaneid prawns, functional mouthparts do not occur until the
fourth naupliar stage, even though the larvae do not actually become self-feeding until zoea stage 1.
Therefore, the larvae are totally dependent on their accumulated yolk for nutrition during the first 36 to
48 hours after hatching. All of the nutritional and other compounds necessary for successful
embryogenesis and early larvae development have all been previously stored during egg maturation
within the mother as her ovaries matured. The yolk and its associated compounds are a major
determinant of egg quality.

This manual has been compiled for hatchery operators to assist in the identification of some broodstock
performance measures. Hatchery operators regularly report poor hatch rates from females that have
average to above average egg production. A critical first step in isolating the reason for poor spawner
performance is to identify the problem so that a treatment can be specified. This manual describes one
approach to accurately determine whether poor hatches are due to the lack of fertilisation, and hence
probably due to poor male quality, rather than poor female performance and egg quality. If fertilisation is
high and hatch rates are poor, then either the female should be replaced or holding conditions of the
female stock need improvement.

This manual is divided into three sections. Section I describes the general breeding biology of P.
monodon. Although this information is not required to determine fertility rates it is included to assist in
the potential trouble shooting of broodstock performance issues. Although much of the information may
already be known to some hatchery personnel, the manual should provide a useful reference document,
particularly when training new staff. Section II details the developmental changes that can be observed
in egg development from the time of spawning and post-fertilisation to hatching. Section III describes the
determination of fertility rates and how to distinguish between fertile and non-fertile eggs.

We are indebted to Don Booth, Matthew Salmon and a host of others who have either worked or
volunteered in the Maturation and Hatchery Unit at AIMS. We acknowledge the assistance of the staff of
the Anton Breinl Centre (Department of Public Health and Tropical Medicine), Townsville, for access to
and assistance with flow cytometry equipment. The photographs in Figures 1.3b, d, f, h are credited to
the PhD thesis work of Carol Fraser and the CRC Aquaculture Ltd. The video of early embryogenesis
was produced by Marc Leutjens while on a visiting research program at AIMS. We thank Kate Wilson for
critically reading and making comments on earlier drafts. The Science Communication section at AIMS
kindly assisted in the final moulding of the text and illustrations. Barry Tobin produced the necessary
transformations for publication on the web. The Fisheries Research and Development Corporation and
AIMS funded the work that made this manual possible.

References:

Lo C-F, Chang Y-S, Cheng C-T, and Kou G-H (1998) PCR Monitoring of Cultured Shrimp for White Spot
Syndrome Virus (WSSV) Infection in Growout Ponds. Pp. 281-286. In "Advances in Shrimp
Biotechnology", Flegel T W (ed). BIOTEC, Thailand.

Walker P J, Cowley J A, Spann K M, and Dimmock C M (1998) The Emergence of Yellow Head-Related
Viruses in Australia. Pp. 263-265. In "Advances in Shrimp Biotechnology", Flegel T W (ed). BIOTEC,
Thailand.
SECTION I

BREEDING BIOLOGY of Penaeus monodon PREFACE

The age of first breeding in P. monodon is unknown. However, it is apparent that they go
through a period of adolescence before passing through puberty and finally becoming sexually
mature. Anecdotal evidence supports the conclusion that it is a combination of age and body
size that influences the onset of sexual maturity. Induced maturation and spawning has been
attained by eyestalk ablation in individuals as young as 5 months of age (Primavera 1978). On
rare occasions intact females of approximately 50 grams have been found in grow-out ponds
with developed ovaries. Since most prawns are harvested by 6 months of age it appears that
only those young females which are also large are capable of successfully spawning. Although
little is known of the life history of wild P. monodon in Australia, the size distribution of adults
sampled at various times of the year in north Queensland indicates that an adult population is
comprised of two age classes from a spring and autumn cohort. Members of the spring cohort
pass through their juvenile stage during summer, when water temperatures are high, and are
thought to grow rapidly during this period. In contrast, members of the autumn cohort pass
through their juvenile state during winter, when estuarine water temperatures are low, and are
thought to grow slowly during this period. This may explain the distribution in body size of
females that is observed from a sampled wild population during autumn when there appears to
be two distinct groups. One group of very large and one of small females, although the exact age
of either cohort has not been conclusively demonstrated. Nevertheless, based on the experience
of specialist trawl operators that supply wild broodstock to hatcheries, it is estimated that the
age of first breeding ranges from 5 – 11 months of age depending upon which cohort they are
from and when they are flushed from estuaries during the summer wet season. It is believed that
thereafter females will breed on a seasonal basis for the rest of their lives. Although the life span
of P. monodon is not known with certainty, it is probable that some females survive to 2 years of
age.

Interested readers are referred to a more in depth account of the life history of Penaeus
monodon in Australian waters.

The Supply of Black Tiger Prawn Broodstock for Aquaculture

This account is based on anecdotal information from the principal broodstock suppliers and is
intended to describe factors that influence the supply of black tiger prawn broodstock for the
aquaculture sector. This paper was first published as a feature article in the journal Austasia
Aquaculture in June/July 1999 (Dr. Tim Walker, Editor-in-Chief, turtle@pop.netspace.net.au).

The average size of P. monodon broodstock varies according to geographic location and may
have a genetic and environmental basis. Large males, 80 grams or larger, should be utilised as
broodstock as matings with large males result in higher fertilisation rates compared to small
males (Pratoomchat et al. 1993). In Australia, sourced female wild broodstock are typically in the
range of 110 to 160 grams whereas in the more equatorial regions of Thailand broodstock range
from 150 to over 200 grams. Total egg production per female per spawning scales to body
weight, so commercial hatcheries prefer larger females, in excess of 100 grams, to smaller ones,
in order to maximise egg production per spawner utilised. However, the very large females, over
150 grams, which are assumed to be older females, often do not perform well in hatcheries.
These very large females typically exhibit high mortality immediately after eyestalk ablation or
develop an intense reddish coloration that is believed to be an indicator of stress and may be in
the final stages of senescence.
SECTION I

BREEDING BIOLOGY of Penaeus monodon OVARIAN DEVELOPMENT 1

The ovary lies dorsal to the gut and extends from the cephalothorax (head and thorax region)
along the entire length of the tail. The determination of ovarian development by hatchery
technicians, by illuminating the internal body organs of the female by means of a bright
underwater torch beam being passed along her side, only reveals the shadow of the ovary in the
tail region and is scored from 1 to 5.

Line drawing outline of female with ovary in situ.

Colour photo of 3 stages of ovarian development.

Figure 1. (a) The ovary extends the entire length of the prawn. (b) Complete ovaries of females
scored from Stages 2 to 4. Note the colour change that is due to an increasing carotenoid
content (ovaries have been fixed in 70% alcohol causing a change from an original greenish ting
to a reddish-brown color).
AIMS Home
About AIMS Manual for the Determination
Research
Facilities of Egg Fertility in Penaeus monodon
News
Search SECTION I
Site map
Site index
Topics index BREEDING BIOLOGY of Penaeus monodon OVARIAN DEVELOPMENT 2

Figure 1.2 The view observed by hatchery operators when female broodstock are
graded for ovarian development by torchlight. The wide saddle of ovarian tissue
directly behind the carapace (Stage IV) is indicative of an immediate pre-spawning
female. A female scored as a Stage IV during the day is most likely to spawn that
night.

The intensity of the ovarian shadow is due to the different density of the ovary and
the pigmentation of the egg mass. Although the majority of the ovary is found within
the cephalothorax area, the intense pigmentation of the shell in this region prevents
the visualisation of any ovarian outline. In immediate post-spawning females a vague
shadow may be seen which is the area the previously enlarged ovary occupied. In
some instances a dark shadow is seen with intermittent areas of vague shadow
outline. This indicates that a partial spawning has occurred and that only a
proportion of the egg mass was ovulated (spawned).
 In penaeid prawns the ovaries are paired, but partially fused in the cephalothoracic
region, and consist of a number of lateral lobes. In an undeveloped state, the ovary
either does not cast any shadow or a thin opaque line is seen along the length of the
tail, and is scored as Stage 1. At this point the ovary is comprised of a connective
tissue capsule surrounding a soft vascular area containing future eggs, called
oogonia, and accessory cells, also called follicle or nurse cells. The internal wall of
the ovary capsule is lined with epithelial cells (called the germinal epithelium)
containing oogonia. Once the female is sexually mature the germinal epithelium will
produce oogonia by mitosis division throughout the reproductive life of the female.
The eggs develop from oogonia in an area known as the zone of proliferation. As the
oogonia develop they increase in size and enter the first stage of meiotic division and
henceforth are irreversibly destined to become haploid, with only one set of maternal
chromosomes. At this point, although the developing eggs are increasing in size they
are not as yet producing yolk, and are known as previtellogenic oocytes. At this
stage the ovary can be visualised with a light beam as a large centrally located
opaque rope-like structure, and classified as Stage 2.

web@aims.gov.au

Last updated - 24 May, 2000

Copyright ©1996-1999 Australian Institute of Marine Science

URL http://www.aims.gov.au
AIMS Home
[ About AIMS ] [ AIMS research ] [ AIMS facilities ] [ AIMS news ] [ AIMS search ]
[ AIMS publications ] [ Doing business with AIMS ] [ What's new ]
[ Site index ] [ Navigating this site ]
AIMS home
About AIMS Manual for the Determination
Research
Facilities of Egg Fertility in Penaeus monodon
News
Search SECTION I
Site map
Site index
Topics index BREEDING BIOLOGY of Penaeus monodon OVARIAN DEVELOPMENT 3

Fig 1.3a Stage I, underdeveloped and/or

spent stage
Fig 1.3b
 Fig 1.3c Stage II, developing stage
Fig 1.3d

Fig 1.3e Stage III, nearly ripe stage

Fig 1.3f
 Fig 1.3g Stage IV, ripe stage
Fig 1.3h

Figure 1.3 The complete ovary extends from the head to the tail. The majority of the
ovarian mass is within the cephalothorax region which cannot be observed by
torchlight.

Typically, it is at Stage 2 that inhibitory hormones in the eyestalk, arising from what
is called the X-organ-sinus-gland (XO-SG) neurosecretory complex, prevent further
ovarian growth, especially if nutritional or environmental conditions – such as those
while females are held in captivity in tanks – are deemed unfavourable by the female.
This block on ovarian development can be removed by eyestalk ablation. However,
since the prawn is bilaterally symmetric, ie with two eyes, eyestalk ablation only
results in the removal of one of the two XO-SG complexes producing an inhibitory
substance. Nevertheless, in most cases this is sufficient to induce further ovarian
development and spawning, albeit under sub-optimal conditions.

As the oocytes develop further they migrate out towards the margins of the ovarian
lobes in preparation for ovulation. During this migration, follicle cells are attached to
the periphery of each oocyte (fig 1.3a, b). It is believed that the follicle cells produce
the yolk that is internalised in the oocytes in a process called vitellogenesis (fig 1.3c,
d). As vitellogenesis proceeds, oocytes mature synchronously as yolk accumulates
and develop a characteristic dark green colour as a result of deposition of carotenoid
pigments. It is the carotenoid pigmentation that mainly causes the dark ovarian
shadow during illumination of the female by torchlight. The female is now in Stage 3
(fig 1.3e, f). By the end of vitellogenesis, the eggs develop cortical granules filled with
a jelly-like substance destined to form part of the egg shell membrane after ovulation.
At this time the shadow cast by the ovary is large, resulting in a very distinct dark
thick region extending the length of the abdomen, with an enlarged bulbous region
directly behind the carapace, called the saddle. The saddle may not be as apparent in
some broodstock, such as those that have made several spawnings after eyestalk
ablation or in captive reared broodstock. The female is now in a pre-spawning state
and is scored as a Stage 4 (fig 1.3g, h).

The culmination of ovarian development is marked by release of the fully mature

oocytes (eggs) into the oviduct at ovulation. Ovulation is typically followed within a
few minutes by oviposition, ie the release of eggs and stored sperm into the water –
the actual spawning event itself (see sections on mating and spawning). Within the
egg the nuclear membrane disappears in readiness for fusion between the two
(haploid) pronuclei, one in the egg originating from the female and the second, which
comes from the sperm at the time of fertilisation.
SECTION I

BREEDING BIOLOGY of Penaeus monodon OVARIAN DEVELOPMENT 4

Figure 1.4. (a) Schematic diagram of the major endocrine organs in P. monodon. The sinus gland
is composed of the terminals from neurons which have their cell bodies in the X-organ and
brain. (b) Electron microscopy section of the sinus gland demonstrating hormone filled vesicles
(dark circles) which fuse and release their contents into the blood. Magnified 8500x.

Eyestalk ablation results in the excision of one of the paired X-organ-sinus gland complexes (fig
1.4a) that are the source of reproductive inhibitory hormones. The sinus gland (fig 1.4b) contains
vesicles filled with hormones which are released into a blood sinus and hence into the general
circulation where the reproductive inhibitory substance prevents spawning.
Manual for the Determination
of Egg Fertility in Penaeus monodon
SECTION I

BREEDING BIOLOGY of Penaeus monodon OVARIAN DEVELOPMENT 5

Partial spawning occurs when a proportion of the mature oocytes are not released
from the ovary are destined to be reabsorbed by the female. It has been reported that
oocytes infected with virus, such as white spot syndrome virus (WSSV), do not fully
mature (Kuo and Lo 1998). Thus prevalent partial spawnings may indicate that the
broodstock are infected with virus. Although infected oocytes are not ovulated, virus
particles released in the ovary as the infected oocytes are reabsorbed may adhere to
uninfected eggs that are ovulated. This may result in the vertical transmission of
virus from mother to offspring and hence potentially produce offspring that are viral
carriers. The washing of eggs from infected mothers can assist in the prevention of
horizontal transmission of viruses.

Partial spawnings may also be due to other factors. Typically 300,000 to 500,000 eggs
are spawned within a 2 minute period (see section below on spawning). If spawning
itself is time limited then a partially blocked oviduct will slow egg release and hence
not all of the fully developed eggs will be released within the spawning period.
Alternatively the chemical trigger that induces ovulation, followed by spawning, may
be below a critical threshold level in some of the ovarian lobes and hence eggs
within these lobes are not ovulated.

Fig 1.5 Figure 1.5. Partial spawnings are typically

only observed in the tail region, where
either the anterior or posterior proportion is
spawned. Partial spawning may also occur
in the cephalothorax region, but this can
only be observed after dissection.

A female can spawn several times within a

single molt cycle. A new population of
primary oogonia are recruited for each
spawning event. If the female does spawn
 repeatedly within a molt cycle the same
reserve of sperm within the thelycum, from
the initial mating after molt, is utilised to
fertilise the eggs. Typically successive
spawnings have progressively decreasing
fertility rates as the sperm mass is
exhausted (Fig 1.4).

If the female does not spawn within a molt

cycle the developed ovarian mass is
reabsorbed a day or two before the next
molt. After each molt the ovary is fully
regressed and the ovary must mature
anew.

The female must mate again immediately after the next molt if she is going to be
capable of fertilising spawned eggs. It is therefore necessary to hold both females
and males in hatchery holding tanks if successful fertilisation is to be achieved from
females held for longer than 2 weeks. The average molt frequency in the 90 to 120
gram body mass range in 28 to 30ºC water in the AIMS Maturation Unit is 16 – 18
days.

Since ovarian maturation may take place entirely in the hatchery, a high quality diet
is necessary for the female to optimise egg quality. The quality of the egg at the time
of formation, ie vitellogenesis, and the packaging of critical molecules into the egg, is
a major determinant of larval quality. The successful development of the embryo,
hatching, and the 6 naupliar stages are dependent on egg quality. The larvae do not
become self feeding until the zoea 1 stage, which is the first time that they become
dependent on the quality of larval feeds.
 Figure 1.6. Fertility decreases with
successive spawnings within a molt cycle
after eyestalk ablation. This possibly
indicates that the sperm reserve within the
thelycum is being exhausted.

http://www.aims.gov.au/pages/research/mdef/mdef-02a-5.html (3 of 3) [31/03/2008 15:53:01 PM]

SECTION I

BREEDING BIOLOGY of Penaeus monodon MATING

Upon reaching sexual maturity the female is typically inseminated by a male each time she molts. The
spermatophores are stored in the closed thelycum of the female, which is a modification to the posterior
thoracic sternal plates and is a flesh covered chamber which can maintain viable sperm throughout the
inter-molt period. The stored spermatophore is lost each time the female molts, so the female must be
inseminated each molt cycle if fertilised eggs are to be produced. Insemination only occurs within a few
hours of molt when the female’s shell and thelycum are still soft. The mechanism by which a male and
female find each other in this short time in turbid estuarine or coastal water is not known. Since P.
monodon occurs at low population densities in North Queensland and throughout most of its
geographical range in Australia, it must be a highly efficient mechanism. In the North Atlantic lobster
(Homarus americanus), the male produces a chemical signal (pheromone), which can attract pre-molt
females from great distances. In the lobster mating takes place within 30 minutes after the female
completes her molt (Cowan and Atema 1990).

Mating in P. monodon has only been observed in captive broodstock. Generally, mating takes place at
night immediately after the female molts. The female is followed by one or more males which swim
parallel to her. The male bends its body so there is ventral-ventral contact and turns perpendicular to the
female. A spermatophore pair is released from the male’s terminal ampullae and inserted through the
slit between the lateral plates of the female thelycum into the chambers underneath; there are two
separated chambers, each for one of the two spermatophores, on either side of the thelycum ridge
(Motoh 1981). Courtship and mating varies from 0.5 to 3 hours and requires parallel swimming over
considerable distances. For mating to occur successfully in captivity large rectangular holding tanks may
be required for uninterrupted swimming during mating (Primavera 1988). Alternatively, successful
matings can be obtained in circular 4 meter diameter tanks having 1 meter water depth.
SECTION I

BREEDING BIOLOGY of Penaeus monodon SPAWNING

Wild female broodstock that have fully developed ovaries may spawn spontaneously on the night, or the
night after, delivery to a hatchery. Generally, however, the females are eyestalk ablated to increase the
likelihood of successful spawning in the hatchery. Obviously females spawn successfully in the wild
without eyestalk ablation. It is not known what factors inhibit the female from spawning once in captivity,
although stress in suspected to be a dominant factor. Eyestalk ablation has the paradoxical effect of
both stimulating the reproductive system while at the same time placing the female under additional
stress. However, it has the beneficial effect of synchronising spawning and is hence of considerable use
in keeping to required post-larvae production cycles. Spawning is generally induced 4-7 days after
eyestalk ablation in females with partially developed ovaries (Stage 2-3).

Spawning itself occurs at night. Normally the pending event is heralded by the female swimming
continually around in the tank. Swimming activity may occur for several hours before spawning or may
only occur a few minutes before. During spawning the last 3 pairs of pereiopods are held tightly together
and are flapped vigorously. The unfertilised eggs are extruded from the paired ovipores located at the
base of the third pereiopods. Simultaneously stored sperm are released from the thelycum. Both are
ejected with considerable force where the eggs appear as a greenish, and the sperm as a whitish, cloud
exiting from beneath the females as she swims. The sperm are non-motile and must come into contact
by passive collision with the surface of the egg at the moment of ejection. It is believed that the vigorous
flapping of the female’s pereiopods creates an intense vortex that increases the likelihood of sperm and
egg coming into contact. The entire process lasts approximately 2 minutes, over which time up to
500,000 eggs of 0.3 mm diameter are shed through the 2 ovipores resulting in a minimum flow rate of
2.25 km/hr. Immediately after spawning the fertilised eggs are either slightly positive of neutrally
buoyant. Within a few minutes the eggs gradually drift down through the water and collect on the bottom
of the tank. Spawning may also occur in females that have not been inseminated. In such cases,
although egg output per female may appear adequate, there will be no fertilisation and hence none of
the eggs will hatch.

Egg coloration is mainly due to the accumulated carotenoids, which are fat-soluable nitrogen free
yellow, orange, blue and red compounds. Carotenoids are only produced by plants and microbes, hence
animals can only obtain them from their diets. Within the prawn or egg the carotenoids are complexed
with proteins as carotenoproteins. Although the predominant carotenoid in eggs is the red carotenoid
astaxanthin, the association with a protein causes a large bathocromic shift in the visual region of the
light absorption spectrum producing different colors, ranging from brown, blue, green and yellow. When
the protein is denatured, such as by heating, the carotenoid is freed revealing the true color of the
underlying carotenoid. Since prawn eggs mainly accumulate astaxanthin the color is orange to red.
Figure 1.7. (a) Individual sperm cells of P. monodon. Note especially the small size (cf egg diameter of
250 – 300 microns) and lack of a flagellum – the sperm are not capable of swimming. The sperm
adheres to the egg at the spike end that afterwards undergoes the acrosome reaction. Magnification
400x. (b) Section of spermatophore. The sperm cells appear darkly stained and are surrounded by
tissue of the spermatophore itself. Magnification 40x.
SECTION I

BREEDING BIOLOGY of Penaeus monodon REFERENCES

Cowan D F and Atema J (1990) Moult staggering and serial monogamy in American lobsters,
Homarus americanus. Anim. Behav. 39:1199-1206.

Kuo G-H and Lo C-F (1998) Practical Strategies for Combating Shrimp White Spot Syndrome
Virus. Joint Taiwan-Aquaculture and Fisheries Management Resources and Management Forum.
Nov. 2-8 1998, Taiwan Fisheries Research Institute, Keelung, Taiwan.

McVey J P (1993) CRC Handbook of Mariculture. Crustacean Aquaculture. Volume 1.

CRC Press, Boca Raton, Florida, USA.

Motoh H (1981) Studies on the fisheries biology of the giant tiger prawn, Penaeus monodon in
the Philippines. Aquaculture Department, Southeast Asian Fisheries Development Center.
Tigbauan, Iloilo, Philippines. Extension Manual No. 7.

Pratoomchat B P, Piyatiratitivorakul S, Menasveta P and Fast A W (1993) Sperm Quality of Pond-

Reared and Wild-Caught Penaeus monodon in Thailand. J. Worl Aqua. Soc. 24:530-540.

Primavera J H (1988) Biology and Culture of Penaeus monodon. Aquaculture Department,

Southeast Asian Fisheries Development Center. Tigbauan, Iloilo, Philippines. Extension Manual
No. 7, Third Edition.

Treece, G D and Yates M E (1988) Laboratory Manual for the Culture of Penaeid Shrimp Larvae.
TAMU-SG-88-202. Marine Advisory Service, Sea Grant College Program, Texas A&M, Texas, USA.

Wyban J A and Sweeney J N (1991) Intensive Shrimp Production Technology: The Oceanic
Institute Shrimp Manual. The Oceanic Institute, Hawaii, USA, 158 pp.
SECTION II

DEVELOPMENT OF THE EGG:

PREFACE
Post-Fertilisation to Hatching

The rate of development during embryogenesis is temperature dependent. The lower the
temperature the slower the rate of development. The video clips included here demonstrate
some of the early cell divisions occurring between the time of spawning and 2 to 3 hours post-
spawning.

The two video clips are of the same material. However, Video 1 is of lower resolution (770 Kb)
while Video 2 is of higher resolution (2.78 Mb). Single photographs with descriptions of the
changes which occur during egg development may be found in the following "Time after
spawning" sections.

Video 1 - Low resolution QuickTime movie (770Kb)

Video 2 - Medium resolution AVI movie (2.78Mb)

Early cell division can be easily distinguished through the hatching envelope under a low
magnifying, 10 x 40, dissecting microscope. A suitable unit can be purchased for a few hundred
dollars. To determine fertility rates it is critical that eggs be collected at specific times after
spawning. If eggs are collected too early after spawning before egg development begins it is not
possible to distinguish between fertilised and non-fertilised eggs. If eggs are collected the
following morning after spawning the eggs are too developed to be able to accurately
distinguish fertile from non-fertile eggs, as the latter also undergo cellular cleavage. The
cleavage pattern of unfertilised eggs, however, is asymmetrical. Although this can be
distinguished a few hours after spawning, it is extremely difficult to distinguish the morning after
a spawning since cell division is so far advanced. By this time the egg is composed of
thousands of eggs and distinguishing a symmetrical compared to a non-symmetrical pattern is
not possible with any accuracy.

Figure 2.0. Time of spawning of P.

monodon broodstock. Based on these data
it is likely that a hatchery operator could
collect eggs from a spawning tank that
 would be in suitable state for the
determination of fertility. Assuming that
spawners are put into darkness at 6 pm, a
collection would be necessary between
mid-night and 1 am.

Estimates of fertility rates of eggs collected the morning following spawning by several
independent observers are highly variable and unreliable. In contrast, the same observers were
able to make consistent and accurate determinations of fertility rates of eggs collected 1-2 hours
post-spawning. At the AIMS hatchery the time of spawning of 186 wild broodstock occurred
approximately 5 hours after lights are turned off (Figure 2.0). If eggs are collected 1 – 2 hours
after spawning they will be between the 2nd (4-cell stage) to 7th (128 cell stage) cleavage.
Approximately 1.25 hours after spawning the 4-cell stage egg can be easily distinguish from non-
fertilised eggs. Non-fertilised eggs have either not begun to divide at all or have undergone 1-2
asymmetric cell cleavages. This pattern clearly differs from the symmetric 4-cell stage of a
fertilised egg. A trained technician can score a sub-sample of 100 eggs in 5-15 minutes. This
information is useful in determining whether poor hatchings are due to the eggs being
unfertilised, and hence there being no embryos to hatch, or due to the embryos dying during
development and not hatching as nauplii.

The rate of development during embryogenesis is temperature dependent. The lower the
temperature the slower the rate of development. The timing of events described here is typical of
those found at a water temperature of 30ºC.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 0-30 MINUTES

All headings are times after spawning. The timing of events described here and in the following
pages are typical of those found at a water temperature of 30oC.

2.1a 2.1b

Figure 2.1a, b Time 0 - 30 minutes

The attachment of sperm to the egg takes place within the first minute of spawning. Sperm are
composed of a central spherical body and a short non-flagellated spike. Attachment to the egg’s
surface is via the spike end of the sperm. By the second minute post-spawning the egg becomes
spherical and a clear membrane is observed beginning to envelop the egg. The eggs are
approximately 250 – 330 µm in diameter. By the third minute the sperm have undergone the
acrosome reaction, where the sperm fuses with the egg membrane and the male pronucleus
enters the egg’s cytoplasm. If the egg has not come into contact with a sperm at the time of
spawning, or the fusion of the pronuclei from the sperm and egg is unsuccessful, those eggs
will be unable to hatch and so hatching rates will be below 100%.
The first and second meiotic cell divisions occur at approximately 2-5 and 8-14 minutes after
spawning, respectively. These divisions result in the production of minute cells called polar
bodies. Although these can be seen under the microscope the egg appears to remain as a single
large 250 – 300 µm diameter cell. The extrusion of the jelly-like substance from the cortical
crypts of the egg is completed within the first 8 minutes (Fig. 2.1a). The soft jelly hardens into a
tough transparent shell, the hatching envelope, by 12 – 15 minutes post-spawning and probably
acts as a shield against microbial attack during the 12-14 hours of embryo development (Fig.
2.1b). During egg incubation numerous ciliates can be observed under the microscopic which
appear to be attracted to the egg and may be searching for perforations in the shell membrane
where they can gain entry into the egg and feed on the nutrient rich yolk. By electron
microscopy microbial organisms can be observed adhering to the shell surface. The fertilised
egg, called a zygote, undergoes its first cell division to clearly defined two-cell stage
approximately 30-60 minutes after spawning. However, some eggs remain undivided for at least
60 minutes. By 90 minutes all of the eggs which have been fertilised are into at least the first cell
division.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 1 HOUR

2.2a 2.2b

Figure 2.2a, b Time 1 hour

The zygote (fertilised egg) has begun its development (embryogenesis). The pronuclei of the egg
and spermatozoa have fused to form a diploid nucleus. Embryogenesis by progressing mitotic
cell division has begun. By 1 hour the egg has cleaved once (2-cell stage) or twice (4-cell stage) -
(Fig. 2.2a, b). The 4-cell stage is perhaps the easiest to distinguish between fertilised and
unfertilised eggs (Fig. 2.2b). One hour after spawning an unfertilised egg either has not divided
at all or has divided asymmetrically. In either case a population of eggs at this time can rapidly
be scored for percentage fertilisation.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 1 HOUR 30 MINUTES

2.3a 2.3b

Figure 2.3a, b, c, d Time 1 hour 30 minutes

Cell division is occurring rapidly. Within a sample of eggs a range of developmental

stages will be seen (Fig. 2.3a, b, c). The majority will be in the 4-cell stage but will range
between the 8-cell and 16-cell stage. At this time it is still possible to distinguish between
fertilised and non-fertilised eggs due to the cleavage pattern on the outside edge of the
egg. Fertilised eggs have an even regular pattern of cell division whereas unfertilised
eggs are uneven and nonsymmetrical (Fig. 2.3d).

2.3c 2.3d
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 2 HOURS

Figure 2.4 Time 2 hours

The egg is well into embryogenesis at this

time. This figure shows a number of stages
of embryo development. The latest stage
of development at this time is
approximately the 6th (64-cell stage) and
7th (128-cell stage) cleavage. By this time
a layer of cells has formed at the periphery
of the egg, called the blastoderm.
Differentiation of cells to form various
tissues required for development of the
nauplius has begun.

2.4
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 3-4 HOURS

Figure 2.5 Time 3 hours

The embryos continue to undergo rapid

cell division, the number of cleavages is
difficult to estimate by this stage. It is
increasingly difficult to distinguish between
a fertilised and unfertilised egg. The edges
of the eggs have a corrugated appearance
and it is difficult to visualise individual cells.
Figure 2.6 Time 4 hours

There appears to be very little difference in

external appearance of the embryo even
though cell division continues at a rapid
rate. At this time a secondary envelope,
within the external hatching envelope, can
be seen. An invagination can be seen on
the surface of the egg that is due a
process called gastrulation. It involves a
dynamic morphological change from a
monolayered to a multilayered embryo,
accompanied by tissue differentiation.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 5-6 HOURS

Figure 2.7 Time 5 hours

A wide range of embryonic development

may be observed. Limb buds may be
distinguishable which will form the major
appendages of the nauplii. Depending on
the orientation of the individual egg the
limb buds may or may not be visible.
 Figure 2.8 Time 6 hours

Further embryonic development has

occurred. Some embryos will appear
similar to those at 5 hours after spawning.

web@aims.
gov.au

AIMS home
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 7-8 HOURS

Figure 2.9 Time 7 hours

The appendages are more defined as the

naupliar body develops into a more
distinguishable form. Depending on
orientation, it may be possible to
distinguish the formation of setae on the
tips of the developing appendages.
Figure 2.10 Time 8 hours

Major developmental changes are

distinguishable. The naupliar body has
thickened and further development,
including setae, may be observed.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 9-10 HOURS

Figure 2.11 Time 9 hours

Embryos progressively develop with little

change in observable form.
Figure 2.12 Time 10 hours

Further differentiation of appendages are

observable on the naupliar body.
Depending on orientation, the pairs of
major naupliar appendages are clearly
distinguishable.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 11-12 HOURS

Figure 2.13 Time 11 hours

The naupliar form is recognisable. A single

reddish pigmented spot can be seen which
is the naupliar eye. Appendages are well
developed.
Figure 2.14 Time 12 hours

The nauplii begin to hatch, typically around

12.5 hours after spawning. In this figure
the bottom of the hatching envelop has
been broken and the nauplii tears their
way out using their appendages.
Commercial hatcheries have reported that
occasionally vigorous active nauplii can be
seen within the hatching envelope but are
unable to ‘hatch’ and die within the shell.
The reason for this is unknown but may be
due to poor quality and weak nauplii that
are unable to tear open the hatching
envelope.
SECTION II

DEVELOPMENT OF THE EGG: TIME AFTER SPAWNING

Post-Fertilisation to Hatching 13 HOURS

2.15a 2.15b

Figure 2.15a, b Time 13 hours

Not all eggs will have hatched by this time. Hatching does not occur synchronously and its timing is
temperature dependent. However, if the eggs do not hatch out within a few hours of each other then the
nauplii will be of questionable quality. In addition, if hatching has not occurred by 15 hours post-
spawning the nauplii may be of inferior quality. This figure (2.15b) shows the 1st naupliar stage with
appendages fully extended.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS PREFACE

Fertilisation rates vary considerably between spawning events, from 0% to 100%. Individual
spawning trials in these studies typically resulted in an average fertilisation rate per group of
females (sample sizes of 25 to 100 females per trial) of 50% to 85%. Some individuals produce
eggs well below, and above, this average. Biologically, poor fertilisation can be due to poor
quality eggs, poor sperm quality, low sperm count per spermatophore or broodstock in a
stressed condition. Physical dimensions of the spawning tank can also have an effect.
Disruption of normal behaviour during the spawning event itself, such as the female colliding
with the side of small tanks, may result in suboptimal mixing of sperm and eggs, resulting in low
fertilisation rates.

Fertilisation can be determined by other methods beside's microscopic observation. Although

high tech and requiring expensive equipment, flow cytometry is one method that lends itself to
automation and would potentially be a useful method for the purpose of monitoring large
commercial operations when problems arose. However, flow cytometry requires specialised
equipment and samples would have to be analysed off-site at a research laboratory rather than
the hatchery. Nevertheless, flow cytometry was investigated as a possible method to determine
fertility since a larger number of samples can be analyzed quickly. The technique relies on
incubating a sub-sample of eggs in the presence of a special nucleus deoxyribonucleic acid
(DNA) fluorescence dye. The intensity of the fluorescence is proportion to the amount of DNA.
An unfertilised haploid egg has half the amount of DNA as a fertilised diploid egg (zygote). A
subsample of several hundred to thousands of eggs from a spawning can be pumped through a
flow cytometer and the relative proportion of unfertilised and fertilised eggs therefore
determined. The technique, however, depends partially on the size and shape of the fluorescing
object. Our research found that the nuclei of prawn eggs are highly irregular in size and shape
(Fig. 3.1). Because of this it was impossible to distinguish between haploid and diploid eggs by
this method.

Figure 3.1 Isolated nuclei of eggs

collected 1 hour after fertilisation stained
with propidium iodide. Nuclei acids are
stained with propidium iodide. Note
variability in size and shape which results
in a very high coefficient of variation of
fluorescence characteristics making the
use of flow cytometry for haploid:diploid
differentiation impractical.
 The most affordable and appropriate
method for a commercial hatchery to
determine fertility rates would be by
observation of eggs under a stereo low-
power (40X) dissection microscope.
Collected eggs need to be examined at
specific times after spawning. Alternatively,
if a permanent collection is desired, the
eggs may be fixed and stored in seawater
with 5% formalin.

Eggs may be added to a Bogorov tray (a plastic tray with routed lanes used for counting
plankton). However, any clear bottom container, such as a glass petri dish will suffice. One to
two hundred eggs should be scored to obtain an accurate reflection of fertility rate. Use a
microscope with illumination from below the sample. It is best to first make a total count of eggs
in the container under low magnification. Afterwards, using a higher magnification, count the
number of eggs that have a symmetrical cleavage pattern.

Three samples of eggs collected from a single spawning. Eggs collected 1

Example:
hour 15 minutes after spawning.

Count 1 Count 2 Count 3

Total Fertilised Total Fertilised Total Fertilised

385 235 430 270 597 385

Average total count (385 + 430 + 597) / 3 = 471
Average fertilised eggs (235 + 270 + 385) / 3 = 297
Fertility rate 297 / 471 x 100 = 63%
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFERENTIATING 1

Differentiating Between Fertilised and Unfertilised Eggs

Fertilised Unfertilised
Characteristic
Egg Egg

Cleavage
Symmetrical Asymmetrical
pattern

1st cleavage
Post-1 hour 2nd-3rd cleavage
undivided or
development 4-8 cell stage
2 cell stage

Rapid cell division Erratic cell division

Other
after 1st cleavage after 1st cleavage

Table 3. Characteristics distinguishing fertilised from unfertilised eggs.

SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFERENTIATING 2

FERTILISED EGGS

Early Cleavage Stages

3.2a 3.2b

Figure 3.2a, b Fig 3.2a shows a typical fertilised egg 1 hour and 15 minutes after spawning. The
egg is at the 1st cleavage stage and is composed of 2 cells all of which are clearly visible and
symmetrical. Most of the eggs 1 hour and 15 minutes after spawning will be at this stage of
development. Fig 3.2b shows a fertilised egg at the 2nd cleavage stage and composed of 4 cells.
It is more difficult to see each individual cell but the pattern appears clearly symmetrical. Within
a sample of eggs at this time some may be anywhere between these stages so an uneven
number of cells is possible. The most important feature is that cell division is symmetrical and
remains so past the first cleavage.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFERENTIATING 3

UNFERTILISED EGGS:

3.3a 3.3b

Figure 3.3a, b If sampling is made less than or near to 1 hour, cell division may not have
occurred even in fertilised eggs and hence it is impossible to distinguish between them (Fig
3.3a). Cell division usually occurs in unfertilised eggs after the 2nd cleavage of fertilised eggs.
When cell division does occur in unfertilised eggs it is clearly asymmetrical (Fig 3.3b). This
pattern is easily recognisable at this time and it is why eggs are best sampled between 1 and 1.5
hours after spawning.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFICULTIES 1

Mixture of Fertilised and Unfertilised Eggs:

Figure 3.4 shows egg development

approximately 8 hours after spawning. It is
difficult to distinguish fertilised from
unfertilised ones. The egg in the lower
middle is probably the only fertilised egg of
the ones shown. Even though the fertilised
eggs have undergone cell division some of
them begin to break down. Overall a
sample collected several hours after
spawning will contain a mixture of fertilised
and unfertilised eggs which cannot be
scored for fertility rates with accuracy.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFICULTIES 2

Egg Membranes:

Figure 3.5 In some spawnings it was

observed that the hatching envelope did
not rise from the egg. These eggs may be
fertile but nevertheless fail to develop
normally. Cleavage patterns in these eggs
both symmetric and asymmetric. However,
they fail to develop normally and will not
hatch. These eggs can be distinguished
from normal fertilised eggs (compare
Figure 2.1c).
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFICULTIES 3

Developmental Abnormalities 1:

Figure 3.6 shows asymmetrical collapsed

eggs. Some spawnings result in a high
proportion of eggs exhibiting such a
pattern. These eggs fail to develop.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFICULTIES 4

Developmental Abnormalities 2:

Figure 3.7a shows abnormal development

of a fertilised egg approximately 10 hours
after spawning. The egg in the lower
middle has suffered some form of
developmental deformity. A sample of
eggs from this spawning would have
resulted in this egg being classified as
fertile since it clearly developed past the 4-
cell stage. This fertility information is
valuable since it will rule out poor fertility, e.
g. due to poor male quality, as the reason
for poor hatches. It is indicative of poor
egg quality, which may be due to a host of
factors.
 3.7b 3.7c

Figure 3.7b and c shows deformed embryos approximately 11 hours after spawning. Note that
the embryo does not have the normal single naupliar eye but rather has two. Although this egg
may have been successfully fertilised it clearly suffered developmental abnormalities.
SECTION III

DISTINGUISHING FERTILISED
AND UNFERTILISED EGGS DIFFICULTIES 5

Developmental Abnormalities 3:

Figure 3.8a shows eggs that have

ruptured egg membranes with leakage of
yolk. These eggs succumb to microbial
attack. These eggs usually have high
bacterial contamination. Ciliates can often
be seen within the outer shell membrane
feeding on the yolk. Figure 3.8a shows
examples of eggs which are in the process
of breaking down, possibly due to bacterial
attack.

3.8a