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Fish Oocyte Ageing and its Effect on Egg Quality

a a b
Azin Mohagheghi Samarin , Tomas Policar & Franz Lahnsteiner
a
Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of
Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and
Hydrobiology, University of South Bohemia in Ceske Budejovice, Vodňany, Czech Republic
b
Federal Agency for Water Management, Institute for Water Ecology, Fisheries and Lake
Research, Mondsee, Austria
Published online: 14 Jul 2015.

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To cite this article: Azin Mohagheghi Samarin, Tomas Policar & Franz Lahnsteiner (2015) Fish Oocyte Ageing and its Effect on
Egg Quality, Reviews in Fisheries Science & Aquaculture, 23:3, 302-314, DOI: 10.1080/23308249.2015.1053560

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 Reviews in Fisheries Science & Aquaculture, 23:302–314, 2015
Copyright Oc Taylor & Francis Group, LLC
ISSN: 2330-8249 print / 2330-8257 online
DOI: 10.1080/23308249.2015.1053560

Fish Oocyte Ageing and its Effect

on Egg Quality

AZIN MOHAGHEGHI SAMARIN,1 TOMAS POLICAR1 and FRANZ LAHNSTEINER2

1
Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity
of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in Ceske
Budejovice, Vodnany, Czech Republic
2
Federal Agency for Water Management, Institute for Water Ecology, Fisheries and Lake Research, Mondsee,
Austria
Downloaded by [New York University] at 08:55 16 July 2015

In fish, delayed spawning in nature, delayed egg collection in capture and delayed fertilization after egg stripping can lead
to oocyte ageing and over-ripening. Oocyte ageing is reported as one of the most important factors affecting the egg
quality. During over-ripening, morphological, physiological, biochemical, histological, cellular, and molecular changes
occur inside the eggs and ovarian fluid. These alterations can negatively affect the egg fertilization capacity and
successive developmental stages. The time period during which eggs remain viable after ovulation or stripping has been
reported from a few minutes to a few weeks depending on the fish species and the storage temperature. Although several
biomarkers that characterize the over-ripening phenomenon have been defined, the reliability of these parameters in the
field of aquaculture is controversial. For future researches, inhibition of oocyte ageing by antioxidants could be a valuable
research topic. In the present article, the morphological, physiological, biochemical, histological, cellular, and molecular
changes that occur during oocyte over-ripening under in vivo conditions and during in vitro storage are reviewed and
possible reasons for the decrease in viability of eggs are presented. Species-specific changes in the egg viability and larval
development are also considered.

Keywords fish egg over-ripening, Larval malformations, Ploidy anomalies, Viability rates

INTRODUCTION refers to the time period between ovulation and fertilization,

Varying egg quality and loss of oocyte viability after ovula-
tion are the limiting factors in the reproduction and mass pro-
duction of several fish species (Furuita et al., 2003; Rizzo
et al., 2003). The egg quality is influenced by a number of
external factors and broodstock management practices (Bobe
and Labbe, 2010). Oocyte over-ripening has been reported as
the most important factor affecting egg quality of several fish
species (e.g., Lam et al., 1978, McEvoy, 1984; Rime et al.,
2004). The time of egg stripping in relation to the time of ovu-
lation is a much more significant determinant of the egg qual-
ity than any of the investigated chemical or physical aspects of
egg composition (Craik and Harvey, 1984). Oocyte ageing
Address correspondence to Azin Mohagheghi Samarin, University of
South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of
Waters, South Bohemian Research Center of Aquaculture and Biodiversity of
Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisı
728/II, 389 25 Vod
nany, Czech Republic. E-mail: mohagheghi@frov.jcu.cz
302
which can occur inside or outside the fish body. The first case
when eggs are stored and aged inside the fish body is termed
in vivo storage of eggs and the second case, when eggs are
stored and aged outside the fish body, is termed in vitro storage
of eggs.
In primitive fishes, matured eggs are released from follicle
cells into the body cavity during ovulation, while in most bony
fishes the eggs are ovulated into the ovarian lumen when they
are in metaphase of the second meiotic division stage (Bobe
and Labbe, 2010), and they remain there until spawning is
stimulated by environmental factors or eggs are collected by
artificial techniques. During this period, ovarian fluid sur-
rounds the eggs. Under farming conditions, environmental and
social stimuli are absent. Therefore, except for a few fish that
spontaneously release the ovulated eggs, oocytes remain in the
fish body until they are manually stripped. If they are not col-
lected, they degenerate and are progressively resorbed
(Aegerter and Jalabert, 2004). In the artificial propagation of
cultured fish, the females are examined for ovulation to avoid
 FISH OOCYTE AGEING AND ITS EFFECT ON EGG QUALITY
the ageing of ovulated eggs. Ovulated eggs can retain their fer-
tilizing ability only for a while after ovulation depending on
the fish species and the storage temperature. Therefore, effi-
cient management of the brood stock depends on predicting
the accurate time for egg ovulation to obtain the best quality
eggs (Schreck and Moyle, 1990). In nature, retention of the
ovulated eggs and consequent over-ripening may occur when
fish cannot find the proper spawning habitat or due to the envi-
ronmental conditions caused by humans, such as dam building
that delays fish spawning (Gaudemar and Beall, 1998).
Delayed spawning in nature, delayed egg collection in capture
and delayed fertilization after egg stripping can lead to exces-
sive oocyte ageing and finally to an over-ripening phenome-
non. Ovulated oocytes retained in the ovarian or body cavity
undergo over-ripening due to major morphological, physiolog-
ical, histological, biochemical, cellular, and molecular changes
(e.g., Nomura et al., 1974; Craik and Harvey, 1984; Forma-
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cion et al., 1993; Lahnsteiner, 2000, Aegerter et al., 2005). In
addition, during storage of the eggs outside the fish body, the
eggs undergo gradual changes (Kjorsvik et al., 1990), resulting
in ageing-associated reduced fertility and larval development.
Until now, there has been only poor understanding about
the processes and underlying mechanism of oocyte ageing in
fish. This is important from the standpoint of basic research
and practical aquaculture purposes. Successful in vivo and in
vitro storage of eggs can not only provide synchronous egg
fertilization for brood fish, they can also be beneficial when
mature male brood fish are unavailable. Improvements in egg
retention methods are strongly desirable to maximize the effi-
cacy of mass production of cultured fish species. In the present
article, morphological, physiological, histological, biochemi-
cal, cellular and molecular data associated with fish oocyte
ageing and over-ripening under in vivo and in vitro conditions
are reviewed. The effects of ageing and over ripening on egg
viability rates and larval developmental success are also
considered.
1. HORMONAL CHANGES AND SPAWNING
BEHAVIOUR THROUGH OOCYTE AGEING
Oogenesis and ovulation are controlled by hormones (Lub-
zens et al., 2010). At ovulation, the single basement mem-
brane breaks and the active germinal epithelium produce post-
ovulatory follicles (Grier et al., 2007). The mechanisms link-
ing ovulation-inducing factors to the processes of follicular
degradation and the expulsion of the matured ovum, however,
are complex and poorly understood (Patino and Sullivan,
2002). Hormones and pheromones act as signals that trigger
the spawning behavior (e.g., Partridge et al.1976; Dulka et al.,
1987; Kobayashi et al., 2002; Stacey et al., 2003). During
oocyte ageing, there are changes in fish spawning behavior
(Gaudemar and Beall, 1998).
In rainbow trout, Oncorhynchus mykiss, the level of 17a-
hydroxy-20b-dihydroprogesterone hormone was highest when
Reviews in Fisheries Science & Aquaculture
303
ovulation occurred; therefore, it can be used as an indicator of
ovulation (Springate et al., 1984). In goldfish, Carassius aura-
tus, both the serum and ovarian fluid progesterone levels were
highest at the time point of ovulation and decreased signifi-
cantly 18 hr post ovulation, when the eggs completely lost
their fertilizing ability (Formacion et al., 1995). The progester-
one levels in the ovarian fluid decreased during oocyte over-
maturation (Formacion et al., 1995). The serum 17a,20ß-
dihydroxy-4-pregnen-3-one levels showed a progressive and
more rapid decline during ageing of oocytes in goldfish, while
the hormone generally increased in the ovarian fluid. The
serum testosterone levels were constant during oocyte ageing
while the ovarian fluid testosterone levels increased signifi-
cantly 3 hr post ovulation and remained high until 18 hr post
ovulation. Thereafter, they declined to a level that was similar
to that measured at the time point of ovulation. No significant
changes in the estradiol-17(E2) levels were observed in both
the ovarian fluid and serum as well as in ovarian fluid E2, T or
17 ß,20-P levels. The role of progesterone in the maintenance
of egg viability after ovulation was suggested (Formacion
et al., 1995). Liley et al. (1986) reported that increased hor-
mone levels might contribute to an acceleration or synchroni-
zation of breeding or be responsible for vigorous sexual
activity.
Ovulation occurs when environmental factors are optimal
for spawning. In salmonids, females do not spawn the ovulated
eggs in unfavorable environments (Crisp and Carling, 1989;
Barlaup et al., 1994). Retention of the ovulated eggs inside the
fish body may occur in nature in response to environmental
changes caused by humans, such as dam development and
non-proper quality of the spawning habitat (Gaudemar and
Beall, 1998). In this case, delays in spawning and, conse-
quently, over-ripening can affect the pattern and efficiency of
reproductive activities, such as spawning alone without the
presence of a mate (Gaudemar and Beall, 1998). The authors
hence proposed that female selectivity of males might also
decrease in overripe fish. They concluded that over-ripening
might be a critical factor in the wild for salmonids, affecting
not only their reproductive success, but probably also their
mate choice and, therefore, the integrity of the population
genetic structure.
In nature, ovulation does not occur simultaneously for all
females, even when they are in the same population. Under
cultured conditions, hormone treatments can significantly
induce ovulation and synchronize ovulation, facilitating hatch-
ery management. A simple coverslip wet-mount method has
been developed for rapid, accurate, low-cost and noninvasive
evaluation of final oocyte maturation and reproductive condi-
tion in common snook, Centropomus undecimalis (Neidig
et al., 2000; Rhody et al., 2013). In addition, a system of pre-
ovulatory oocyte maturity has been proposed in Eurasian perch
Perca fluviatilis (Zarski et al., 2011) and pikeperch Sander
lucioperca (Zarski et al., 2012a) based on the nucleus position
and the coalescence of oil droplets to improve the synchroni-
zation of ovulation. Some of the external signs of egg ripeness,
vol. 23 2015
 304 A. M. SAMARIN ET AL.
such as softening belly, while applying a gentle pressure on the
fish abdomen, or observing spawning small batches of eggs
may be helpful for determining the time point of ovulation.
However, to determine the exact time when ovulation occurs,
females should periodically be examined for ovulation from a
few hours to a few days, depending on the fish species and
retention temperature.
2. EGG MORPHOLOGICAL CHANGES THROUGH
OOCYTE AGEING
The morphology of unfertilized eggs and consistency of the
ovarian fluid have been reported to be helpful for detecting the
over-ripened eggs in some species. In rainbow trout, a dark
orange spot can easily be observed on the over-ripened eggs,
while in kutum, it is not possible to find any morphological
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indication for the over-ripening (Personal observations). The
over-ripened eggs turn white after water contact, while normal
eggs remain orange. While normal eggs swell within a short
period after water contact, over-ripened eggs do not. Over-rip-
ened eggs of the pike, Esox lucius, change their shape from
round to oval (Personal observations). However, such visual
criteria are difficult to apply to species with a pigmented cho-
rion, such as sturgeons (Gisbert and Williot, 2002). Over-rip-
ened eggs are larger in diameter than freshly ovulated eggs in
goldfish (Formacion et al., 1993) and Arctic char, Salvelinus
alpinus (Mansour et al., 2008). The water content and absolute
weight are also significantly increased through oocyte ageing
in salmonids (Craik and Harvey, 1984; Lahnsteiner, 2000;
Mansour et al., 2008). Over-ripened eggs are characterized by
increased transparency in some fish species, which can be
attributed to the accumulation and coagulation of oil droplets.
In normal eggs or freshly ovulated eggs, the distribution of oil
droplets is uniform, while in over-ripened eggs oil droplets
concentrate at one pole in rainbow trout Oncorhynchus mykiss
(Azuma et al., 2003) and brown trout (Mansour et al., 2007).
Therefore, the oil droplet distribution can be used to determine
the oocyte ageing stage in some fish species. The morphology
of the first polar body indicates the postovulatory age of the
human oocyte (Lasiene et al., 2009). However, this has not yet
been studied in fish.
Oocyte ageing is also accompanied by an increase in the
amount of ovarian fluid in common carp Cyprinus carpio
(Personal observations) and in its turbidity in walleye Sander
vitreus. The increase in turbidity is considered to be due to
the elevated protein concentration (Dietrich et al., 2012).
Nomura et al. (1974) divided the over-ripening phenomenon
in rainbow trout into four stages. The first stage, which is
immediately after ovulation, includes normal eggs. In the sec-
ond stage, the eggs show the first signs of over-ripening. In
both the third and fourth stages, the eggs are over-ripened.
However, in the fourth stage, the eggs have an abnormal
shape and their composition has changed. Only eggs in the
first and second stages can develop to eyed-stage embryos.
Reviews in Fisheries Science & Aquaculture
The time that is needed for observing the over-ripening is
species specific and lasts from a few minutes to several
weeks.
The use of morphological characteristics as markers for the
occurrence of over-ripening is controversial. In many cases,
postovulatory ageing can induce a significant decrease in egg
developmental capacities without any noticeable morphologi-
cal changes in the appearance of the egg (Bobe and Labbe,
2010). Therefore, the egg and ovarian fluid morphology or
appearance may not be sufficient markers for detecting over-
ripening phenomenon in some fish species.
3. EGG PHYSIOLOGICAL CHANGES THROUGH
OOCYTE AGEING
The egg cortical reaction is activated by contact with water.
It leads to the extrusion of the cortical vesicles into the perivi-
telline space, secretion of their contents and to an increase in
the osmolality in the perivitelline space. The water absorption
and egg weight increase due to water influx can consequently
be observed.
The cortical reaction is delayed or inhomogenous in over-
ripened eggs compared to the freshly ovulated eggs. In fact, in
high quality eggs, the cortical reaction and formation of the
perivitelline space are homogenous, rapid and clearly visible
within a few minutes after fertilization (Kjorsvik and Lonning,
1983; Lahnsteiner, 2000; Zarski et al., 2012b). By contrast, in
over-ripened eggs, the extrusion of cortical vesicles is not
homogenous and only partial. When freshly ovulated eggs of
rainbow trout were incubated in water, the cortical reaction
was detectable within minutes; in over-ripened eggs, hardly
any extrusion of cortical vesicles was visible and the width of
the perivitelline space was very irregular (Lahnsteiner, 2000).
Nearly similar results were obtained in cod, Gadus morhua
(Kjorsvik and Lonning, 1983). The intensity of the cortical
reaction in high quality eggs of pikeperch occurs immediately
after water contact, and the chorion deformation becomes visi-
ble within 3–5 min (Zarski et al., 2012b). In addition, the cor-
tical reaction in this species is asynchronous and slow in low-
quality eggs.
The egg wet weight increase during water hardening is
closely related to the intensity of the cortical reaction. In rain-
bow trout, the water uptake is higher for high-quality eggs due
to the complete extrusion of cortical vesicles into the perivitel-
line space than for low-quality eggs (Lahnsteiner et al., 1999).
As the velocity of the cortical reaction differed between
freshly ovulated and over-ripened eggs, the assay on egg water
hardening might have been more sensitive if not applied as
assay monitoring the completed reaction after 120 min but if
applied as a short time assay after 30 or 60 min (Lahnsteiner,
2000). In herrings, over-ripened eggs have decreased dry
weight and increased water content compared to freshly ovu-
lated eggs (Schreck and Moyle, 1990).
vol. 23 2015
 FISH OOCYTE AGEING AND ITS EFFECT ON EGG QUALITY 305

4. EGG HISTOLOGICAL AND ULTRASTRUCTURAL
CHANGES THROUGH OOCYTE AGEING
After egg activation, the cortical vesicles are completely
secreted into the perivitelline space (Figure 1B), increasing its
thickness. Subsequently, the tension of the chorion increases,
which is morphologically reflected by a decrease in its thick-
ness. By contrast, the chorion thickness did not decrease in
over-ripened rainbow trout eggs due to incomplete extrusion
of cortical vesicles (Figure 1E) (Lahnsteiner, 2000). In addi-
tion, the thickness of the perivitelline space was very variable
in over-ripened eggs differing up to 10 times (Figure 1D)
(Lahnsteiner, 2000). In the Caspian brown trout, the chorion
diameter did not decrease in over-ripened eggs 40 days after
ovulation (egg viability almost 0%), while the enlargement of
perivitelline space was quite visible (Bahrekazemi et al.,
2010). However, in over-ripened goldfish eggs, the chorion
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appeared to be affected since the ooplasmic segregation was
altered (Rizzo et al., 2003).
In addition, changes in the structure of yolk components
were observed. Over-ripening herring eggs revealed yolk
decomposition (Schreck and Moyle, 1990). The structure
of yolk was homogenous in the freshly ovulated eggs of
the rainbow trout (Figure 1A) (Lahnsteiner, 2000) and of
the Caspian brown trout (Bahrekazemi et al., 2010), while
it became heterogeneous in over-ripened eggs, character-
ized by vesicular inclusions of various diameters (Fig-
ures 1C–E). In the case of rainbow trout, fibrous-like
structures also were found in the yolk (Lahnsteiner, 2000).
Yolk degradation is tightly controlled by specific protease
inhibitors and proteolytic activities increase in poor-quality
eggs of rainbow trout (Rime et al., 2004). In the freshly
ovulated eggs of curimata, basophilic granules accumulated
in the micropylar region and then distributed among the

was thinner than in freshly ovulated eggs and revealed surface
modifications, especially with the recessing or disappearance
of the pore plugs (Formacion et al., 1993). No differences in
the surface structure were detected between freshly ovulated
and over-ripened eggs in curimata Prochilodus marggravii
(Rizzo et al., 2003). However, the cytoskeleton of the oocyte
yolk globules in oocytes after 2 hr of in vitro storage at
26 C (Rizzo et al., 2003). In addition, after complete loss
of egg viability due to over-ripening, the micropyle still
remained open in curimata eggs; therefore, micropyle clo-
sure reported not to cause the loss of oocyte fertilizing
ability (Rizzo et al., 2003).

Figure 1. Morphology of eggs of the rainbow trout during over-ripening. C—chorion; P—perivitelline space; V—cortical vesicle, Y—yolk. (A) Freshly ovu-
lated egg with cortical vesicles before incubation in water. Magnification: 225£. (B) Freshly ovulated egg during extrusion of cortical vesicles, 5 min after
immersion of eggs into water. Magnification: 150£. (C) Over-ripened egg before incubation in water. Cortical vesicles are located at the plasma membrane. Mag-
nification: 225£. (D) Over-ripened egg, before incubation in water. Note, enlargement of the perivitelline space and the non homogenous composition of yolk.
Magnification: 65£. (E) Over-ripened egg. Extrusion of cortical vesicles (arrowhead), 5 min after immersion in water. Magnification: 130£ (obtained from
Lahnsteiner, 2000).

Reviews in Fisheries Science & Aquaculture vol. 23 2015

 306 A. M. SAMARIN ET AL.
5. EGG AND OVARIAN FLUID BIOCHEMICAL
CHANGES THROUGH OOCYTE AGEING
During oocyte ageing, biochemical changes occur both
inside the eggs and in the ovarian fluid, which may be used as
egg quality indicators. The most important parameters associ-
ated with oocyte ageing are summarized as follows:
5.1. Ovarian Fluid pH
During oocyte ageing, the ovarian fluid pH decreases in
rainbow trout (Lahnsteiner, 2000; Aegerter and Jalabert,
2004), lake trout (Salmo trutta fario lacustris) (Lahnsteiner
et al. 1999), Caspian brown trout (Bahrekazemi et al., 2010)
and turbot (Scophthalmus maximus) (Fauvel et al., 1993),
while it increases in grass carp (Ctenopharyngodon idella) and
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common carp (Lahnsteiner et al., 2001). Lahnsteiner (2000)
found that in vivo retention of rainbow trout eggs for 21 days
resulted in a pH decrease from 8.16 to 7.95. In parallel, egg
viability rates decreased from 85% to 25%. In another 21-day
long experiment on oocyte ageing in rainbow trout, the pH of
the ovarian fluid decreased from 8.2 to 8.0 and 7.9 at storage
temperatures of 12 and 17 C, respectively (Aegerter and Jala-
bert, 2004). Lahnsteiner et al. (1999) demonstrated that in lake
trout egg, the viability rate determined as the percentage of
embryos reaching the eyed stage was highest when the ovarian
fluid pH was between 8.44 and 8.57. Increasing the time inter-
val between ovulation and stripping from 0–10 days postovu-
lation to 30–40 days in Caspian brown trout resulted in an
ovarian fluid pH decrease from 8.3 to 7.7 (Bahrekazemi et al.,
2010). The pH of ovarian fluid was approximately 8.1 in turbot
eggs stripped shortly after ovulation and decreased to 7.1 when
overripening occurred (Fauvel et al., 1993). When eggs of
grass carp were stored for 4 hr in vitro in ovarian fluid at 4 C,
the fertilization rate decreased from 91% to 2%, while the pH
increased from 8.85 to 9.19. In vitro storage of common carp
eggs under the same conditions resulted in decreasing the fer-
tilization rate from 84% to 20% and increasing the ovarian
fluid pH from 8.85 to 8.97 (Lahnsteiner et al., 2001). Determi-
nation of the pH should be standardized for practical use as the
time interval between ovarian fluid collection and pH mea-
surement, and the storage temperature of the sample influence
the results (Aegerter and Jalabert, 2004).
The ovarian fluid pH may change due to HC diffusion or
transport between ovarian fluid and eggs or between ovarian
fluid and epithelium of the ovarian cavity or due to leakage of
metabolites, e.g., lactic acid, amino acids, or proteins (Lahn-
steiner et al., 2001). During oocyte ageing, pH changes in the
reported cyprinid species were in the opposite way compared
to salmonids. This can be interpreted in the following way. In
cyprinids, ovulated eggs remain in the ovarian cavity and ovi-
duct; the epithelia may continue secreting ions or other com-
ponents, leading to the observed effect. However, in
salmonids, eggs are released into the abdominal cavity, which
Reviews in Fisheries Science & Aquaculture
does not have active secretory function; therefore, the changes
are different from cyprinids. The ovarian fluid pH may influ-
ence the resting potential and conductivity of the oolemma
and, subsequently, the exchange processes between eggs and
ovarian fluid (Lahnsteiner et al., 1999). A low ovarian fluid
pH may affect fertilization, at least partially, via its negative
effect on sperm motility parameters as has been shown in rain-
bow trout (Dietrich et al., 2007). However, the idea needs to
be studied regarding to the over-ripening as well.
5.2. Ovarian Fluid Osmolality
In rainbow trout, the ovarian fluid osmolality significantly
decreased during 21 days post-ovulatory oocyte ageing and
the significant effect was observed earlier at 17 C than at
12 C (Aegerter and Jalabert, 2004). The authors suggested
that this trend might result from either water inflow into the
coelomic cavity through the genital papilla after successive
forced egg release or changes in the ovarian secretions. They
concluded that in both cases, decreased ovarian fluid osmolal-
ity could promote egg hydration and lower egg fertilizing abil-
ity. However, regarding the fact that rainbow trout eggs could
retain their fertilizing ability as quite high after at least three
times of egg stripping (Samarin et al., 2008), the probability
of entering water into the body cavity through genital papilla
remains speculative. Moreover, the genital papilla has a ring
muscle and connective tissue fibers that effectively close the
porus (Lahnsteiner, unpublished data). As the ovarian fluid
osmolality was significantly correlated with egg viability rates,
it can be a good indicator of egg quality in rainbow trout
(Aegerter and Jalabert, 2004).
5.3. Ovarian Fluid Protein Concentration
The ovarian fluid protein concentration and its turbidity
have been reported as indicators of oocyte ageing. Oocyte age-
ing is accompanied by a significant increase in the ovarian
fluid protein concentration (Lahnsteiner, 2000; Lahnsteiner
et al., 2001; Aegerter and Jalabert, 2004; Rime et al., 2004),
which can also lead to increased turbidity (Wojtczak et al.,
2004; Johnston et al., 2008).
In turbot, the ovarian fluid of over-ripened eggs has high
protein levels (Fauvel et al., 1993). Lahnsteiner et al. (1999)
observed a significant correlation between the ovarian fluid pH
and protein concentration in lake trout and proposed that pro-
tein may influence the egg viability via its influence on pH. In
rainbow trout, ovarian fluid protein concentration is signifi-
cantly correlated with the egg viability rates (Lahnsteiner,
2000). This is also evident for turbot (Fauvel et al., 1993). In
rainbow trout, the ovarian fluid protein concentration
increased during 21 days of oocyte ageing for almost 22%
(Lahnsteiner, 2000). The authors proposed that these proteins
are most probably derived from the eggs. Aegerter and Jalabert
vol. 23 2015
 FISH OOCYTE AGEING AND ITS EFFECT ON EGG QUALITY
(2004) measured increased protein concentration 14 days post-
ovulation and they were higher at 17 than at 12 C. The authors
reported that this increase is more likely due to changes in the
secretory activity of the post-ovulatory ovary rather than to
egg damage after sequential stripping and handling of fish.
Rime et al. (2004) suggested that some protein fragments
accumulate in the ovarian fluid of rainbow trout during post-
ovulatory oocyte ageing. Using electrophoretic techniques,
they found that the number of detected proteins progressively
increased in the ovarian fluid and approximately 20 protein
spots appeared between ovulation and 21 days post-ovulation.
More specifically, 8 protein spots increased strongly between
7 and 21 days post-ovulation. Their abundance exhibited a
260% and sometimes up to 500% increase between 7 and
21 days post-ovulation. Several ovarian fluid proteins were
undetectable at the time of ovulation, but they exhibited a pro-
gressive and strong increase during post-ovulatory ageing, and
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vitellogenin fragments originating from the ovulated oocytes
accumulated in the ovarian fluid. The authors suggested that
post-ovulatory ageing might be associated with a leakage of
oocyte components into the ovarian fluid due to changes in the
cellular membrane. Therefore, the level of detected protein
fragments in the ovarian fluid was proposed to be a useful
marker of the oocyte quality in rainbow trout. The protein con-
tent of the ovarian fluid in Caspian brown trout was increased
almost 3-fold during post-ovulatory oocyte ageing for 30–
40 days (Bahrekazemi et al., 2010). Coffman and Goetz
(1998) reported that a group of so-called ovulatory proteins
produced by the trout ovary and secreted in the ovarian fluid
during ovulation act as protease inhibitors and are responsible
for maintaining oocytes in a nonactivated state.
5.4. Ovarian Fluid Enzyme Activities
Aspartate aminotransferase activity has been reported to
increase during oocyte ageing (Lahnsteiner et al., 1999; Lahn-
steiner, 2000; Bahrekazemi et al., 2010). The enzyme cataly-
ses the reversible transfer of an a-amino group between
aspartate and glutamate and, as such, is an important enzyme
in amino acid metabolism. Aspartate aminotransferase is an
indicator of the integrity of the egg plasma membrane (Lahn-
steiner, 2000) because this enzyme is highly soluble in the
cytoplasm and leaks out of the cell when the membrane is
damaged (Lahnsteiner et al., 2001). In lake trout, an activity <
31.65 mmol min¡1 L¡1 ovarian fluid characterized egg
batches with high viability (Lahnsteiner et al., 1999). In rain-
bow trout, the level of aspartate aminotransferase was two
times higher for eggs retained in the coelomic cavity for
21 days after ovulation compared to the freshly ovulated eggs
(Lahnsteiner, 2000). The mentioned enzyme was almost three
times higher in both grass carp and common carp eggs stored
for 4 hr in vitro compared to the recently ovulated eggs (Lahn-
steiner et al., 2001). In Caspian brown trout eggs, which were
collected 30–40 days postovulation and had completely lost
Reviews in Fisheries Science & Aquaculture
307
their viability rates, the activity of aspartate aminotransferase
was 16 times higher than in eggs collected 0–10 days after
ovulation (Bahrekazemi et al., 2010). As the egg viability rates
were significantly correlated with ovarian fluid aspartate ami-
notransferase in lake trout (Lahnsteiner et al., 1999), rainbow
trout (Lahnsteiner, 2000), grass carp and common carp (Lahn-
steiner et al., 2001) and Caspian brown trout (Bahrekazemi
et al., 2010), the enzyme activity has been proposed as an egg
quality indicator in the mentioned fish species.
5.5. Egg ATP Concentration
When common carp eggs were stored in vitro for 4 hr, a
decrease in the ATP concentration was observed followed by a
drop in the ADP. It is proposed that the deleterious effects of age-
ing can be related to the oocyte ATP depletion, leading to an irre-
versible shortage of available metabolic energy that could alter
the cytoskeletal organization and, therefore, chromosomal reparti-
tion and cell divisions (Boulekbache et al., 1989). During in vivo
oocyte ageing, there were no changes in the oocyte ATP concen-
tration up to 14 days postovulation in rainbow trout (Aegerter
and Jalabert, 2004). Longer oocyte ageing periods resulted in
decreasing ATP concentration, which was observed faster at
17 C than at 12 C (Aegerter and Jalabert, 2004). During in vitro
storage of Chinook salmon (Oncorhynchus tschawytscha) eggs in
various media for 5 days, the ATP concentration was constant
(Wending et al., 2000). The ATP concentration in the two men-
tioned salmonids was approximately 100 times higher than in
carp eggs (Boulekbache et al., 1989). Therefore, it is considered
that ATP is not a limiting factor during postovulatory ageing in
salmonids (Aegerter and Jalabert, 2004).
5.6. Egg Biochemical Content and Enzyme Activities
Craik and Harvey (1984) showed that in rainbow trout,
over-ripening was accompanied by the following significant
changes when expressed absolutely (as the weight of compo-
nent per egg): increase in water content, free lipid, iron, and
calcium and decrease in bound lipid, precipitable protein, pro-
tein phosphorus, lipid phosphorus, total lipid, and chorion
weight. When expressed relatively (as the percentage of egg
dry weight), there were increases in the water content, free
lipid, iron, calcium, total lipid, and chorion weight and
decreases in the bound lipid, precipitable protein, protein
phosphorus, and lipid phosphorus (Craik and Harvey, 1984).
In over-ripened rainbow trout eggs, the levels of esterified and
of non-esterified fatty acids were significantly decreased, but
the levels of nucleic acids and the ratio of the DNA to RNA
levels were not correlated with the egg viability (Lahnsteiner
et al., 2000). In Caspian brown trout, the level of protein and
triglycerides decreased 30 days post ovulation, while the glu-
cose, cholesterol, calcium and iron concentration did not show
statistically significant changes (Bahrekazemi et al. 2010).
vol. 23 2015
 308 A. M. SAMARIN ET AL.

Decreasing egg sediment protein content was observed during
over-ripening in herring eggs (Schreck and Moyle, 1990).
In vitro storage of common carp and grass carp eggs for 4 hr
revealed that the investigated biochemical parameters of the eggs
(protein, peptides, fructose, galactose, glucose, non-esterified
fatty acids, esterified fatty acids, and total DNA and RNA) and
egg enzyme activities (phosphofructokinase, pyruvate kinase pro-
tease, lipase, NAD-dependent malate dehydrogenase, respiration
rate, NADP-dependent isocitrate dehydrogenase, and aspartate
aminotransferase) remained constant and were not correlated
with the fertilization rates (Lahnsteiner et al., 2001). The authors
inferred that mechanisms influencing the egg viability in stored
samples differ from those in fresh samples; however, the distinct
metabolic processes are still unknown.
6. EGG CELLULAR AND MOLECULAR CHANGES
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oocytes. Most findings in this field have been obtained from
studies on higher vertebrates.
It has not yet been clearly determined whether the oocyte
ageing process is controlled by an initiating factor and is fol-
lowed by a series of events that finally lead to loss of the fertil-
izing ability or whether different cellular and molecular events
start separately and then join each other that finally result in
the over-ripening phenomenon. Most authors suggest that an
increase in reactive oxygen species (ROS) is involved in post-
ovulatory oocyte ageing (Figure 2). ROS are chemically reac-
tive molecules containing oxygen, such as oxygen ions and
peroxides, which may act as an initiator for over-ripening in
human (Lord and Aitken, 2013; Takahashi et al., 2013) and
mouse oocytes (Lord et al., 2013). Therefore, an increase in
the ROS levels of the oocyte could be associated with the
progress of ageing (Takahashi et al., 2003; Lord et al., 2013;
Tatone et al., 2011). ROS induce mitochondrial oxidative

THROUGH OOCYTE AGEING stress and lipid peroxidation in different membrane systems
(Takahashi et al., 2003). Increasing oxidative stress can lead
Few studies have been performed on the cellular and to mitochondrial dysfunction and consequently decreasing
molecular changes occurring during over-ripening in fish ATP production. This can have consequences for different

Figure 2. Schematic of the mechanism of poor embryo development in post-ovulatory oocyte ageing (obtained from Takahashi et al., 2013).

Reviews in Fisheries Science & Aquaculture vol. 23 2015

 FISH OOCYTE AGEING AND ITS EFFECT ON EGG QUALITY
intracellular activities. In particular, the pattern of calcium
oscillations during fertilization is impaired in over-ripened
oocytes during fertilization as it has higher frequency but
lower amplitude. Collectively, the mechanism of postovula-
tory oocyte aging might be caused by ROS-induced mitochon-
drial injury followed by disturbance in intracellular Ca2C
regulation in the endoplasmic reticulum (Takahashi et al.,
2013). The intra-cytoplasmic level of glutathione (GSH),
which has a major role in protecting oocytes from damage by
ROS, decreases in aged mouse oocytes (Boerjan and Boer,
1990). In addition, the level of lipid peroxidation, which is an
indicator of the degree of oxidative stress, increases in the in
vivo-aged mouse oocytes (Takahashi et al., 2003).
To delay or even prevent the ageing process in oocytes
stored in vitro, treating them with some chemicals and antioxi-
dant agents, such as caffeine in pigs (Kikuchi et al. 2002),
nitric oxide (Goud et al., 2005 a, b) and DL-dithiothreitol in
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mice (Rausell et al., 2007) and humans (Tarin et al., 1998)
and trichostatine A in pig (Jeseta et al., 2008) and mice
(Huang et al., 2007) has been proposed and applied. In addi-
tion, melatonin has been reported to reduce oxidative stress in
aged mouse oocytes and to delay the onset of apoptosis, which
is the end point of the oocyte ageing process (Lord et al.,
2013). As the expression of the anti-apoptotic protein BCL2 is
decreased during oocyte ageing in mice (Gordo et al., 2002)
and pigs (Ma et al., 2005), the oocytes and developing
embryos are more prone to undergo apoptosis (Takahashi
et al., 2013; Lord et al., 2013).
Ageing has also been reported to affect the DNA of the
oocyte, e.g., epigenetic changes (Liang et al., 2008) and mis-
aligned chromosomes in mouse (Wakayama et al., 2004) and
premature chromosome separation in human (Spielmann et al.,
1985) and mouse oocytes (Mailhes et al., 1998). For rainbow
trout oocytes, IGF1mRNA is considered to be necessary for
proper embryonic development (Aegerter et al., 2005).
Aegerter et al. (2005) and Bonnet et al. (2007) have reported
different quantities of mRNAs between over-ripened and
freshly ovulated rainbow trout eggs, and over-ripened eggs
have low mRNA levels of Npm2, IGF1, and beta-tubulin
(Aegerter et al., 2005).
7. EGG VIABILITY RETE CHANGES THROUGH
OOCYTE AGEING
The time period during which eggs remain viable inside the
fish body has been reported for a variety of species: 30 min for
striped bass (Morone saxatilis) (Piper et al. 1982), 1.5 hr for
tilapia (Sarotherodon mossambicus) at 18–20 C (Harvey and
Kelley 1984), 2 hr for Asian catfish (Pangasius hypophthal-
mus) at 28–29 C (Legendre et al. 2000), 2 hr for the neotropi-
cal teleost fish, curimata, at both 18 and 26 C (Rizzo et al.
2003), at least 3 hr for Senegalese sole (Solea senegalensis) at
16 C (Rasines et al., 2012), 9 hours at 20 C and 5 hr at 24 C
for the South American catfish (Rhamdia sapo) (Espinach
Reviews in Fisheries Science & Aquaculture
309
et al. 1984), and 12 hr for goldfish (Formacion et al. 1993).
Complete loss of egg viability in kutum (Rutilus frisii kutum)
was observed 72–96 hr postovulation at 11 C and 60–72 hr
postovulation at 14 C (Samarin et al., 2011a). Successful in
vivo egg retention time has been reported to be 5 to 15 days
for rainbow trout at 8–17 C (Sakai et al., 1975; Bry, 1981;
Springate et al., 1984; Lahnsteiner, 2000; Azuma et al., 2003;
Bonnet et al., 2003; Aegerter and Jalabert, 2004; Samarin
et al., 2008) and 30–40 days for Caspian brown trout (Salmo
trutta caspius) at 7 C (Bahrekazemi et al. 2010).
In vitro storage of eggs in the ovarian fluid seems to result
in a more rapid decrease of egg fertility compared to in vivo
egg storage. The time period during which eggs remain viable
after stripping is species-specific and depends on storage tem-
perature and ranges between some hours and some days. For
example, in the curimata, the fertilization rate was »20% for
eggs stored in vitro for 30 min at 18 C. Then, the fertility
decreased rapidly and it was almost 0% after 2 hr of storage.
The storage time also depended on the temperature as storage
at 18 C caused a more drastic reduction in the fertilization
rates than storage at 26 C (Rizzo et al., 2003). In vitro storage
of cyprinid fish eggs is unsuccessful because of auto-activation
after ovulation (Stoss and Donaldson, 1983). For instance, in
vitro storage of common carp and grass carp oocytes in ovar-
ian fluid at 4 C for 4 hr resulted in a significant decrease of the
fertilization rate to 20.7% and 2.8%, respectively (Lahnsteiner
et al., 2001). Kutum eggs stored in vitro in ovarian fluid at
26 C almost completely lost their viability within 4 hr, while
eggs stored at 4, 10, and 12 C retained their viability up to
approximately 80%, 70%, and 50%, respectively, during the 8
hr of storage (Samarin et al., 2011b). Gisbert and Williot
(2002) observed that the ovulated eggs of Siberian sturgeon,
Acipenser baeri, and sterlet, Acipenser ruthenus, stored in
vitro in ovarian fluid at 15 C retained their viability for 2–4 hr.
Thereafter, their fertilizability and hatchability were dramati-
cally reduced. Ovulated eggs of Persian sturgeon, Acipenser
persicus, could be kept in the ovarian fluid at 18 C for up to 3
hr without a significant reduction in the fertilizability and
hatchability (Hajirezaee and Niksirat, 2009). After 3 hr, the
viability significantly decreased (Hajirezaee and Niksirat,
2009). In vitro storage of salmonid eggs is more successful
because egg activation occurs only after contact with water
(Stoss and Donaldson, 1983). Toleration of low storage tem-
perature appears to be another main reason. For sockeye
salmon (Oncorhynchus nerka) and pink salmon (Oncorhyn-
chus gorbuscha) eggs stored in vitro in the ovarian fluid at 8–
9 C for 72 hr, the fertility was approximately 35% (Withler
and Morley, 1968). Fertility of chum salmon (Oncorhynchus
keta) eggs was maintained for 40 hr when stored in vitro in
ovarian fluid at 12 C, but it decreased to 28% when stored for
82 hr (Jensen and Alderdice, 1984). Caspian brown trout eggs
could be stored for 2 days in vitro at 2–3 C in ovarian fluid
(Niksirat et al., 2007a). Rainbow trout eggs have been
reported to lose fertility after 24 hr of storage in ovarian fluid
at 19 C and after 52 hr at 10 C (Billard and Gillet, 1981).
vol. 23 2015
 310 A. M. SAMARIN ET AL.
Azuma et al. (2003) showed that the hatching rate of rainbow
trout eggs decreased to almost zero after 5 days of incubation
in artificial ovarian fluid at 10 C. Bonnet et al. (2003) reported
»50% fertilization of rainbow trout eggs stored in vitro for
9 days at 12 C in coelomic fluid. Rainbow trout eggs stored
for 7–9 days in ovarian fluid at 1–3 C showed no decrease in
fertilization rates compared to freshly collected eggs (Babiak
and Dabrowski, 2003; Niksirat et al., 2007b). The underlying
physiological and biochemical processes for oocyte viability
lose during in vitro storage are not yet known (Rizzo et al.,
2003).
Because in poikilothermic organisms the rate of physiologi-
cal processes is affected by temperature, it is more appropriate
to express the optimal egg storage time in terms of degree-
days or degree-hours rather than days or hours as a practical
unit for hatcheries. In rainbow trout, the optimal postovulatory
stripping time was within a range of 30 to 40 degree-days, and
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over-ripening of the eggs occurred after 224 degree-days of
egg retention inside the fish body (Samarin et al., 2008). In
kutum, egg stripping should take place within 168 degree-
hours after ovulation and complete loss of egg viability occurs
after 672 degree-hours of egg in vivo retention (Samarin et al.,
2011a). In salmonids, the in vivo and in vitro storage times of
eggs can be improved at lower temperatures (e.g., Aegerter
and Jalabert, 2004; Samarin et al., 2008). However, in other
species, such as turbot (Psetta maxima) (Suquet et al., 1999)
and curimata (Rizzo et al., 2003), storage of eggs at low tem-
peratures, deviating widely from the natural optimum, resulted
in decreased egg viability rates. The viability of curimata
oocytes was greatly reduced when stored in vitro at 18 C com-
pared to 26 C (Rizzo et al., 2003). The viability of in vitro
stored eggs of turbot was higher at 8 and 13 C compared to
3 C.
Some studies show that the viability rates of the eggs fertil-
ized for some time after ovulation (after a few hours to a few
days depending on the fish species) are slightly higher than
those of the eggs fertilized immediately after ovulation (Sakai
et al., 1975; Bry, 1981; Springate et al., 1984; Mylonas et al.,
1992; Linhart and Billard, 1995; Aegerter and Jalabert, 2004;
Samarin et al. 2008; Rasines et al., 2012). However, other
studies have reported conflicting data (e.g., Espinach et al.,
1984; Lahnsteiner, 2000; Rizzo et al., 2003; Samarin et al.,
2011a). The contradictory effects in the latter studies might be
ascribed to a masking effect due to longer time intervals
between successive strippings. A slight asynchrony between
the processes of meiotic maturation and ovulation has been
reported as the most likely reason for this trend (Mylonas
et al., 1992). There may also be some delay in the reorganiza-
tion of chromosomes after sperm chromatin decondensation
and in the process of the second meiotic division (Linhart and
Billard, 1995). In rainbow trout, increases in the abundance of
both cyclins A1 and A2 mRNA were observed simultaneously
with increasing egg viability rates at 5 days after ovulation
(Aegerter et al., 2005). This topic appears to be interesting for
future studies.
Reviews in Fisheries Science & Aquaculture
8. LARVAL MALFORMATION RATE AND PLOIDY
LEVEL CHANGES THROUGH OOCYTE AGEING
Oocyte ageing has been reported to cause malformations in
the larvae of different fish species. The proportion of deformed
larvae in the African catfish, Heterobranchus longifilis,
increased significantly from 4 to 20% 2 hr after the completion
of ovulation (Legendre and Oteme, 1995). In European catfish,
Silurus glanis, after 6 hr of storage of the eggs inside the fish
body, up to 50% of the hatched larvae were malformed (Lin-
hart and Billard, 1995; Varkonyi et al., 1998). When ova were
stripped 3 hr after ovulation in Asian catfish, the proportion of
deformed larvae increased significantly compared to those
observed at the moment of ovulation and almost all of the
hatched larvae originating from ova collected 5 hr post ovula-
tion were considerably deformed (Legendre et al., 2000). For
the curimata, the rate of deformed larvae increased to almost
60% when eggs were stored for 2 hr in the ovarian cavity at
26 C before fertilization (Rizzo et al., 2003). In addition, in
rainbow trout, oocyte ageing significantly affected the inci-
dence of larval malformations (Aegerter and Jalabert, 2004;
Rime et al., 2004) and increased to 50% 16 days postovulation
at 12 C (Bonnet et al., 2007). The increased eyed-egg mortal-
ity and larval malformation rates can be interpreted as a conse-
quence of biochemical changes (Craik and Harvey, 1984;
Lahnsteiner, 2000) as well as of the leakage of essential oocyte
components into the ovarian fluid (Rime et al., 2004). These
components might be required for the normal development of
embryos and larvae.
Reports in salmonids (Yamazaki et al., 1989; Aegerter and
Jalabert, 2004; Aegerter et al., 2005), European catfish (Var-
konyi et al., 1998) and tench (Flajshans et al., 2007) indicate
that ploidy anomalies are frequent in larvae of aged oocytes.
In European catfish, 3–20% of the eggs fertilized 6 hr postovu-
lation resulted in larvae with failures in the chromosome distri-
bution, such as aneuploidy, triploidy, and tetraploidy, while no
chromosomal abnormality was found in the larvae originating
from eggs fertilized immediately after ovulation (Varkonyi
et al., 1998). The incidence of triploid larvae increased with
the post-ovulatory ageing time and increasing temperature in
rainbow trout (Oncorhynchus mykiss) (Aegerter and Jalabert,
2004). In tench (Tinca tinca), the incidence of triploid larvae
increased significantly after 5 hr in vitro storage at 24 C as
well as after 3 hr in vitro storage at 22 and 17 C. During in
vivo storage, a significant triploid larval yield appeared after 5
hr of storage at 22 C (Flajshans et al., 2007). Changes in
oocyte cytoskeletal organization during ageing can be associ-
ated with a failure of second polar body extrusion and finally
with the occurrence of ploidy anomalies in the larvae
(Aegerter and Jalabert, 2004; Flajshans et al., 2007). The rate
of triploid larvae increased in over-ripened oocytes of
the pike, Esox lucius. By contrast, all larvae obtained from the
eggs fertilized at different hours post ovulation were diploids
in common carp Cyprinus carpio (Samarin, un-published
data). As malformed larvae may include a number of ploidy
vol. 23 2015
 FISH OOCYTE AGEING AND ITS EFFECT ON EGG QUALITY
anomalies (Varkonyi et al., 1998; Aegerter and Jalabert,
2004), measurement of the larval ploidy levels should be per-
formed during the first days of hatching when malformed lar-
vae are still alive.
9. CONCLUSIONS AND FUTURE DIRECTIONS
Oocyte ageing and over-ripening is an important issue both
in nature and under farming conditions. In nature, very limited
information is available about oocyte ageing and the probable
effects on fish reproductive behavior and the mate selection by
females. No study has yet been performed on the effect of fish
oocyte ageing on the growth and genetic structure of progeny.
This topic should be addressed in the future studies.
For cultured fish species, post-ovulatory oocyte ageing has
been reported as one of the most important factors affecting
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the egg quality and hence, it can be a limiting factor for suc-
cessful mass production. To collect high-quality eggs, it is
essential to determine the oocyte developmental stage, the
time point for hormone injection and ovulation, the best post-
ovulatory stripping time as well as the time needed for the
occurrence of over-ripening phenomenon. The optimal time
period for egg collection has been studied and determined in
some fish species as in the Salmonidae and Cyprinidae; in
many other species, it has yet to be investigated. Because the
time interval between ovulation and over-ripening of the eggs
is greatly affected by temperature, it is more appropriate to
express the optimal egg storage time in terms of the degree-
hours or degree-days (depending on the fish species) rather
than hours or days as a practical unit for hatcheries.
Cryopreservation and vitrification of fish oocytes has been
unsuccessful until now due to the difficulties associated with
the removal of intercellular water during cooling, toxicity of
cryoprotectants, and inhomogeneous freezing and thawing
rates (e.g., Haga, 1982; Harvey and Ashwood-Smith, 1982;
Stoss and Donaldson, 1983; Rana, 1995; Guan et al., 2008).
Therefore, in vitro storage of unfertilized eggs has been the
only alternative until now. In addition, during in vitro storage,
fish eggs gradually undergo changes that are partly similar to
the effects observed during over-ripening. In higher verte-
brates, treatment of unfertilized eggs with antioxidants may
delay, prevent or even reverse the ageing process during in
vitro storage (Miao et al., 2009; Lord and Aitken, 2013; Lord
et al., 2013; Takahashi et al., 2013). The effect of antioxidants
and oxidative damage on oocyte ageing and over ripening in
fish has not yet been examined.
Although some morphological, physiological, and bio-
chemical parameters that characterize the over-ripening phe-
nomenon have been determined for different fish species, the
reliability of these biomarkers is still under discussion. Very
little is known about the cellular and molecular mechanisms
involved in the ageing process of oocytes in fish and higher
vertebrates. With respect to the fact that fish display a vast
diversity of reproductive modes and produce a high number of
Reviews in Fisheries Science & Aquaculture
311
oocytes, studying cellular and molecular changes through
oocyte ageing and also mechanisms controlling the phenome-
non in fish could contribute valuable information for aspects
of basic research and practically for aquaculture purposes.
ACKNOWLEDGEMENTS
We would like to thank the Elsevier Language Editing Service
for the English improvement of the text.
FUNDING
This study was financially supported by the Ministry of Educa-
tion, Youth and Sports of the Czech Republic—projects
‘CENAKVA’ (No. CZ.1.05/2.1.00/01.0024), ‘CENAKVA II’
‘(No. LO1205 under the NPU I program)’ and POSTDOC JU
(No. CZ.1.07/2.3.00/30.0006).
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