Egg and sperm quality in sh

Julien Bobe
*
, Catherine Labb
INRA, UR1037 SCRIBE, IFR140, Ouest-Genopole, F-35000 Rennes, France
a r t i c l e i n f o
Article history:
Received 23 October 2008
Revised 16 February 2009
Accepted 20 February 2009
Available online 9 March 2009
Keywords:
Gamete
Teleost
Aquaculture
Developmental competence
Oocyte
Environment
a b s t r a c t
Fish egg quality can be dened as the ability of the egg to be fertilized and subsequently develop into a
normal embryo. Similarly, sperm quality can be dened as its ability to successfully fertilize an egg and
subsequently allow the development of a normal embryo. In the wild or under aquaculture conditions,
the quality of sh gametes can be highly variable and is under the inuence of a signicant number of
external factors or broodstock management practices. For these reasons, the topic of gamete quality
has received increasing attention. Despite the signicant efforts made towards a better understanding
of the factors involved in the control of gamete quality, the picture is far from being complete and the
control of gamete quality remains an issue in the aquaculture industry. Some of the factors responsible
for the observed variability of gamete quality remain largely unknown or poorly understood. In addition
very little is known about the cellular and molecular mechanisms involved in the control of egg and
sperm quality. In the present review, the molecular and cellular characteristics of sh gametes are pre-
sented with a special interest for the mechanisms that could participate in the regulation of gamete qual-
ity. Then, after dening egg and sperm quality, and how can it can be accurately estimated or predicted,
we provide an overview of the main factors that can impact gamete quality in teleosts.
2009 Elsevier Inc. All rights reserved.
1. Introduction
The control of reproduction is a key issue in aquaculture and
one of the limiting factors of the reproductive success is the quality
of male and female gametes. Gamete quality in the wild, or in cap-
tivity, is inuenced by many factors and is sometimes highly vari-
able. For this reason, gamete quality has received increasing
attention and many studies have characterized the effect of specic
factors on egg or sperm quality. The issue of sh egg (Kjrsvik et al.,
1990; Brooks et al., 1997) and sperm (Billard et al., 1995) quality
has previously been reviewed. Yet, the picture of the factors that
can signicantly affect gamete quality remains incomplete. The
relative effects of each factor on gamete quality can be highly var-
iable and are not always well characterized. In addition, the deni-
tion of gamete quality is not always consistent among existing
studies and several types of indicators have been used to assess
gamete quality. Finally, the knowledge of the role of some intracel-
lular gamete components on the quality of the gamete has bene-
ted from recent studies, including genomic-based investigations
that have provided new hints on the long-term process of under-
standing what is and what makes a good egg and a good
spermatozoon.
The present review is therefore aiming at (i) briey presenting
the molecular and cellular characteristics of sh gametes with spe-
cial attention paid to the molecular actors that could play a key
role in the regulation of gamete quality, (ii) dening what is egg
and sperm quality and how can it can be accurately estimated or
predicted, and (iii) providing an up to date review of the factors
that can impact gamete quality in teleosts.
2. Molecular and cellular characteristics of sh gametes
2.1. Egg
The unfertilized egg or female gamete is an oocyte arrested
in metaphase of the second meiotic division. This metaphase 2
oocyte is the nal product of the oogenetic process that oc-
curred within the ovary throughout oogenesis (Tata, 1986).
As a consequence, the coordinated assembly of the egg can
last for a very long time, up to several years in some species.
Hence, the incorporation, synthesis, and processing of egg com-
ponents that occur during oogenesis play a key role in the
coordinated assembly of a good quality oocyte that will, once
fertilized, develop into a normal embryo. The main characteris-
tics of egg assembly, including the increase in egg volume
resulting from massive incorporation of yolk proteins during
vitellogenesis have been extensively studied and previously re-
viewed (Wallace and Selman, 1981, 1985; Brooks et al., 1997;
Patino and Sullivan, 2002; Mommsen and Korsgaard, 2008)
0016-6480/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2009.02.011
* Corresponding author. Address: Fish Reproduction Group, INRA SCRIBE UR1037,
Campus de Beaulieu, 35042 Rennes Cedex, France. Fax: +33 2 23 48 50 20.
E-mail address: Julien.Bobe@rennes.inra.fr (J. Bobe).
General and Comparative Endocrinology 165 (2010) 535548
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(see also the oogenesis review in this issue). Besides yolk pro-
teins, the ovulated oocyte (unfertilized egg) contains many
other components such as maternal mRNAs, proteins, vitamins
and hormones. To date, our knowledge of the hormonal con-
tent of unfertilized eggs is limited. Several studies reported
the presence of thyroid hormones (Tagawa and Hirano, 1987),
cortisol (Hwang et al., 1992) and several sex steroids (Feist
et al., 1990). Despite several studies aiming at manipulating
the hormonal content of the egg, the function of the hormonal
egg content remains poorly investigated to date. Maternally-
inherited mRNAs and proteins accumulate in the oocyte
throughout oogenesis (Tata, 1986; Howley and Ho, 2000; Pele-
gri, 2003). After fertilization, those maternal factors support
early embryonic development until activation of zygotic tran-
scription and thus play a pivotal role during early embryogen-
esis. The initiation of zygotic transcription occurs during the
maternal-embryo transition (MET). In sh, as in other lower
vertebrates, MET occurs at the mid-blastula stage and is also
known as mid-blastula transition (MBT) (Kane and Kimmel,
1993). In rainbow trout (Oncorhynchus mykiss), inhibition of
maternal mRNA translation using cycloheximide resulted in de-
layed embryonic cleavage (Nagler, 2000). In addition, the func-
tion of maternal factors in the developing embryo depends on
a precise plan of storage and localization in the oocyte that is
initiated during oogenesis. Recent molecular analyses using
newly available genomic tools have shown that some maternal
mRNAs exhibited a differential abundance in eggs of varying
quality (Aegerter et al., 2005; Bonnet et al., 2007b). The nature
and abundance of maternal mRNAs stored in the eggs thus ap-
pears to be important to ensure a full developmental compe-
tence of the oocyte. Similarly, the spatial distribution of
specic maternal mRNAs within the oocyte (e.g. animal pole)
is important to specify the dorso/ventral axis of the embryo
(Bally-Cuif et al., 1998; Howley and Ho, 2000). It was also
shown in zebrash that the micro RNA miR-430 could acceler-
ate the deadenylation of target mRNAs, including numerous
maternal mRNAs thus suggesting its participation in maternal
mRNA clearance during embryogenesis (Giraldez et al., 2006).
These recent studies have provided new hints on the molecular
mechanisms involved in the regulation of egg quality in sh.
For instance, a sharp decrease in the mRNA levels of nucleo-
plasmin (npm2) is observed during egg post-ovulatory ageing
in rainbow trout (Oncorhynchys mykiss), a period when egg
quality progressively decreases (Aegerter et al., 2005). Interest-
ingly, nucleoplasmin is a maternal-effect gene critical for nu-
clear and nucleolar organization and embryonic development
(McLay and Clarke, 2003; Burns et al., 2003). Similarly, a dif-
ferential abundance of prohibitin 2 (phb2) mRNA was observed
betweens eggs of varying quality in rainbow trout (Bonnet
et al., 2007b). Phb2 is a highly conserved protein originally
know as a chaperone protein also involved in a broad range
of cellular functions (Mishra et al., 2006). In addition to mater-
nal mRNAs, the ovulated oocyte also contains proteins that
have been synthesized during oogenesis. Recently, proteomic
analysis of the developing oocyte has led to the identication
of protein in the full-grown zebrash (Danio rerio) and sea
bream (Sparus aurata) oocyte (Knoll-Gellida et al., 2006; Ziv
et al., 2008). These studies have shed light on the protein rep-
ertoire of the unfertilized egg. However, the contribution of
identied proteins in the regulation of egg quality requires fur-
ther investigation. Very recently, a protein differentially ex-
pressed in European sea bass (Dicentrarchus labrax) eggs of
poor and high quality was identied (Crespel et al., 2008). Fur-
ther investigations are now required to functionally character-
ize the participation of this candidate protein in the
regulation of sh egg quality.
2.2. Sperm
2.2.1. Sperm membrane characteristics
The plasma membrane of the spermatozoa head tightly overlays
the nucleus and only a thin cytoplasmic layer remains between the
plasma membrane and the nucleus. At the midpiece level, some
folding of the plasma membrane along the axoneme root results
in the superposition of membrane layers. In some species, includ-
ing Salmonidae and Percidae, the plasma membrane surrounding
the axoneme presents paired lateral extensions resembling a heli-
coid n all along the tail. Such lateral ribbons are not observed in
Labridae, for example, whereas in Sparidae, 2, 1 or no lateral rib-
bons are reported, depending on the species (reviewed by Lahnste-
iner and Patzner, 2008).
The spermatozoa plasma membrane plays a major role in motil-
ity activation. The sensing of ionic changes, responsible for the ini-
tiation of agellar beating upon sperm release in water, (see
Section 2.2.5.) takes place across this membrane. Many ion chan-
nels are described in sh sperm plasma membrane and progestin
receptors involved in motility were characterized in seatrout
(Cynoscion nebulosus) (Tubbs and Thomas, 2008). As spermatozoa
do not bear an acrosome in teleosts, sperm plasma membrane is
also a key component of gamete fusion, and some components
were described in rainbow trout: GM3, a ganglioside localized in
the sperm head, was shown to be involved in sperm binding to
eggs (Yu et al., 2002). Some uncharacterized proteins localized in
the head region were also shown to be involved in fertilization
(Beck et al., 1992). At the biophysical level, membrane lipids will
determine membrane uidity whereas both proteins and lipids
will contribute to the overall permeability to water and ions. Lipids
in rainbow trout sperm plasma membrane were extensively stud-
ied (Muller et al., 1994; Labbe et al., 1995; Pustowka et al., 2000;
Muller et al., 2008). The molar cholesterol to phospholipid ratio
ranges between 0.4 and 0.6. Among phospholipid classes, phospha-
tidyl choline represents 50% of the total phospholipids, with a
localization in the outer leaet, whereas phosphatidyl serine
(10%) and phosphatidyl ethanolamine (30%) are enriched in the in-
ner leaet. The polyunsaturated fatty acid 22:6n 3 is well repre-
sented (more than 10% of the total fatty acids), leading to an
unsaturated to saturated ratio as high as 1.30. From the low plasma
membrane permeability to water assessed in Labbe and Maisse
(2001), it can be extrapolated that rainbow trout spermatozoa do
not possess aquaporins which would facilitate water penetration.
2.2.2. Cytoskeleton
Most studies on sperm cytoskeleton are focused on the agellar
axoneme. In most sh species, the axoneme bears the typical
arrangement of nine pairs of peripheral microtubules and one pair
of central microtubules, although some species do not possess the
central microtubules such as the eel (Anguilla anguilla) (Gibbons
et al., 1983). Each microtubule is organized into a cylinder whose
walls are made by 13 adhering protolaments, the later being com-
posed of polymerized tubulin dimeres. The sliding of one doublet
from the adjacent doublet is mediated by inner and outer rows
of dynein arms acting as force-generating ATPases with rotating
arms. When the dynein arms attached to a given microtubule dou-
blet undergo an ATP-dependent bindingbendingunbinding to
the adjacent microtubule doublet, the resulting sliding-induced
tension initiates agellar curvature. This sequence is proposed to
be the basic step towards agellar beating (see the extensive
description by Cosson, 2008a). Actin is also found closely associ-
ated to the plasma membrane.
2.2.3. Nucleus
Sperm nucleus organization is very different depending on sh
species. Most nuclei show an invagination in which the axoneme
536 J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548
will be anchored. As a consequence, nucleus shape will determine
the strength of agellar attachment to the head. In rainbow trout
(Billard, 1983) and in other Salmonidae, the elongated nucleus pre-
sents an invagination whose depth is about one third of the nu-
cleus length. In other species, this invagination can reach more
than two third of the nuclear length (Sweetsh (Plecoglossus altiv-
elis) (Ohta et al., 1993); red mullet (Mullus barbatus) (Lahnsteiner
and Patzner, 1998)) or it can be only a minor depression as in loach
(Misgurnus anguillicaudatus) and goldsh (Carassius auratus) (Ohta
et al., 1993).
Nuclear proteins in sh sperm will also vary according to the
species. As an example, sea bass (Dicentrarchus labrax) and North-
ern pike Esox lucius nuclei contain only protamines whereas striped
red mullet (Mullus surmuletus), carps (Cyprinus carpio, Ctenophar-
yngodon idella), goldsh and sea bream (Sparus aurata) nuclei con-
tain only histones (Munoz-Guerra et al., 1982; Saperas et al.,
1993a, 1993b), and rainbow trout nuclei contain both protamines
and histones (Avramova et al., 1983; Christensen et al., 1984). It
is accepted that nucleus protein types will determine the extent
of chromatin condensation. Chromatin condensation in turn is
known to enhance DNA resistance to chemically or mechanically-
induced damages. It is likely that species differences observed in
sperm DNA stability are linked to species differences in chromatin
organization, although this issue has not been extensively studied
in sh spermatozoa.
2.2.4. Mechanism responsible for the initiation of sperm motility
In Salmonidae, motility of testicular spermatozoa cannot be
activated (Morisawa and Morisawa, 1986) whereas during sperma-
tozoa migration down the sperm ducts, spermatozoa acquire the
potential for motility activation (Morisawa et al., 1993; Koldras
et al., 1996). This maturation was shown to be caused by the semen
pH increase (Morisawa and Morisawa, 1988), leading to an in-
crease in intracellular AMPc (Miura et al., 1992; Morisawa et al.,
1993). This maturation event was poorly investigated in other spe-
cies, but it is often admitted that storage of testicular spermatozoa
in a buffer, at or above pH 8, favours the sperms ability to respond
to the motility initiation signal.
When mature spermatozoa are released in the external med-
ium, extracellular ionic changes induce motility activation (re-
viewed by Morisawa, 1994). In most species, difference in
osmolality between seminal plasma and water is the main trigger
of sperm motility. In Salmonidae however, the decrease in extra-
cellular K
+
is the sperm motility activator (Morisawa and Suzuki,
1980). Some egg factors can also stimulate motility hyper activa-
tion as shown in herring (Clupea palasii) (Morisawa et al., 1992;
Oda et al., 1998). The molecular events making a link between io-
nic changes and motility initiation were recently reviewed by Ina-
ba (2008). The cascade was especially well studied in Salmonidae,
but also in Cyprinidae. Dilution of external K
+
induces intracellular
K
+
efux and intracellular Ca
2+
increase. This triggers membrane
hyperpolarization. Adenylate cyclase activation, and cAMP in-
crease were reported in Salmonidae, but not in Cyprinidae (Krasz-
nai et al., 2000). All these events have been demonstrated to be
involved in motility activation (Morisawa and Okuno, 1982; Mor-
isawa and Ishida, 1987; Boitano and Omoto, 1992; Kho et al.,
2001). Again, the link between cAMP increase and motility initia-
tion at the axoneme level was mainly investigated in Salmonidae.
Such a link involves a complex phosphorylation/dephosphoryla-
tion sequence whose actors have been only partially characterized
so far. This includes the cAMP-dependant phosphorylation of the
15 kDa MIPP (movement-initiating phosphoprotein, (Morisawa
and Hayashi, 1985; Hayashi et al., 1987; Jin et al., 1994a, 1994b),
of the PKA (Itoh et al., 2003), and of the 22 kDa dynein light chain
(Inaba et al., 1998, 1999). Regulation of protein phosphorylation by
proteasomes was also demonstrated (Inaba et al., 1998; Ohkawa
et al., 1997; Inaba et al., 1993). How the whole network will ulti-
mately lead to microtubule sliding and movement initiation is still
under investigation.
2.2.5. Sperm energetics
Spermatozoa energy metabolism provides for the basal energy
demand in quiescent conditions (storage in the male ducts, in vitro
storage) and for the high energy demand upon motility activation
(reviewed by Ingermann, 2008). Spermatozoa capacity to metabo-
lize exogenous and/or endogenous substrates will mostly depend
on the sh reproductive strategy. Spermatozoa fromspecies under-
going internal fertilization will be able to metabolize extracellular
substrate, a feature similar to that found in mammals and birds.
One reason is that some substrates can be found in the female uid
in which spermatozoa are released. This is true for Guppy (Poecilia
reticulata) and Surfperch (Cymatogaster aggregata) spermatozoa
where glycolysis in the presence of extracellular glucose was dem-
onstrated (Gardiner, 1978). On the contrary, spermatozoa fromspe-
cies undergoing external fertilization cannot rely on the medium to
provide for their energy supply. This may explain why these cells
present a poorer ability to metabolize extracellular substrate. In
rainbow trout spermatozoa, no extra cellular glucose metabolism
was detected (Terner, 1962). However, this poor afnity for glucose
can be compensated for by a more efcient tricarboxylic acid incor-
poration, as demonstratedinseveral species withextracellular pyru-
vate, lactate or glyoxylate (Terner andKorsh, 1963a, 1963b; Mounib,
1967). Spermatozoa inability to metabolize extracellular glucose is
proposed to be due to a poor membrane permeability to glucose
(Gardiner, 1978) rather than to some deciency in glucolysis en-
zymes, as enzymatic capacity for intracellular glycolysis was dem-
onstrated in Salmonidae and Cyprinidae (Lahnsteiner et al., 1992,
1993). Upon motility activation when spermatozoa are released in
water, spermatozoa face a huge energy demand to induce and sus-
tain agellar movement. Adenylate cyclase will mediate cAMP in-
crease at the onset of motility activation and dynein ATPase
activity will allow axonemal microtubules sliding during move-
ment. These processes are greatly responsible for the rapid ATP con-
sumption described in most species such as rainbowtrout (Christen
et al., 1987), commoncarp(Cyprinus carpio) (Perchec et al., 1995), sea
bass, and turbot (Scophthalmus maximus) (Dreanno et al., 1999a,
1999b, 2000). ATP stores accumulated in quiescent spermatozoa
are the most readily available energy supply for sustaining motility
after activation (see the recent review by Ingermann, 2008), espe-
cially in species whose sperm mitochondria present a low basal
capacity for oxidative phosphorylation, such as in Salmonidae.
Maintenance of ATP level during motility can also be provided by
creatine phosphate (Robitaille et al., 1987; Dreanno et al., 1999b)
and to some extent by monosaccharides as shown by Lahnsteiner
et al. (1992) in the chub (Leuciscus cephalus).
At the morphological level, the number of mitochondria is var-
iable depending on the species. Salmonidae spermatozoa possess a
single ring-shaped mitochondria, resulting from the fusion of sev-
eral mitochondria, whereas Blenniidae and Labridae have up to 6
mitochondria (see the very well documented review of Lahnsteiner
and Patzner (2008)). However, as reported by Lahnsteiner and
Patzner (2008) no relationship between the number or arrange-
ment of mitochondria and functional differences in energetic
capacity of the spermatozoa could be highlighted in interspecies
comparisons.
3. What is egg/sperm quality and how can it be estimated or
predicted?
From a biological standpoint, the quality of a gamete can be de-
ned as its ability to fertilize or to be fertilized, and subsequently
develop into a normal embryo. However, the quality of gametes
J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548 537
can also be dened differently depending on the specic biotech-
nological applications that the gametes are to be used for, such as
cryobanking, nuclear transfer or androgenesis. Several types of crite-
ria can thus be used to quantitatively estimate egg and sperm qual-
ity; some of them being species-specic or application-specic. In
the present chapter we will reviewthe biologically relevant and bio-
technologically relevant parameters that can be used to estimate
gamete quality. We will also discuss criteria that can be used, for in-
stance in aquaculture, to tentatively predict egg and sperm quality.
3.1. Quality estimators prior to fertilization
3.1.1. Egg
The size and appearance of unfertilized eggs can tentatively be
used to evaluate or estimate the overall developmental potential of
the eggs after fertilization. For instance, the size of the egg was
sometimes considered to be benecial for the future development
of the embryo, especially in ecological studies. However, despite
the large variations in egg weight that can be observed in sh with-
in the same species, very little data can support this hypothesis. In
rainbow trout for instance, it was shown that under normal condi-
tions of temperature, post-ovulatory ageing, and husbandry prac-
tices, small eggs produce similar rates of fertilization than larger
ones (Bromage et al., 1992).
The appearance or morphology of unfertilized eggs has also
sometimes been used to estimate the developmental potential of
the egg. In brown trout (Salmo trutta) females caught in the wild
and transferred to an aquaculture facility, the distribution of lipid
droplets in the unfertilized eggs was shown to be reective of em-
bryo survival at the eyed stage (Mansour et al., 2007). However, the
use of such estimators is limited under normal hatchery conditions
and the lack of a consistent relationship between the distribution
of lipid droplets and egg quality in hatchery-raised rainbow trout
was recently stressed (Ciereszko et al., 2009). Indeed, while major
morphological changes can routinely be observed in specic cases,
such as excessive post-ovulatory ageing times, signicant differ-
ences in egg quality are also observed that can not be linked to
simple morphological criteria.
Egg quality can also tentatively be predicted using indirect mea-
surements such as physico-chemical parameters of ovarian or coe-
lomic uid in which the eggs are bathed. Several studies have
shown that low pH values of coelomic or ovarian uid have been
associated with reduced egg quality. In turbot (Scophtalmus maxi-
mus), the drop of egg quality during egg post-ovulatory ageing is
associated with a drop of ovarian uid pH (Fauvel et al., 1993).
Similar observations were made in rainbow trout (Lahnsteiner,
2000; Aegerter and Jalabert, 2004). Indeed, proteomic analyses
have shown that vitellogenin fragments originating from the eggs
accumulate in coelomic uid during post-ovulatory ageing (Rime
et al., 2004). The drop in coelomic uid pH during post-ovulatory
ageing could therefore be induced by the presence of egg content
in the uid. It was indeed shown in rainbow trout that broken eggs
could decrease the pH of coelomic uid (Dietrich et al., 2007). It is
however noteworthy that the pH variation range during the post-
ovulatory ageing process is limited (Lahnsteiner, 2000; Aegerter
and Jalabert, 2004). In addition, no signicant linear regression be-
tween pH and embryonic survival could be identied (Aegerter and
Jalabert, 2004). In turbot, however, ovarian pH was correlated with
the fertilization rate but not with survival rate at the 4-cell stage.
Taken together these observations suggest that egg quality can
not be accurately predicted using ovarian or coelomic uid pH,
especially when low egg quality is not induced by post-ovulatory
ageing or when other factors are involved (e.g. temperature)
(Aegerter and Jalabert, 2004). In contrast, low pH could be useful
to estimate post-ovulatory ageing, especially in some species such
as the turbot (Fauvel et al., 1993).
3.1.2. Sperm
3.1.2.1. Sperm motility. Apart from fertilization ability, sperm
motility is an integrative quality parameter which will combine
the quality of several cellular compartments responsible for motil-
ity activation and progressive movement sustaining. Among these,
plasma membrane mediates the ionic motility activation signal
and maintains the permeability barrier in order to prevent leakage
of important intracellular components. Mitochondria activity re-
sults in adequate energy stores, and axoneme structure and com-
position is responsible for the efciency of spermatozoa
movement. Motility analysis is very useful to compare different
experimental conditions such as collecting procedures, sperm dilu-
tion medium, and sperm storage conditions. Sperm motility is also
extensively used to assess the effect of biotechnologies such as
cryopreservation. Methods used to analyze sh sperm motility
were recently reviewed by Cosson (2008b) and the reader is re-
ferred to this detailed book chapter for a comprehensive descrip-
tion of the important steps in the determination of sperm
motility. The simplest method for a rapid screening of sperm qual-
ity is the direct observation of the percentage of motile spermato-
zoa with a phase contrast or a dark eld microscope using low
magnication (10 to 20 lenses). One possible source of discrep-
ancies in this kind of analysis arises from sperm density during
observation. Too many or too few cells in the microscope eld will
lead to respectively overestimating or underestimating the motil-
ity percentage. Another source of underestimation lies in the stick-
ing of the spermatozoa to the glass slide, and this can be easily
prevented by adding proteins (such as BSA) to the activating med-
ium. In all, this simple motility assessment is reliable for analysis of
sperm motility duration and that of global sperm motility capacity.
In computer-assisted sperm analysis (CASA) (Kime et al., 1996;
Rurangwa et al., 2001), video recording of the sperm movement
and analysis of several movement parameters will allow sperm
morphology to be assessed, together with sperm velocity, head
movement and overall percentage of motile sperm. Such analyses
produce a great deal of data, which is often difcult to correlate
to specic sperm function. The method is however very useful in
comparing samples, especially using the clustering analysis devel-
oped by Martinez-Pastor et al. (2008) where changes in sperm
cluster distribution within ejaculates are analyzed. Stroboscopic
analysis of agellar beating using dark eld microscope and higher
magnication lenses (Cosson et al., 1985) allowed for the explora-
tion of the nest events along the axoneme (see the recent reviews
of Cosson, 2008a, 2008b) together with the pattern of agellar beat
frequency over motility duration. However, incase some cellular
defects should take place, parameters other than motility will have
to be explored in order to describe more precisely the affected cel-
lular functions.
3.1.2.2. Other estimators. Other sperm quality estimators are often
based on morphological, biochemical and subcellular analyses. At
the subcellular level, plasma membrane quality is one estimator
of sperm quality. Plasma membrane integrity can be analyzed indi-
rectly by the assessment of cytoplasmic components (enzymes,
metabolites) whose release into the seminal uid will reect some
alteration in the membrane permeability barrier (Ciereszko and
Dabrowski, 1994; Zilli et al., 2004). One more direct measurement
of plasma membrane quality is based on the measurement of plas-
ma membrane impermeability to hydrophilic dyes. When the plas-
ma membrane is altered, these dyes, such as propidium iodide (PI)
or ethidium homodimer, will enter the cell and label the nucleus
DNA. The percentage of cells with a labeled nucleus (altered plas-
ma membrane) can be assessed by a ow cytometric analysis of
the sperm population (Ogier de Baulny et al., 1997, 1999). Another
plasma membrane quality test is based on membrane resistance to
osmotic shock (Marian et al., 1993). This test indistinctly reects
538 J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548
plasma membrane mechanical resistance to stretching upon water
entry and plasma membrane permeability to water. In this test,
exposure of spermatozoa to a hypo-osmotic media will induce
plasma membrane swelling and ultimately PI penetration. This
estimator was used to study the effect of broodstock rearing salin-
ity (Labbe and Maisse, 2001), or that of changes in plasma mem-
brane cholesterol content (Muller et al., 2008).
At the biochemical level, energy metabolism is also used as a
sperm quality estimator. Most studies explored nucleotide concen-
tration (ATP-ADP-AMPc, creatine phosphate (Geffen and Frayer,
1993; Saudrais et al., 1998; Zietara et al., 2004; Zilli et al., 2004),
and respiration activity (Lahnsteiner et al., 1998). Presence of ac-
tive mitochondria was also measured on live spermatozoa with
uorescent labeling of the negatively charged mitochondria and
cell sorting analysis (Ogier de Baulny et al., 1995, 1997).
Quality of the nucleus DNA is increasingly explored with re-
gards to the risk raised by sperm handling or sperm exposure to
damaging chemical agents (Labbe and Maisse, 2001; Zilli et al.,
2003; Ciereszko et al., 2005). Comet assay is the most commonly
used method to assess DNA stability in sh. This method is based
on the ability of damaged DNA strands to migrate faster than intact
DNA in an acidic electrophoresis gel, leading to the occurrence of a
comet-like tail preceding the nucleus DNA bulk. Therefore, the
more DNA that is damaged, the bigger the comet tail is whereas
nuclei with an intact DNA show a homogenous round shape (Labbe
et al., 2001).
It should however be stressed that whatever the sperm quality
parameter, it is often very difcult to correlate sperm grading in a
quality test to the fertilization rate obtained with the same sperm.
One reason lies in the fact that fertilization of one egg requires only
one good spermatozoon taken from a huge sperm population. In-
deed, 2002000 eggs are often fertilized with a suspension of
10
4
10
10
spermatozoa. Sperm quality estimators give a picture of
the whole sperm population in which the weight of few spermato-
zoa is virtually detectable. As a consequence, improving the quality
of a small fraction of the sperm population will be sufcient to sig-
nicantly improve the fertilization success, although such
improvement may not be detectable when assessing quality esti-
mators such as sperm motility or membrane quality. Another rea-
son is that sperm fertilizing ability depends upon many cellular
functions. It is therefore difcult to determine which combination
of estimators can faithfully integrate the weight of every required
cellular parameter. However, this lack of correlation between the
fertilization rate and quality estimators should not conceal the
importance of sperm quality analyses. Quality estimators focus
on specic cellular functions whose improvement can thereby be
experimentally and individually investigated. This should ulti-
mately increase the size of the sperm population in which all the
requirements for sperm fertilization are met (Lahnsteiner et al.,
1998; Zilli et al., 2004). To conclude, in vitro parameters should
not be used as substitute estimators of sperm fertilization ability,
but as partial descriptors of this ability.
3.2. Fertilization success and early estimators
Fertilization success is probably one of the earliest estimators
that can be recorded to accurately estimate egg quality and it is
the most integrative estimator of sperm quality. Indeed, the ability
to fertilize or be fertilized is one of the key components of gamete
quality. In some species, recording of fertilization rates is relatively
easy. This is especially true for species with transparent eggs such
as cod (Gadus morua), turbot and yellowtail ounder (Limanda fer-
ruginea). This is, in contrast, much more difcult in sh species
with opaque eggs. In rainbow trout for instance, it is necessary to
x the egg and subsequently stain it with specic dyes (Fig. 1).
In the past, many studies have used the term fertilization rate
in an improper manner as it corresponded in fact to embryonic sur-
vival at much later stages. This is especially true in sperm studies
where sperm fertilizing ability is assessed by the percentage of
developing embryos recorded at the easiest rather than at the ear-
liest developmental stage. Thus, the fertilization rate is a good esti-
mator of early developmental success but, depending on the
species, not necessarily easy to monitor.
It is also obvious that fertilization success is not necessarily
reective of further developmental success, as shown in several
marine species (Shields et al., 1997). After fertilization, the embry-
onic cell starts to divide. The shape of the rst embryonic cells and
the occurrence of abnormal cell division patterns have also been
used as quality estimators in species with transparent eggs. In yel-
low tail ounder, abnormal cleavage resulted in early embryonic
mortalities (Avery and Brown, 2005). It was also shown that blas-
tomere morphology was correlated with developmental success at
later stages (Shields et al., 1997; Kjrsvik et al., 2003). In Atlantic
cod, a recent study showed that abnormally cleaving embryos
had signicantly higher mortality than normal embryos. However,
both normal and abnormal eggs had similar mortality-rate trends
and hatching success was not signicantly different between nor-
mal and abnormal eggs. Together, these results indicate that an
abnormal cleavage pattern does not necessarily result in embry-
onic failure (Avery et al., in press). Finally the buoyancy of pelagic
eggs is often better for eggs able to develop normally as shown in
the red sea bream (Pagrus major) (Sakai et al., 1985) and other spe-
cies (Kjrsvik et al., 1990) even though this does not hold true for
all species (Brooks et al., 1997).
3.3. Embryonic survival at several key steps
Embryonic survival at a specic embryonic step is one of the
most common ways of characterizing the ability of the fertilized
egg to successfully develop. Survival can thus be assessed at spe-
Fig. 1. Normal (A) and abnormal (B) early embryonic cleavage in rainbow trout (Oncorhynchus mykiss).
J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548 539
cic stages such as the eyed stage, hatching, and yolk-sac resorp-
tion stage, which can be monitored in most sh species. It is also
noteworthy that monitoring survival at successive developmental
steps can be extremely valuable to characterize the timing of
embryonic mortalities that can signicantly differ between differ-
ent experimental treatments (e.g. broodstock breeding conditions,
effect of sperm cryopreservation) (Kopeika et al., 2003; Bonnet
et al., 2007a).
3.4. Embryonic malformations
The occurrence of embryonic or larval malformations is a valu-
able tool to fully characterize the developmental potential of fertil-
ized eggs. In rainbow trout, it was shown that specic breeding
conditions of brood sh induce specic or predominant types of
malformations in the offspring (Bonnet et al., 2007a). For instance,
long-term post-ovulatory ageing of the eggs has been associated
with a high occurrence of the cyclop malformation (Aegerter
et al., 2004; Bonnet et al., 2007a). Larval malformation can also re-
veal sperm damage, especially after cryopreservation, as shown by
Horvath and Urbanyi (2000) in African catsh (Clarias gariepinus).
Such an effect was however not shown in the rainbow trout (Labbe
et al., 2001). One explanation is that some species may have more
resistant DNA than others. It is also likely that, in many cases,
sperm DNA defects were repaired after fertilization by the egg re-
pair system, as demonstrated by Kopeika et al. (2004) in the loach
(Misgurnus fossilis).
4. What are the factors that can affect egg or sperm quality?
4.1. Nutrition
Broodstock nutrition is an important factor susceptible to affect
not only fecundity and gametogenesis but also gamete quality, and
existing work has been extensively reviewed (Kjrsvik et al., 1990;
Brooks et al., 1997; Izquierdo et al., 2001). It is well accepted in the
literature that broodstock nutrition can signicantly impact repro-
ductive performance and this topic has been specically reviewed
by Izquierdo and co-workers (Izquierdo et al., 2001). The denition
of reproductive performance is very broad and includes many
types of parameters including egg size, larval size, timing of repro-
duction season, puberty, fecundity, plasma levels of reproductive
hormones, and spawning frequency. In contrast, data on the spe-
cic effects of broodstock diet on gamete quality are more limited,
and sometimes controversial. Indeed, Brooks and co-workers have
previously indicated that the evidence that diet can directly affect
egg quality is very limited (Brooks et al., 1997). According to these
authors, the different methods used to evaluate egg quality and the
different timings of provision of the studied diet during the shs
life, make it difcult to identify any real improvements that are a
function of the diet alone. In the present section, we will focus
on the effects of broodstock nutrition on the developmental capac-
ities of the egg once fertilized, and on the fertilization ability of the
sperm.
4.1.1. Eggs
While specic data on egg fertilization and/or developmental
success are scarce, many studies have shown that food restriction
can have major effects on spawning success, gametogenesis, egg
and larvae size as previously reviewed (Izquierdo et al., 2001). Sim-
ilarly, many studies have shown signicant effects of diet compo-
sition on fecundity, spawn quantity, spawn frequency, eicosanoid
production, steroid hormone levels, and gonadotropin-induced
maturation. Other investigations (also reviewed in Izquierdo
et al., 2001) have shown that some components of broodstock diet
were necessary to ensure a normal development of the embryo. For
instance, suboptimal levels of vitamin E resulted in reduced larval
survival and increased developmental abnormalities (reviewed in
Izquierdo et al., 2001). The detrimental effect of a diet with insuf-
cient amounts of vitamin A and E has been recently reviewed
(Palace and Werner, 2006) and will not be discussed further here.
It seems however well accepted that vitamin A is important for
embryo and larval development (Izquierdo et al., 2001; Palace
and Werner, 2006). Similarly, suboptimal levels of ascorbic acid
in the diet can lead to low embryonic survival at the eyed stage
in rainbow trout (Blom and Dabrowski, 1995). In contrast, experi-
mental data on the effect of the carotenoid content of broodstock
diet on egg quality are controversial (Izquierdo et al., 2001) and
will not be further discussed here. Finally, essential fatty acids
are also required to ensure normal embryonic and larval develop-
ment and deciencies or unbalanced diets generally result in re-
duced egg viability, low embryonic survival, and embryonic
malformations (Leray et al., 1985; Izquierdo et al., 2001).
In summary, it seems clear that major food restriction or long-
term deciencies in essential components, such as vitamins or
essential fatty acids, will impair broodsh fecundity, egg quality
and embryo development. However, some of the studies aiming
at characterizing the effect of broodstock nutrition on egg quality
have been conducted in species for which nutritional requirements
were not fully characterized, thus leading to reproductive perfor-
mances lower than what had been reported using natural diets.
This is especially true for newly domesticated species, or for spe-
cies that require specic breeding conditions (e.g. photoperiod
and temperature) to complete their gametogenesis and spawning
in captivity. In this case, it is likely that the use of nutrients by
brood sh and their incorporation into the yolk will be affected
by external factors such as photoperiod and temperature that are
known to control vitellogenesis (see Section 4.2 below). For all
the above reasons, it should thus be stressed that many existing
observations on the effect of a specic component of the diet on
reproductive performance could be species-specic and/or experi-
ment-specic.
4.1.2. Sperm
It is an obvious statement that nutrient requirements of the
broodstock are to be met in order to sustain reproductive perfor-
mance, and this includes the provision of essential fatty acids,
mainly unsaturated ones, that the sh cannot synthesize. Gener-
ally, fresh water species including Salmonidae, Cyprinidae, and
eel need both 18:2(n 6) and 18:3(n 3) fatty acids in the diet
while marine species need the more elongated and unsaturated
fatty acids 20:5(n 3) and 22:6(n 3) (Takeuchi and Watanabe,
1982; Watanabe, 1982). It was shown that lipid composition of
the diet affects sperm lipid composition even when the essential
nutritional requirements are met (Labbe et al., 1995; Bell et al.,
1996; Pustowka et al., 2000; Asturiano et al., 2001). Nevertheless,
preferential retention of some polyunsaturated fatty acids (namely
22:6(n 3)) was observed, as if the most important components
were maintained at a specic level in sperm membranes (Labbe
et al., 1995). This stability of a basal fatty acid composition in sper-
matozoa may explain why lipid diet little affected spermatozoa
quality, as shown after cryopreservation of rainbow trout sperma-
tozoa (Labbe et al., 1993; Pustowka et al., 2000). It should be noted
however that in European sea bass (Asturiano et al., 2001),
although sperm motility and fertilization ability was not affected
by the male diet, survival of embryos and larvae was affected. This
indicates a unique and striking long-term effect of diet-mediated
sperm quality.
Vitamins are also components that the sh cannot synthesize
and which should be added to the diet. Ascorbic acid (vitamin C)
is one of these vitamins whose effect on sperm quality was ex-
plored to some extent. Dabrowski and co-workers showed in rain-
540 J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548
bow trout and in yellow perch (Perca avescens) that seminal plas-
ma concentration of ascorbic acid reects vitamin C dietary sup-
plementation. They further demonstrated that low ascorbic acid
levels in the seminal uid impaired sperm concentration and
motility (Ciereszko and Dabrowski, 1995), sperm fertilization abil-
ity (Dabrowski and Ciereszko, 1996) and the hatching rate of em-
bryos sired by spermatozoa from the decient males (Dabrowski
and Ciereszko, 1996; Lee and Dabrowski, 2004).
4.2. Environmental factors: temperature, photoperiod, salinity
4.2.1. Temperature
Temperature is known to have a major impact on egg quality.
This is especially true during the reproductive season and spawn-
ing. Both high and low temperature can have a negative impact
on egg quality, depending on the species. In rainbow trout, tem-
peratures of 15 C and above can signicantly reduce egg quality
(Pankhurst et al., 1996; Aegerter and Jalabert, 2004) and in brook
trout (Salvelinus fontinalis) temperatures of 12 C and above sig-
nicantly reduced hatching rate (Hokanson et al., 1973). In those
salmonid species, high temperatures signicantly increased the
incidence of embryonic deformities (Hokanson et al., 1973;
Aegerter and Jalabert, 2004) and triploid fry (Aegerter and Jala-
bert, 2004). In contrast, the quality of Arctic charr (Salvelinus alpi-
nus) eggs obtained from females held at 10 C is signicantly
reduced in comparison to eggs obtained from females held at
5 C (Gillet et al., 1996). In the Atlantic halibut (Hippoglossus hip-
poglossus) high water temperature during the reproductive season
resulted in signicantly lower fertilization and hatching rates
(Brown et al., 1995). Similarly, the temperature experienced by
female Wolfsh during the breeding season signicantly inu-
enced egg quality (Tveiten et al., 2001). It has also been shown
in Atlantic salmon (Salmo salar) that suboptimal temperatures
during vitellogenesis can negatively impact egg quality (King
et al., 2003). In addition to the negative impact on egg quality
by itself, high temperatures also increase the drop of egg quality
resulting from the post-ovulatory ageing process. In several sal-
monid species, high temperatures result in a much faster and
stronger decrease of egg quality in comparison to optimal tem-
perature (Gillet, 1991; Aegerter and Jalabert, 2004). Finally, out
of season spawning resulting in ovulation at suboptimal temper-
atures has led to the production of signicantly reduced egg qual-
ity in several species (Devauchelle et al., 1988; Davies and
Bromage, 2002; van der Meeren and Ivannikov, 2006). In these
studies, it is however difcult to separate the specic effects of
temperature and photoperiod on egg quality.
There are little reports to our knowledge on sperm quality
changes in response to broodstock rearing temperature. The rear-
ing temperature of the sh is known to modulate their metabo-
lism, and some adaptive responses to abrupt temperature
changes were described in organs bearing a key role for sh sur-
vival. Erythrocytes, brain, liver or muscle membranes respond to
temperature changes by changing lipid composition (see the re-
view by Hazel and Williams, 1990). In rainbow trout, spermatozoa
from sh transferred to a temperature lower than their rearing
temperature (cold acclimation) displayed a lipid composition
slightly different from that of warm-acclimated sh (Labbe and
Maisse, 1996). Additionally, sperm quality after freezethawing
was better in cold-acclimated sh in comparison to warm-accli-
mated sh. The effect of this temperature acclimation was no long-
er detected 2 months after the temperature change. However,
these temperature effects were not tested on fresh spermatozoa.
Whether the positive temperature effect was due to the lipid
changes, namely cholesterol content changes, was explored (Mul-
ler et al., 2008), but no straightforward role of cholesterol was
reported.
4.2.2. Photoperiod
As discussed above, the effect of photoperiod on egg quality is
difcult to study, because manipulation of photoperiod often re-
sults in the modication of other parameters throughout oogene-
sis, including during late oogenesis. For instance, age and body
size of the broodsh, water temperature, and length of oogenesis
can be different from the normal-photoperiod control. In many sh
species, including salmonids, the decrease of egg quality observed
after out of season spawning has often been attributed to subopti-
mal temperature as discussed in the previous section (Breton et al.,
1983; Devauchelle et al., 1988; Davies and Bromage, 2002; van der
Meeren and Ivannikov, 2006). Nevertheless, it was shown in rain-
bow trout that articial photoperiod resulting in an advanced
spawning in JuneJuly resulted in a signicant decrease of egg
quality even if the water temperature was kept at 12 C during
the reproductive season (Bonnet et al., 2007a). Egg quality defects
were characterized by signicantly lower survival of the progeny
at eyeing and yolk-sac resorption and a signicantly increased inci-
dence of deformities, especially abnormal yolk-sac resorption.
Some evidence also suggested that the timing of ovulation during
the rst reproductive season had an impact on egg quality ob-
served after photoperiod-advanced spawning (Bonnet et al.,
2007a). A study conducted in sea bass using 5 different articial
photoperiod regimes showed that egg quality (estimated by the
percentage of oating eggs), hatching rate and survival to rst
feeding were affected by at least some photoperiod regimes (Car-
rillo et al., 1989). Indeed, while mean hatching rate expressed as
a percentage of oating eggs was 84.8 3.3 (mean SE) in the con-
trol group, the 5 different photoperiod regimes resulted in
21.4 2.8, 64.5 9.7, 78.9 7.5, 81.2 6.3, and 83.2 6.7 hatching
respectively. In addition, at least one spawn in which less than 5%
of the eggs were oating (i.e. were of good quality) was found for
each articial photoperiod regime while that never happened in
the control group (Carrillo et al., 1989). In summary, available data
indicate that photoperiod-induced manipulation of spawning data
can negatively impact egg quality. The importance of this negative
effect on egg quality seems highly dependant on the type of photo-
periodregime used. It seems also clear that suboptimal temperature
around spawning time will further increase any negative impact of
the photoperiod regime on the overall quality of the eggs.
4.2.3. Salinity
For species that spend a part of their life in sea water and mi-
grate to fresh water for reproduction, water salinity during the
reproductive season can signicantly affect reproductive success.
In Atlantic salmon, holding females in sea water during the repro-
ductive season resulted in either delayed or blocked ovulation
(Haffray et al., 1995). However, eggs stripped from females held
in sea water and freshwater exhibited similar eyeing rates. In con-
trast, when eggs were fertilized two weeks after ovulation, egg
quality was signicantly reduced in eggs stripped from females
held in sea water. Earlier studies conducted in Coho salmon
(Oncorhynchus kisutch) produced in similar results (Sower et al.,
1982). In contrast, another study showed that transferring Pink
and Coho salmon females captured in estuary or freshwater into
seawater resulted in a limited but signicant decrease in the eye-
ing rate (Werthemer, 1984).
It was reported that the spermatozoa of brown trout reared in
sea water had a higher membrane permeability and/or a lower
membrane resistance to hypo-osmotic stress than spermatozoa
of sh kept in freshwater (Labbe and Maisse, 2001). Interestingly,
sperm motility just after collection was better in the fresh water
group whereas it was better in the sea water group after 3 days
of storage. The role of seminal uid osmotic pressure is proposed
as one parameter involved in the sperm response to the salinity
in the male rearing environment.
J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548 541
4.3. Husbandry practices including spawning induction, egg post-
ovulatory ageing, and gamete handling post-stripping
4.3.1. Spawning induction
Hormonal induction of spawning is widely used in aquaculture
to induce ovulation in species that do not spontaneously ovulate in
captivity, or to synchronize ovulation for practical reasons in other
species. A great deal of information is thus available on spawning-
induction techniques and their efciency in terms of ovulation
rate. In contrast, the impact of spawning-induction techniques on
gamete quality is less documented and sometimes limited to
embryonic survival at early stages (up to eyed stage). During the
nal stages of oogenesis (also referred to as nal oocyte matura-
tion), the follicle-enclosed oocyte progressively acquires its ability
(i) to resume meiosis, and (ii) to subsequently develop into a nor-
mal embryo after fertilization (Bobe et al., 2008). In vertebrates,
these two partially overlapping processes are referred to as oocyte
maturational competence acquisition and oocyte developmental
competence acquisition respectively. It is thus possible to arti-
cially induce meiosis in a meiotically competent oocyte that will
not necessarily result in a fully developmentally competent meta-
phase II oocyte. Indeed, the ovarian stage at the time of spawning
induction will have a major effect of the developmental capacities
of subsequently ovulated oocytes. A study in European sea bass
showed that the quality of eggs obtained using 3 different types
of spawning-induction techniques was always lower (hatching rate
below 50%) than for eggs obtained after natural ovulation (75%
hatching rate) (Fornies et al., 2001). Similarly, in Senegal sole (Solea
senegalensis), the percentage of buoyant eggs used as an estimator
of egg quality was lower (2661%) in hormonally-stimulated fe-
males than in saline-injected females (88.997.8%) (Agulleiro
et al., 2006). In yellowtail ounder, it was concluded that GnRHa-
induction of ovulation tends to decrease egg quality (Avery et al.,
2004). These studies, along with previous work in other species,
clearly show that developmental capacities of the eggs obtained
after spawning induction can be extremely variable and range from
0% hatching rate for some females to hatching rates close to 90% for
other females subjected to similar hormonal induction protocols
(Marino et al., 2003). This high variability is probably linked to
existing differences between females in terms of oogenetic stages
at the time of stimulation and the lack of an accurate morpholog-
ical estimator that could be used to identify the best timing for
hormonal stimulation.
In contrast to what was observed in some marine shes (see
above), the impact of spawning induction seems to have a milder
effect on egg quality in salmonids. In rainbow trout, a study con-
ducted at 14 C showed that hormonal induction of ovulation using
GnRH analogs resulted in rather low fertilization rates (Breton
et al., 1990). Even though the differences were not signicant, fer-
tilization rates were lower in hormonally-stimulated sh (2440%)
than in control females (53%). It is however noteworthy that the
experimentation was conducted at the beginning of reproductive
the season at a time when females are still far from having com-
pleted oocyte developmental competence acquisition. Similarly,
another study carried out before the onset of the spawning season
showed that treatments that could most efciently advance rain-
bow trout ovulation resulted in low embryonic survival at eyed
stage (around 55%) while the progeny of control females and fe-
males subjected to a less efcient treatment resulted in higher sur-
vival rates (7985%) (Billard et al., 1984). In total agreement with
these results, a study carried out in brown trout using several
doses of GnRHa showed that the highest GnRHa (10 lg/kg bw) that
could signicantly advance ovulation resulted in signicantly low-
er embryonic survival at both eyeing and hatching whilst lower
concentrations had no signicant effects on ovulation and survival
rates (Mylonas et al., 1992). In contrast, an experiment conducted
on Arctic charr where 30% of the population had naturally ovu-
lated, resulted in a limited advancement of ovulation and compa-
rable survival at the eyed stage in hormonally-stimulated and
control groups (Gillet et al., 1996). Finally, a study conducted on
rainbow trout at 11.5 C did not show any differences in fertiliza-
tion, eyeing and hatching percentages between the progenies of
control and hormonally-stimulated females (Arabaci et al., 2004).
However, authors did not test differences between cumulative sur-
vival rates at each stage, the maximum being observed in the con-
trol group. These results were conrmed by a recent study
conducted at 12 C in which the lower survival rates observed in
the progeny of hormonally-stimulated females were signicantly
different fromthe control at yolk-sac resorption but not at the eyed
stage (Bonnet et al., 2007a). Interestingly, in contrast to what has
been seen for other factors known to impact egg quality, spawning
induction does not seem to signicantly increase the percentage of
malformed alevins (Bonnet et al., 2007a). Together, these studies
indicate that spawning induction can induce egg quality defects
in salmonids, the strongest effects being observed when the hor-
monal stimulation protocol is applied early in the reproductive
season. The egg quality defects subsequently observed are charac-
terized by lower embryonic survival up to yolk-sac resorption, but
with no specic embryonic malformation. Finally, as previously
hypothesized (Mylonas et al., 1992), existing data indicate that
the physiological status or state of maturity of the female at the
time of hormonal induction is critical for the quality of the subse-
quently ovulated eggs. This physiological status probably corre-
sponds, at least in part, to the process of intrafollicular oocyte
developmental competence acquisition.
As recently reviewed by Alavi et al. (2008), spawning inductors
are very efcient at increasing sperm volume production and den-
sity. Spawning inductors either maintain or increase sperm motil-
ity in all species tested, most likely because the hormonal
stimulation is favorable to sperm maturation in the testis and in
the sperm ducts. Improved fertilization ability was subsequently
observed after hormonal stimulation in both tench (Tinca tinca)
and goldsh (Zheng et al., 1997; Caille et al., 2006).
4.3.2. Egg post-ovulatory ageing
Post-ovulatory ageing occurs between the release of the meta-
phase II oocyte from the follicle at ovulation, and fertilization. Dur-
ing this time period, the oocyte undergoes an overall decrease in its
ability to be fertilized and to subsequently develop into a normal
embryo. Excessive post-ovulatory ageing can lead to major mor-
phological and biochemical changes of the egg. This process has
been referred to as over-ripening. However, in many cases,
post-ovulatory ageing can induce a signicant decrease in egg
developmental capacities without any noticeable morphological
changes in the appearance of the egg. This decrease in egg quality
occurs more or less rapidly depending on the species and is also
highly dependent on external factors (e.g. temperature) and sub-
ject to high inter-female variability. In most sh species, the drop
in egg quality occurs very rapidly after ovulation. In goldsh, fertil-
ization and hatching rates are dramatically reduced within a few
hours after ovulation and after 10 h hatching rate is 0% (Formacion
et al., 1995). In turbot, freshly ovulated eggs showed fertilization
rates above 90%, while eggs retained in the lumen for 24 h exhib-
ited 0% survival at hatching (McEvoy, 1984). In Japanese ounder
(Paralichthys olivaceus), hatching rate reached a signicant maxi-
mum for eggs fertilized 24 h after ovulation and subsequently de-
creased for ageing times greater than 24 h (Hirose et al., 1979). A
rapid decrease in egg quality was also observed in other species
such as bighead catsh (Clarias macrocephalus), cod, Ayu (Plecoglos-
sus altivelus), Oriental weathersh (Misgurnus anguillicaudatus),
and striped bass (Roccus saxatilis) as previously reviewed (Kjrsvik
et al., 1990; Bromage et al., 1994). By contrast, it has been known
542 J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548
for a long time that salmonids are able to hold their eggs for several
days without losing their developmental capacities (Sakai et al.,
1975; Escaffre et al., 1976, 1977; Escaffre and Billard, 1979). In
rainbow trout, eggs can be held in the body cavity for 24 weeks,
but maximum egg quality is reached around 5 days after ovulation
at 1012 C (Sakai et al., 1975; Escaffre et al., 1977; Bromage et al.,
1994; Aegerter and Jalabert, 2004; Bonnet et al., 2007a). In this
species a slight but signicance increase in egg quality is observed
within a few days after ovulation. At 10 C, maximum fry survival
was observed from eggs stripped 46 days after ovulation (Sprin-
gate et al., 1984; Bromage et al., 1994) while a signicant increase
in egg quality was observed from eggs stripped 5 days after ovula-
tion, in comparison to eggs stripped at ovulation, from females
held at 12 C (Aegerter et al., 2005). At this temperature, a limited
but signicant increase in malformed embryos was observed when
eggs were stripped 7 days after ovulation. For longer holding times,
post-ovulatory ageing induces not only a dramatic drop in survival
at the eyed stage, hatching, and yolk-sac resorption stages, but also
a dramatic increase in malformed embryos (Aegerter and Jalabert,
2004; Aegerter et al., 2005; Bonnet et al., 2007a). Indeed, the age-
ing process specically induces an increase in the frequency of the
Cyclops malformation (Bonnet et al., 2007a). Finally, long-term
post-ovulatory ageing is also associated with a signicant increase
in ploidy anomalies of the developing embryo, including an in-
creased frequency of triploids (Aegerter and Jalabert, 2004).
4.3.3. Time of sperm stripping during the breeding season and
stripping frequency
In species exhibiting an annual rhythm of reproduction such as
rainbow trout or turbot, sperm production starts earlier and ends
later, during reproductive season, than egg production (Bykha-
tipoglu and Holtz, 1984; Baynes and Scott, 1985; Suquet et al.,
1998). Most studies have reported a lower total sperm production
at both the beginning and the end of the reproductive season, in
contrast to the steady sperm production observed in the middle
of the season. A limited loss in sperm motility at the beginning
and at the end of the spermiation period was sometimes reported.
Besides, a reduction in sperm fertilizing ability was reported at the
end of the season in sea bass (Dicentrarchus labrax) (Dreanno et al.,
1999c). In contrast, no inuence of stripping frequency on sperm
quality was reported, apart from variations in total sperm produc-
tion (Bykhatipoglu and Holtz, 1984). Nevertheless, it should be
kept in mind that frequent stripping could stress the males and
subsequently induce sperm production and/or quality problems.
4.3.4. Gamete handling post-stripping
After ovulation, eggs can be collected from the females by strip-
ping for the purpose of articial fertilization. The ability of the egg
to be fertilized and subsequently develop into a normal embryo
after ex-vivo holding is highly variable depending on the species
(see post-ovulatory ageing section above). Within a sh species,
the success of egg holding procedures will be highly dependant on
several parameters such as temperature, oxygenation, and charac-
teristics of the medium used. In chum salmon (Oncorhynchus keta)
and Sockeye salmon (Oncorhynchus gorbuscha), unfertilized eggs
could be successfully stored for a few days at approximately 3 C
(Withler and Morley, 1968; Jensen and Alderdice, 1984). In both
rainbow and brown trout, unfertilized eggs could be held in vitro
in coelomic uid at 02 C for at least 57 days (Carpentier and Bil-
lard, 1978; Billard and Gillet, 1981; Babiak and Dabrowski, 2003).
At a higher temperature (12 C) comparable egg qualities were ob-
served when eggs were held for 3 days in vivo, for the broodsh, or
in vitro in coelomic uid (Bonnet et al., 2003). For warm-water spe-
cies such as tilapia (Sarotherodon mossambicus) unfertilized eggs
can successfully be held in coelomic uid for 1.5 h at temperatures
above 16 C (Harvey and Kelley, 1984). In contrast, holding tilapia
eggs at 13 C or below resulted in signicantly lower fertilization
rates. Similarly, eggs held in coelomic uid for 19 h exhibited fer-
tilizations rates below 35%, the maximum being obtained at 20
22 C. In common carp, short-term (5, 30 and 60 min) exposure
of non-activated eggs to low temperatures induced a marked de-
crease of embryonic survival at hatching (Dinnyes et al., 1998).
In another study carried out using koi carps, storage of pre-acti-
vated eggs at 7 C resulted in a clear decrease in embryonic sur-
vival at hatching for holding times greater that 2 h (Rothbard
et al., 1996). In European catsh (Silurus glanis), in vitro holding
of ovulated eggs at 8, 19 and 25 C for 3.5, 8.5 or 12 h resulted in
a dramatic decrease in hatching rates. No survival was observed
at 8 C whilst at both 19 and 25 C holding temperatures resulted
in low embryonic survival and an increased occurrence of mal-
formed larvae at hatching (Linhart and Billard, 1995).
The issue of sperm handling and chilled storage after stripping
was recently reviewed (Bobe and Labbe, 2008). It is generally ob-
served that whatever the sh rearing temperature, sperm handling
at 04 C is preferred in order to reduce cell metabolism. Besides,
most sh spermatozoa are not sensitive to chilling injury (Labbe
et al., 1997), in contrast to mammalian spermatozoa, and they
can therefore be stored at sub-zero temperatures without protec-
tive agents. Dilution of spermatozoa is not always necessary for
short-term storage, but addition of antibiotics will always improve
sperm quality during storage lasting for 1 day or more. Sperm from
most species can be diluted in an appropriate medium which will
satisfy the required pH and osmolality. The ionic composition of
these media is usually based on the seminal plasma characteristics.
For species whose motility duration is short, the dilution medium
is also formulated to maximize the inhibition of motility activation.
4.4. Stress
Data on the effect of broodstock stress on subsequent gamete
quality are scare and different conclusions have been reached
depending on the type and intensity of stressor, the species,
and the period when the stressor was applied. In rainbow trout,
repeated acute stress induced by exposure to emersion during
the 9 month period prior to spawning resulted in lower egg vol-
ume, lower sperm density in milt and lower survival at the eyed
stage, hatching and swim-up (Campbell et al., 1992). Similar re-
sults on egg weight, egg volume and progeny survival were ob-
tained in brown trout and rainbow trout males and females
subjected to one or two episodes of connement stress in the
months immediately prior to ovulation (Campbell et al., 1994).
In contrast, sperm counts did not signicantly vary between
stressed and control groups. Finally, signicantly reduced circu-
lating vitellogenin plasma levels were observed in females. In a
later study conducted on female Coho salmon during the nal
two weeks of oogenesis, a stress induced by net chasing resulted
in a higher number of ovulating females in comparison to the
control group and higher cortisol levels in eggs from the stressed
group (Stratholt et al., 1997). In rainbow trout, stress applied dur-
ing early vitellogenesis resulted in smaller eggs while stress ap-
plied during late vitellogenesis-nal maturation resulted in
advanced ovulation. However, no effect on progeny survival was
observed (Contreras-Sanchez et al., 1998). Together, these data
suggest that stress applied during oocyte growth can result in sig-
nicantly smaller egg size, while stress applied during the nal
oogenesis stages can result in advanced ovulation. In addition, a
severe stress (Campbell et al., 1992, 1994) can induce signi-
cantly lower embryonic survival while no or little effect is in-
duced by a milder stress (Contreras-Sanchez et al., 1998).
The acute stress applied to mature wild sh during capture
and transportation may be responsible for the decrease in sperm
motility observed in white bass (Morone chrysops) (Allyn et al.,
J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548 543
2001). In rainbow trout, we also observed that males conned
alone in small tanks had cortisol levels 4 times higher than the
controls, and sperm motility decreased to less than 10% of the
control values.
4.5. Other factors
Many other factors are susceptible to affect gamete quality,
including exposure to xenobiotics and pollutants, or physiochemi-
cal properties of the water (reviewed in Brooks et al., 1997). In
addition, it has been shown in mammals that parental genotypes
can strongly inuence fertility, fecundity and oocyte quality. By
contrast, the effects of broodsh genetics on gamete quality remain
poorly documented. Existing fertility differences in rainbow trout
female broodsh have been attributed to genetic differences (Stod-
dard et al., 2005). Further investigations are however required to
evaluate the heritability of fertility observed during the rst repro-
duction. In addition, the genetic inuence of parental genomes on
egg quality is difcult to study as the onset of zygotic transcription
does not occur until mid-blastula transition. Indeed, the very early
steps of embryonic development rely on gene products (maternal
mRNAs and proteins) supplied to the embryo by the mother. How-
ever, the abundance and processing of maternal mRNAs and pro-
teins in the female gamete are susceptible to be impacted on by
non-genetic factors such as environmental variables (e.g. photope-
riod) (Bonnet et al., 2007b).
Finally, in many sh species, social factors or inter-individual
interactions are highly susceptible to inuence reproductive suc-
cess. In general, rearing conditions can strongly inuence gameto-
genesis and gamete quality. The overall impact of broodstock
breeding conditions is probably different in species that have been
domesticated for a long time in comparison to new aquaculture
species. Within each species, the domestication level of broodsh
(i.e. captured form the wild or originating from captive reproduc-
tion) is also likely to have an impact on reproductive success in re-
sponse to specic rearing conditions.
5. Conclusions and future directions
Egg quality can be dened as the ability of the egg to be fertil-
ized and to subsequently develop into a normal embryo. Similarly,
sperm quality can be dened as its ability to successfully fertilize
an egg and subsequently allow the development of a normal em-
bryo. The identication of predictive estimators or markers of gam-
ete quality would have major applications in the eld or in the
industry. For eggs, it would help prevent the risk of mixing, for
practical reasons, egg batches of poor and good quality. For sperm,
it would prevent the risk of poor fertilization rates resulting in the
loss of whole egg batches. However, to date, it seems clear that no
effective predictive marker or estimator of gamete quality exists
even though non-viable gametes can sometimes be identied in
some species, through the assessment of simple parameters such
as buoyancy, appearance or motility. Thus, apart from markers of
extremely low quality, it is still very difcult to accurately estimate
the quality of the gametes prior to fertilization. For instance, nor-
mal eggs that do no show any sign of non viability could in fact
have low developmental capacities. Hence, the only biologically
relevant way to accurately estimate gamete quality is to perform
fertilization and/or monitor embryonic and larval survival. Fur-
thermore, monitoring embryonic malformation or larval deformi-
ties will further help characterize gamete quality. Specic
malformations are associated with specic gamete quality prob-
lems induced by specic factors (Bonnet et al., 2007a). It is thus
important not only to record survival throughout development
Factors that can influence gamete quality How to estimate gamete quality?
Egg
Spermatozoa
Oogenesis
Spermatogenesis
Motility
Membrane integrity
Energy metabolism
DNA compaction
buoyancy
Development
Survival
Malformations
Eyeing Hatching Yolk-sac
resorption
Early cleavage
patterns
Oocyte maturation
Ovulation
Fertilization success
F
e
r
t
i
l
i
z
a
t
i
o
n
temperature
post-ovulatory ageing
photoperiod
spawning induction
stress
nutrition
sperm handling
stress
time in the
reproductive season
Egg
Sperm
egg handling
Fig. 2. Main factors that can inuence gamete quality in sh and main parameters than can be recorded to fully characterize gamete quality. The time period during when
factors can inuence gamete quality is indicated by dotted lines.
544 J. Bobe, C. Labb / General and Comparative Endocrinology 165 (2010) 535548
but also to monitor embryonic and larval deformities to completely
assess gamete quality.
Many investigations have successfully led to the identication
of external, experimental or rearing factors that can signicantly
impact gamete quality. Despite these efforts, the environmental
control of gamete quality is far from being fully understood and
existing research efforts dedicated to a better characterization
and understanding of the effect of external factors and broodstock
management conditions on gamete quality should be pursued. In
order to be fully informative, such studies have to thoroughly as-
sess gamete quality in order to avoid the drawing of biased conclu-
sions based on a partial evaluation of gamete quality. It should also
be stressed that not only species-specic characteristics but also
domestication level and adequacy of rearing systems and brood-
stock management conditions have to be taken into account when
drawing conclusions on the effect of a specic factor on gamete
quality. Ideally, a control resulting in almost 100% viable and nor-
mal individuals will be a good indicator of the full adequacy of the
experimental system used to test the effect of a specic factor on
gamete quality. This is especially true for factors that have a mild
effect on gamete quality. In suboptimal systems, in which gamete
quality is intermediate and/or variable, only major effects will be
detected. Existing work conducted on the main aquaculture spe-
cies on the inuence of external factors and husbandry practices
on gamete quality (mostly eggs) has led to the identication of
key factors that should be controlled in order to obtain good qual-
ity gametes (Fig. 2). Some specic factors (e.g. post-ovulatory age-
ing) probably have general effects that can be extended to other
species. For other factors, or for less documented species, the
amount of available data is scarce and specic efforts should be
made to address these questions. This is especially true for newly
domesticated species or for future aquaculture species. For these
new species, signicant efforts should also be dedicated to the
determination of appropriate gamete handling procedures after
collection. The procedures include storage conditions (medium,
temperature, and duration), in vitro fertilization methods, and em-
bryo rearing conditions.
In the past few years, several studies have aimed at identifying
cellular and molecular mechanisms that could play important roles
in controlling the developing potential of the oocyte and early em-
bryo (Aegerter et al., 2005; Bonnet et al., 2007b; Ziv et al., 2008;
Crespel et al., 2008). These studies have successfully identied can-
didate mRNAs and proteins that could participate in the regulation
and/or control of egg quality. However, the intracellular picture of
a developmentally competent egg or early embryo is far from being
complete and additional genomic and functional investigations are
required to address this question.
Similarly, it should be stressed that very little is known about
the effect of ongoing genetic selection on the quality of the ga-
metes. In many farmed animals (e.g. dairy cows) a decrease in fer-
tility is often associated with the genetic selection for non-
reproductive phenotypes (Weigel, 2006). This is also a possible is-
sue for aquaculture that will require specic investigations in the
future.
Acknowledgment
The authors thank Miranda Maybank for critical reading of the
manuscript.
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