State of the art and need for applied research in fish

reproduction:

OBTAINING TOP QUALITY EGGS

Julien Bobe

INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE

Fish Reproduction Group • SCRIBE
Julien.Bobe@rennes.inra.fr
http://www.rennes.inra.fr/scribe/
 …Definition…

• Egg quality can be defined as the

ability of the egg to be fertilized
and subsequently develop into a
normal embryo
 …Introduction…

• Egg quality is a major issue in

aquaculture
– Existing aquaculture species
• Controlling the quality of the eggs is
important for the sustainability of aquaculture
• Global warming can become an issue for egg
quality
– For, new aquaculture species, the
availability of “fresh” good quality eggs is
often a limiting factor
 …Outline…

• Overview of egg quality problems in fish based

on existing scientific literature
– What we know
– What we think we know
– What we don’t know
• Scientific questions and main research axes
being investigated
– What we are doing
– What we should be doing
– What we need
 1-Overview of the
factors that can affect
egg quality
 …Egg quality defects…

• Embryonic mortalities
• Abnormal development

• Ploïdy defects (triploid fry, aneuploid

fry)
• Buoyancy problems
 …Post-ovulatory ageing…
• Major effect on egg quality 100%
Embryonic survival (%)

– Low embryonic survival 80%

60%
– Specific malformations 40%
20%
– Molecular and biochemical changes 0%
0 5 14 21
– High differences among females 100% Malformed embryos (%)
– Very rapid in most species 80%
60%
• Optimal post-ovulatory ageing for egg 40%
20%
collection is not always well characterized 0%
0 5 14 21
• Female-dependent ability to maintain good 60%
Triploids (%)

egg quality for a long time driven by 40%

genetics, environment, or both ? 20%

Npm2 1.5 a
0%
• Molecular mechanisms? 0 5 14 21
mRNA levels

1 Post-ovulatory ageing (days)

b

b b
0.5
Aegerter et al.,
Molecular Reproduction
0
0 5 14 21
and Development, 2005
Post-ovulatory ageing
 …Temperature…

• Major effect on egg quality

– Low survival
– High frequency of triploids
– Faster decrease of egg quality
after ovulation
• Time window before ovulation
• Relationship between
temperature and egg quality
defects
• Molecular mechanisms ?

Aegerter and Jalabert, Aquaculture, 2004

 …Photoperiod…
• Photoperiod is very useful to obtain out of
season spawning
• When spawning occurs at suboptimal
temperature: detrimental effect on egg quality
• Some mild problems can occur, even if
temperature is kept within a normal range (e.g.
yolk-sac resorption problems)
• Mechanisms at the ovarian level
– Shorter oogenesis: what effect on the oocyte ?
– Ovarian stage when artificial photoperiod starts: what
effect on the oogenetic process?
– Previous ovulations?
 …Nutrition…

• Existing aquaculture species

– Apart from extreme situations (e.g. insufficient
feeding, lack of specific vitamins), little
evidence of a direct effect of nutrition on egg
quality (Brooks et al., 92)
– What about new diet sources (e.g. vegetal
proteins) or contaminants?
• Newly domesticated species or new
species: adapted diet must be provided to
allow a normal oocyte growth
 …Salinity…

• Migrating species (e.g. salmon)

– Keeping the broodfish in sea water can block
spawning or induce a delayed spawning
– Direct effect on egg quality is limited
• Very little information available
• Mechanisms?
 …Spawning induction…

• Spawning induction techniques are useful

– Sometimes necessary to obtain eggs in captivity
– Synchronize ovulation of broodstock
• Mechanisms: trigger hormone-induced meiosis
resumption
• Reduce the oocyte developmental competence
acquisition process (distinct from the meiotic
process)
• For species that naturally ovulate in captivity
– Mild negative impact on embryonic survival can be
observed
– The clearest effects are observed when SI
procedures are used early in the pre-ovulatory period
 …Stress…

• Results are different depending on the

type and intensity of the stressor used
• Acute and repeated stress (over 9 months
prior to spawning) can reduce embryonic
survival
• Stress can stimulate ovulation and result
in advanced spawning
 …Genetics…

• The genotype of the female is susceptible

to have an impact
– On the overall fertility of the female
– On the fertility in response to specific
environmental factors (e.g. temperature)
• Available data are scarce
 …Other factors…

• Social factors
• Xenobiotics
• Water composition
• Timing during reproductive season
• …
 …Be cautious…

• Approx 30000 fish species

– Many species-specific differences
• Possible experiment-specific effects
• Limited number of studies specifically
designed to monitor egg quality
– Appropriate control
– Egg quality assessment method used
 …Conclusion (1)…

• Different levels of incidence on egg quality

– The effect of some factors is very clear (e.g. post-
ovulatory ageing)
– Limited effect of other factors (e.g. photoperiod) that
should not be underestimated
– Other factors (e.g. genetics): question remains to be
answered
• In all cases a thorough characterization should
be carried out (especially for mild factors and
new species)
2- Scientific questions
and main research axes
being investigated
How to assess egg
quality ?
 …Quality assessment methods…
• Several types of parameters can be recorded
to assess egg quality (=ability to be fertilized and subsequently
develop into a normal embryo)
– Indirect (correlated) parameters
• Egg buoyancy
• pH of coelomic fluid
– Direct parameters
• Fertilization rate
• Shape of embryonic cells
• Survival at specific developmental stages
• Malformations, including the types of malformations
• Ploidy
– A parameter alone is not always relevant/sufficient
• Species-specific differences
• Condition- or external-factor specific relevant parameters
 …Example…
• Spawning induction in rainbow trout
– Survival at eyed stage: no significant difference
– % of normal alevins at YSR: mild but significant
effect
• Importance of a good control to detect a mild
effect 100
– Good quality
– Low variability 80
*
60

40

20

0
Eyed stage YSR

Bonnet et al., Theriogenology, 2007 Natural ovulation Spawning induction

 …Specific defects…
30

20 Control (Natural ovulation)

• Some farming practices can 10

induce specific egg quality 0

defects 30

Post-ovulatory ageing
– Mortalities at specific 20

embryonic stages 10

– Specific types of embryonic 0

30
malformation
20 Spawning induction
• In contrast, some embryonic 10

malformations are female- 0

dependent and not linked to 30

20
Photoperiod
a specific farming practice 10

– Siamese 0
Cy T YSD P Redrawn from Bonnet et al.,
Theriogenology. 2007

Cyclops Prognathia
Torsion Yolk-sac resorption defects
 How to assess egg
quality ?
• Use a complete method to estimate egg quality
– Survival at several stages
– Types of malformations
• Adapt the method
– To species
– To the studied factors
What are the cellular and
molecular factors defining the
developmental competence of
the oocyte ?
 …Mechanisms…

• What are the mechanisms triggering

egg quality problems ?
• First step: describing what is a good
egg
– Genes expressed during oogenesis (can
be affected by external factors)
– Genes expressed during early
development (maternally-inherited)
 …Maternal mRNAs…
• Support very early development
• Inherited from the mother
• In trout, genomic studies have shown
– Accumulation of maternal mRNAs can modified by external factors
– Abundance of some mRNAs within the egg is reflective of egg
quality 100

Normal alevins
80

at YSR %
60

PM HI N 40
20
0
ZNF261
BX082249
TLE1
LYPA3
TF
PKP1
LRTM1
HCK
ALG2
THP
GTF2B
ING1
FGD5
RPN2
CF188
RBM5
PDGFRA
BX878405
CUL5
PYC
BMP7
APOC1
VWF
OSBPL5
NEK1
RLP24
CASZ1
NTAN1
KHK
CTNNBL1
LAMB2
PGH2
RAB31P
DBNL
KIF4A
MR-1
OBSCN
DDAH2
HCFC1
DAB2
tcbr0001.b.08
HNRPK
PDCL3
CA388269
DUSP24
SEC22
AGM1
HSPA9B
APOB
MYO1B
TGFBR2
RL10
MCF2L
DCPS
ZNF16
_ +
PHB2
GMCL1

Bonnet et al., BMC Genomics, 2007 _ +

 …Proteins…

• Proteins present in the egg support

early development
• In seabass, protein expression is
different in poor and good quality eggs

0% survival 80% survival

Crespel et al.,Cybium, 2008

What are the cellular and
molecular factors defining the
developmental competence of
the oocyte ?
• Still a long way to go
• We have identified strong candidate genes
(RNA and protein levels) that could be key
molecular actors of egg quality
What are the important
factor to obtain top quality
eggs ?
 …Important factors…

• Overview in the first part of the talk

• Amount of fully informative data on the
effect of external factors and husbandry
practices remains limited
– Especially true
• Newly aquacultured species
• Specific factors
• Monitor egg quality under a wide variety
of broodstock management conditions
using a standardized egg quality
assessment method
…Negative effect of selection…

• Loss of fertility
associated with selection
for non-reproductive
phenotypes
– Observed in dairy cows and
chicken
• What about aquacultured Weigel, Animal Reproduction Science,
2006
fish?
– No or little feedback on the
fertility of the broodstock in
selection programs
 …A lot remains to be done…

• Characterization of the effects of external factors and husbandry practices

– New factors (protein sources in diet)
– Use appropriate (complete/accurate) methods to assess egg quality
– Better characterize the effect of existing practices
• Feedback, monitoring reproductive success
• Identify any negative effect of existing genetic selection programs on reproductive
success
– Output: adapt broodstock management techniques an selection programs
• Molecular mechanisms
– Molecular studies in progress in some species
– Existing studies should be extended
• Other species
• Other experimental factors
• Thorough functional analyses, possibly in model species
• Other cellular/molecular processes (e.g. metabolism)
– Output: what is a good egg (at cellular/molecular level)
• Ultimate goals
– Limit any negative effect of specific factors including long-term effects through
genetic selection
– Optimization of broodstock management techniques and/or genetic selection to
obtain top quality eggs
– Use existing background to obtain top quality in newly domesticated species