Procedures To Estimate Fecundity of Marine Fish Species in Relation To Their Reproductive Strategy

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J. Northw. Atl. Fish. Sci., Vol. 33: 3354
Procedures to Estimate Fecundity of Marine Fish Species in

Relation to their Reproductive Strategy
H. Murua
AZTI Foundation, Herrera Kaia Portualde z/g, 20 110 Pasaia, Basque Country, Spain
G. Kraus
Institute for Marine Science, Düsternbrooker Weg 20, D-24105 Kiel, Germany
F. Saborido-Rey
Instituto de Investigaciones Marinas, Eduardo Cabello, 6. 36 208 Vigo, Spain
P. R. Witthames
Centre for Environmennt, Fisheries and Aquaculture Science, Lowestoft Laboratory
Lowestoft, Suffolk NR33, 0HT, England
A. Thorsen
Institute of Marine Research, P. O. Box 1870 Nordnes, Nordnesgaten 50, N-5817 Bergen, Norway
S. Junquera
European Commission, Directorate-General Fish, Joseph II 99, B-1049 Brussels, Belgium
Abstract
Appraisal of reproductive strategy and fecundity is necessary to evaluate the reproductive
potential of individual fish species. To estimate reproductive potential, one needs to consider a
variety of attributes including onset of maturity, fecundity, atresia, duration of reproductive season,
daily spawning behaviour and spawning fraction. In this contribution, we review several methods
currently used to estimate fecundity of marine fishes collected in the field in relation to their
reproductive strategy. The advantages and disadvantages of each method are provided. Requirements
are given to appropriately sample gonadal tissue that will enable researchers to establish incidence
of sexual maturity and estimate fecundity.
Key words: atresia, fecundity estimation, marine fish, maturity ogive, North Atlantic, oocyte
histology, reproductive potential, reproductive strategy, sampling, spawning fraction
Introduction commercially valuable fish species. Moreover,

establishment of extensive data bases on reproductive
Descriptions of reproductive strategies and the parameters with corresponding data on abiotic factors
assessment of fecundity are fundamental topics in the enables the study of causal relationships between
study of the biology and population dynamics of fish reproductive potential and environmental variation.
species (Hunter et al., 1992). Studies on reproduction, This leads to a better understanding of observed
including the assessment of size at maturity, fecundity, fluctuations in reproductive output and enhances our
duration of reproductive season, daily spawning ability to estimate recruitment (Kraus et al., 2002).
behaviour and spawning fraction, permit quanti-
fication of the reproductive capacity of individual fish. Marked differences in fecundity among species
This information in combination with estimates of egg often reflect different reproductive strategies (Pitcher
production at sea enable estimation of spawning stock and Hart, 1982; Wootton, 1984; Helfman et al., 1997;
biomass (Saville, 1964; Parker, 1980; Lasker, 1985). Murua and Saborido-Rey, 2003). Within a given
This increases our knowledge about the state of a stock species, fecundity may vary as a result of different
and improves standard assessments of many adaptations to environmental habitats (Witthames et
34 J. Northw. Atl. Fish. Sci., Vol. 33, 2003
al., 1995). Even within a stock, fecundity is known to distribution of individuals in relation to spawning
vary annually, undergo long-term changes (Horwood activity for all areas considered in the study. Sampling
et al., 1986; Rijnsdorp 1991; Kjesbu et al., 1998) and should span the entire range in body length, the total
has been shown to be proportional to fish size (and distribution area and account for variations in the
hence, age) and condition. Larger fish produce more timing of maturation and spawning of different stock
eggs, both in absolute and in relative terms to body components (e.g., sub-stocks and age classes).
mass. For a given size, females in better condition
exhibit higher fecundity (Kjesbu et al., 1991). Fish Species, and even different populations within a
size and condition are, thus, key parameters to species, show differences in size at maturity, growth
properly assess fecundity at the population level. In rate, and spawning time. Hence, it is beyond the scope
heavily exploited populations, large old fish will be of the present contribution to provide reasonable
eliminated more rapidly because they are exposed to guidelines for each stock. Investigators interested in
size-selective fishing mortality (Trippel, 1999). In this pursuing this should scan the relevant assembled
situation, population fecundity not only declines as a literature specific to a stock before designing a specific
consequence of the reduced abundance of spawners, sampling strategy (for example, Tomkiewicz et al.
but also due to the disproportionate reduction in large, (2003a): data available on 42 fish stocks of the
highly fecund females. Values of condition indices Northwest Atlantic).
vary among individuals, and may vary annually within
individuals. Changes in environmental factors, such Although the main purpose of this contribution
as temperature, may affect condition by influencing is to provide guidelines to estimate fecundity of marine
fish behaviour and metabolism, as well as food species exhibiting different reproductive strategies,
availability. Declines in fecundity due to reduced data on sex ratio and sexual maturity are also
condition can be reflected in a lower number of oocytes fundamental to the estimation of population fecundity.
that develop in a given breeding season or through A brief description of requirements for sampling to
atresia. In extreme cases, low condition can induce assess sexual maturity is thus presented followed by
reproductive failure and lead to skipped spawning sections on procedures for fecundity sampling.
seasons (Bell et al., 1992; Livingston et al., 1997).
Fecundity and atresia can also be affected by Sampling for maturity ogive
environmental pollution (Johnson et al., 1998). In The maturity ogive is based on a classification of
light of these issues, regular estimation of fecundity, the population into mature (fish that certainly will or
in conjunction with environmental and other have spawned) and immature individuals in relation
biological data, would not only help to understand to age or length. It has been reported in the case of
the underlying mechanisms regulating annual sole (Solea solea) (Ramsay and Witthames, 1996), that
variability in fecundity, but could possibly help to some females are partially mature by commencing
explain variability in recruitment. vitellogenesis amongst the leading oocyte cohort but
later aborting the development without producing
The aim of this review is to provide a protocol to batches of eggs. It has been hypothesized that this
estimate the annual fecundity (see Table 1 for phenomenon may occur in other teleosts, being most
definitions) of commercially important marine fish abundant among individuals in the year-class
species from field samples. The protocol is divided maturing for the first time. These females have been
into two sections. The first deals with fish sampling defined as mature inactive (Hunter et al., 1992). The
and preservation of gonads. The second part is focused fact that some mature females fail to spawn in a given
on methods to estimate annual fecundity in relation breeding season should be considered when applying
to reproductive strategy as described in Murua and maturity ogives to estimate spawning stock biomass.
Saborido-Rey (2003). The suitability of different
methods specific to each reproductive strategy are During the spawning period, samples from the
discussed and recommendations are made. spawning aggregations may primarily consist of adults
because of different spatial separation or catchabilities
of mature and immature individuals. Consequently,
Sampling
sampling the spawning aggregation may result in
Sampling of fish from a population for maturity under representation of immature fish of maturing
and fecundity studies should provide an accurate cohorts; and therefore the estimated maturity ogive
representation of the seasonal and regional (e.g., size or age at 50% maturity) will be biased low.
MURUA et. al: Estimate Fecundity of Marine Fish 35
TABLE 1. Definitions related to reproductive biology and fecundity terms used within this review (for further details see
Murua and Saborido-Rey, 2003).
Fecundity terms Definition Reference
Total spawner species The whole clutch of developed oocytes is shed in an unique Holden and Raitt
event or over a short period of time but as part of a single episode. (1974)
Batch spawner species The eggs are released in batches usually over a protracted Holden and Raitt
spawning period (weeks or months). Only a portion of the (1974)
yolked oocytes is selected to be spawned and hydrated in
each batch.
Synchronous ovary All oocytes, once formed, grow and ovulate from the ovary Marza (1938);
development in unison; further replenishment of one stage by Wallace and
an earlier stage does not take place. Selman (1981)
Group-synchronous At least two cohorts of oocytes can be distinguished in Marza (1938);

development the maturing ovary; a fairly synchronous population of larger Wallace and
oocytes (defined as a "clutch") and a more heterogeneous Selman (1981);
population of smaller oocytes from which the clutch is recruited.
The former are the oocytes to be spawned during the current
breeding season, while the latter are the oocytes to be spawned
in future breeding seasons.
Asynchronous ovary Oocytes of all stages are present in the ovary without Marza (1938);
development dominant populations. The ovary appears to be a random mixture Wallace and
of oocytes, at every conceivable stage. Selman (1981)
Determinate fecundity In fishes with determinate fecundity, the standing stock of Hunter et al.
yolked oocytes (total fecundity) prior to the onset of spawning (1992)
is considered to be equivalent to the potential annual fecundity.
Indeterminate fecundity This term refers to species where potential annual fecundity is Hunter et al.,
not fixed before the onset of spawning and unyolked oocytes (1992)
continue to be matured and spawned during the spawning season
De novo vitellogenesis The process of producing vitellogenenic oocytes from Hunter and
previtellogenic oocytes during the spawning season, and Goldberg (1980)
consequent recruitment into the standing stock of yolked oocytes
Annual fecundity or annual The total number of eggs released per female in a year. Hunter et al.
realized fecundity (1992)
Annual population fecundity The total number of eggs produced by a population in a Bagenal (1978)
breeding season
Potential annual fecundity The total number of advanced yolked oocytes matured per Macer (1974);
female and year, uncorrected for atretic losses Hunter et al.
(1992)
Total fecundity The total number of vitellogenic or advanced yolked oocytes Hunter et al.
at any time in the ovary. (1992)
Residual fecundity or The number of vitellogenic or advanced yolked oocytes in Murua and Motos
remnant fecundity ovaries showing postovulatory follicles. This indicates that (1998)
these females had already spawned some eggs.
Batch fecundity The number of eggs spawned per batch. The sum of batch De Vlaming
fecundities represents the realized annual fecundity. (1983)
Spawning fraction Fraction of mature females spawning per day Alheit (1985)
Postovulatory follicle After ovulation, the follicle tissue that encapsulated each Hunter and
hydrated oocyte collapses, and remains in the ovary as an Macewicz (1985)
evacuated follicle. These structures are used as indicators of
previous spawning activity.
TABLE 1. (Continued). Definitions related to reproductive biology and fecundity terms used within this review (for further
details see Murua and Saborido-Rey, 2003).
Fecundity terms Definition Reference
Atresia The process of oocyte and follicle resorption altering the Bagenal, (1978)
oocyte structure as an indicator for the destruction and
resorption of oocytes.
Alpha atresia The initial phase of oocyte atresia. In this phase, the oocyte Hunter and
is resorbed, leaving only the follicular layers. Macewicz (1985)
Turnover rates of Duration of specific atretic stages of oocyte resorption. Hunter et al.
atretic oocytes (1985); Kjesbu et
al. (1991)
In order to obtain representative ogives, sampling Thus, regarding maturity assessment, we strongly
should be conducted over the entire stock's spatial recommend sampling the entire distributional area of
distribution, which may be characterized according a given stock (including adult and juvenile areas) and
to age and length (ICES, MS 1997). Additionally, the the use of histological criteria for the classification
maturity ogive, determined after sampling of of gonads to replace (or at least verify) a less costly
spawning and juvenile areas, should be weighted more extensive survey based on macroscopic criteria
according to differences in population density (Table 2). Samples should be taken during pre-
(Armstrong et al., 2001). spawning, preferably when the population is about to
spawn, and during the spawning season but before
Computation of accurate maturity ogives requires peak spawning time to evaluate mature inactive fish.
accurate maturity staging (Table 2). There is Accurate assessment of maturity outside the spawning
considerable concern about the reliability of period could be conducted during the vitellogenesis
macroscopic gonadal grading, and hence it is desirable period long before spawning begins (see Murua and
to increase the precision and confidence in the Saborido-Rey, 2003) or even during the resting period.
assessment of reproductive status and resulting But the latter case is only applicable after extensive
maturity ogives. In this regard the separation of fish evaluation of the parameters used (Saborido-Rey and
classified as immature or partially mature from those Junquera, 1998; Murua and Motos, 2000). The latter
adults staged as post-spawning or resting, and the authors and others (Woodhead and Woodhead, 1965;
determination at which time of the maturation cycle Zamarro et al., MS 1993) reported that postovulatory
such discrimination can be made (i.e., in prespawning, follicles can be identified in post-spawning fish up to
peak spawning, etc.) is of particular importance. The 3 months or longer after the spawning season has
use of histological techniques to study gonadal finished in wild populations, but laboratory studies
maturation and to estimate the length or age at are also needed to evaluate this methodology.
maturity has proven to have greater precision than
traditional macroscopic techniques and is When first undertaking maturity assessment of a
recommended as a possible substitute or augmentation stock, it may be necessary to collect a large sample
to visual classification (Murua and Motos, 1998; size of each age group. After this intensive study, it
Saborido-Rey and Junquera, 1998; Tomkiewicz et al., may be possible to further refine sampling strategy
2003b). by age, season and area in order to reduce the overall
effort, without a reduction in accuracy.
When establishing a maturity sampling scheme,
it is crucial to include those length and/or age groups Sampling for fecundity
within the transition from immature to mature. The
length or age transition range from immature to Estimation of fecundity involves a number of
mature, where the slope of the maturity curve is underlying assumptions, and these may differ
steepest, requires fine resolution and collection of depending on the species' reproductive strategy
sufficiently large sample sizes to estimate reliable (Hunter et al., 1992; Murua and Saborido-Rey, 2003).
proportions of mature individuals. Fish reproduction is also highly adaptive with locality
TABLE 2. General macroscopic maturity classification criteria of fish ovaries and corresponding histological descriptions (for
histological definitions see Appendix 2). To avoid misclassification, due to the difficulty to classify ovaries correctly
based on gross anatomical examination, a simple six scale macroscopic classification is presented. Macroscopic
staging is most accurate when stages 35 predominate within a population which is just before or during the first
part of a spawning season.
Females:
Probability of correct
classification into mature
Maturity or immature based on Macroscopic Microscopic
stages macroscopic criteria Characteristics Characteristics
Immature High Small ovaries without visible oocytes. Ovarian All the oocytes in the previtel-
1 wall thin. Separating this stage from the stage logenic stage (primary growth
of onset of maturity is often difficult. stage)
Maturing Low Medium size ovaries occupying 1/4 to 3/4 body The maturation process has
2 Confused with stages cavity. Visible opaque oocytes (0.2 mm to 0.5 mm started and the most advanced
5 and 6 resolution of human eye). oocytes are in cortical alveoli
stage or early vitellogenic
stages.
Mature High Large ovaries occupying 3/4 to almost filling The most advanced mode of
3 body cavity with blood capillaries. Yellow/ oocytes within the ovary is
orange colored. Visible opaque oocytes in vitellogenesis stages.
(0.5 mm resolution of human eye), without
bruised areas.
Spawning High Translucent oocytes that may flow or not on Spawning is imminent and the
4 applying pressure. Hydrated oocytes are oocytes are either in migratory
larger than the opaque oocytes. nucleus stage or hydration
stage.
Resting Low Bruised ovary. Purple in color and very flaccid. Some eggs have been released
5 Confused with stages Occasionally with remaining translucent and post-ovulatory follicles are
2 and 6 oocytes. But still the advanced oocytes present.
for the next batches are visible opaque Some remaining hydrated
oocytes (0.5 mm resolution of human eye, oocytes, from the previous
as big yellow oocytes). batch, may appear.
Further batches of hydrated
oocytes will be produced.
Spent Low Bruised ovary. Purple in color and very flaccid. The last batch has been
6 Confused with Ovary wall thick and blood capillaries are big. released. Oogonia and
stages 5 and 2 There are no more advanced oocytes remaining chromatin nuclear stages
in the ovary. oocytes are present or if more
advanced oocytes are present
they will undergo generalized
atresia and will be resorbed.
(Witthames and Greer Walker, 1995) and before to make the most informed choice of method. We
commencing any routine study a more detailed emphasize some important requirements regarding
investigation of the dynamics of oocyte growth, fecundity sampling for the two main reproductive
maturation and egg production should be undertaken strategies: determinate and indeterminate fecundity.
for the population in question. It is therefore necessary
to adjust the fecundity estimation approach and the For species with determinate fecundity a basic
sampling program according to reproductive strategy assumption is that the fecundity is fixed before the
onset of spawning, so that the potential fecundity is criteria it is important to ensure that the POF persist
equivalent to the annual egg production after at least as long as the time interval between batches.
accounting for atretic losses (Hunter et al., 1992).
Consequently, atresia needs to be discounted from One of the main goals of estimating fecundity,
potential fecundity to determine realized annual independent of reproductive strategy, is to establish a
fecundity. fecundity-size (length/weight/age) relationship, which
can be used to scale estimates of spawning stock
For individuals exhibiting determinate fecundity, biomass or spawner abundance to population egg
the oocytes that constitute the potential annual production. For this purpose, it is very important to
fecundity have to be identified with certainty. Time ensure that the entire length range of females present
of sampling in relation to the annual maturation cycle in the population is covered providing consistent
is important in this regard. When an ovary is sampled sampling among subcomponents over years. This is
too early in the season, it may not be sufficiently best achieved through length-stratified sampling over
developed to allow the identification of all oocytes the stock's entire size distribution.
destined to be spawned, i.e., the gap in the oocyte
size frequency distribution may not be clear and a bias The term indeterminate fecundity refers to species
may be introduced because not all oocytes have been in which the potential annual fecundity is not fixed
recruited into the annual stock of advanced oocytes, before the onset of spawning (Hunter et al., 1992). In
or, on the contrary, some oocytes that will not develop such species, previtellogenic oocytes may develop and
further become included in the counts. When sampling recruit into the standing stock of yolked oocytes at
during the spawning season for those species that any time during the spawning season (vitellogenesis
exhibit determinate fecundity and batch spawning, de novo; Hunter and Goldberg, 1980). Therefore, the
undetected spawned batches may result in estimation of potential fecundity in the ovary prior to
underestimated fecundity. To exclude both possible the onset of spawning is meaningless. In these species,
sources of bias, the timing of sampling has to be annual fecundity should be estimated from the number
adjusted so that only ovaries in the optimal of oocytes released per spawning (batch fecundity),
developmental stage are considered. the percentage of females spawning per day (spawning
frequency), and the duration of the spawning season
These optimal stages for determinate spawners (Hunter et al., 1985).
are pre-spawning individuals in late vitellogenesis
(stage 3, Table 2) (May, 1967; Hunter et al., 1992; Clearly, for species characterized by indeter-
Kraus et al., 2000). Inherent with this approach is minate fecundity, a different sampling strategy has to
that one should account for body size-specific be applied in order to obtain samples to estimate batch
differences in spawning schedules within a stock. fecundity, spawning fraction and sex ratio. Again, a
Although for most stocks a spawning peak can be length-stratified sampling scheme is recommended to
identified, the entire spawning population will also estimate batch fecundity, as it would cover the entire
contain early and late spawning sub-components (e.g., size distribution of a stock. The sampling for batch
based on genetics, body size, etc.). A possible bias in fecundity analysis would also assure hydrated females
fecundity estimates may arise, when these sub- in adequate numbers to cover the entire size range of
components vary in their size-specific fecundity and a stock. For estimating the spawning fraction and sex
are not sampled representatively. A sampling ratio, mature females should be selected at random.
campaign should, thus, either cover the entire pre- In the latter approach, fishing gear and timing of
spawning and spawning period or should at least be sampling should be designed in order to provide a
scheduled to meet the spawning peak so that fecundity representative sample of the reproductive population.
is representatively assessed for the entire stock. An examination of fish movements (e.g., using
Because fecundity has to be estimated from pre- tagging) may be required to investigate whether
spawning ovaries, histological screening to assess if individuals remain in the survey area during the period
a female has already started spawning should be of investigation. Mackerel (Scomber scombrus) in the
performed. The presence of postovulatory follicles eastern Atlantic, for example, include members of the
(POF) or hydrated oocytes in the ovary (see Appendix same population that spawn south of 40 o N in January
2 for further details on histology) indicate that the and north of 60o N in July. Dawson (1986) showed a
spawning process has already begun. To apply these succession of mackerel females by size in the central
spawning area and proposed an annual migration Estimation Methods

cycle in which adults moved into and out of the According to the species under investigation and
spawning area. available laboratory facilities, investigators should
judge which of the methods described below is
The number of individuals per size class necessary applicable. Depending on the peculiarities of a given
to obtain reliable size-specific fecundity estimates is species small changes in the methodology may be
dependent on the fecundity modality, i.e., for required. For example, it is possible to use the
indeterminate species, a high number of samples per gravimetric method to estimate the potential fecundity
size class is required to account for the relatively large for any species, but the threshold diameter at which
seasonal variation in batch fecundity (i.e., sampling the developing oocytes could be separated from pre-
should be repeated on a regular basis over the vitellogenic oocytes will be variable. Consequently,
spawning season). However, sample sizes are highly prior knowledge of the reproductive biology of the
dependent on the degree of individual variation in species is essential.
fecundity, which could differ strongly between stocks
or species, and within a given stock among sizes. Prior to the application of any fecundity
Thus, no general rule is recommended for sample estimation method, homogeneity in oocyte distribution
sizes, but sampling should be adequately planned to in the ovary should be investigated to ensure that the
ensure significance of fecundity-size relationships. sub-sample to be analysed represents the entire ovary,
e.g., test for differences in oocyte density within an
Reliable estimates of the spawning fraction not ovary lobule and between ovary lobules. For example,
only depend on the number of individuals sampled at it has been shown that oocyte size, and hence fecundity
each station, but also on the total number of stations. per unit volume, in yellowfin sole (Limanda aspera)
According to Picquelle (1985), it is generally more (Nichol and Acuna, 2001) and plaice (Pleuronectes
efficient to increase the number of trawl stations and platessa) (Witthames, unpubl. data) vary along the
reduce the sample sizes per station than vice-versa. axis of each ovarian lobe.
The appropriate number of fish at each station and
the necessary number of stations to compute the Gravimetric method. The gravimetric method is
spawning frequency with sufficient precision are currently the most common method used to estimate
discussed in detail in Picquelle (1985). fecundity. It is based on the relation between ovary
weight and the oocyte density in the ovary. This
In summary, sampling gonads for fecundity method can be used to estimate batch fecundity, total
analysis has to be adjusted according to the repro- fecundity and potential annual fecundity. For more
ductive strategy of the species under investigation, details see Hunter and Goldberg (1980) and Hunter
and even within a specific reproductive strategy it will et al. (1989).
depend on the parameter to be estimated. The
sampling protocol provided in Appendix 1 covers the Using this method, fecundity (F) is determined
different reproductive strategies. However, fine as the product of gonad weight and oocyte density.
adjustments may be required for each stock under Oocyte density is the number of oocytes per gram of
investigation. ovarian tissue, and it is determined by counting the
number of oocytes (oi) in a weighed sample of ovarian
Fecundity Estimation tissue. After weighing the ovaries (W ovary ), 35
In this section we review methods for estimating subsamples of known weight are extracted from
fecundity (total potential fecundity, batch fecundity), different parts of the ovary lobe. The accuracy and
spawning fraction and atresia from field samples. precision of fecundity estimation should be evaluated,
First, different methods to estimate fecundity are listed especially regarding the number of sub-samples (for
including a short description followed by a summary further details see Hunter et al., 1985). As a rule-of-
table of the advantages and disadvantages of each thumb, a sufficient number of subsamples is reached
method (Table 3). Second, problems in estimating the when the CV of the no of oocytes per unit weight is
amount of atresia and spawning fraction are discussed. less than 5% (Kjesbu, 1989). Each subsample is
Finally, combining procedures to estimate fecundity, weighed (wi) to the nearest 0.001 g and then dispersed
spawning fraction and atresia, we focus on estimating with a fine paint brush, or light air pressure created
the realized annual fecundity (see Table 1 for by repeatedly sucking in and out of a Pasteur pipette,
definitions). to identify and count all the vitellogenic oocytes.
Oocytes can be counted (and at the same time To estimate potential or batch fecundity, ovaries
measured) using a stereoscopic microscope with a grid should be screened histologically to check for the
or using any image analysis system software. occurrence of post-ovulatory follicles (POF) (Fig. 3).
Ovaries containing POFs should be eliminated from
 R 
∑ potential fecundity calculations, since the presence of
L

Z
)=  :
L
L POFs indicate that spawning has already started and
Q
RYDU\
the number of oocytes in the ovary has consequently
Variations of this method are related to whether decreased. For batch fecundity estimations, only
total fecundity, batch fecundity or potential annual hydrated ovaries which do not contain early stage
fecundity is estimated and the type of oocytes POFs (Hunter and Macewicz, 1985) should be used
enumerated. To estimate batch fecundity the hydrated because the presence of these follicles indicate that
oocytes within the subsamples are counted, while for some eggs have been already released.
calculations of total fecundity or potential annual
fecundity the advanced yolked oocytes (including the Volumetric method. The volumetric method is
hydrated oocytes) are counted. As we stated above, in based on the same principles as the gravimetric
the case of species where no clear gap between method, but uses ovarian volume and the subsample
previtellogenic and vitellogenic oocytes is present volume instead of ovary weight and subsample weight
(Fig. 1 and 2), e.g., hake (Merlucius merlucius) or (Simpson, 1951).
mackerel, only oocytes larger than a critical minimun
diameter are included for potential or total fecundity Combined gravimetric and automated particle
estimations. This diameter marks the transition from counting method. This method is a variation of the
previtellogenic to vitellogenic oocyte (Khoo, 1979) gravimetric method; the major difference being that
from where the oocytes comprising the fecundity an automated particle counter is used to enumerate
become steadily larger and more opaque as yolk and the number of oocytes in a subsample (Witthames and
cortical alveoli accumulate. Greer Walker, 1987; Kraus et al., 2000).
ca
Vit
Vit
pg
Fig. 1. Histological sections of ovaries showing oocytes at various stages of
development: previtellogenic primary growth oocytes (pg), cortical alveoli
oocytes (ca), and vitellogenic oocytes (Vit.) (for further details see Fig. 1 in
Murua and Saborido-Rey, 2003).
25 automated from stereomicroscope counts of 1.4%

(Kraus, 1997).
Oocyte frequency (%)
20
Vitellogenic
Previtellogenic
Stereometric method. From histological sections
15 it is possible to determine the number of cells in
different categories, i.e., oocytes that are either
10 previtellogenic, vitellogenic or atretic; by applying the
principles of stereology (Emerson et al., 1991). The
5
stereological method of Emerson et al. (1991) is based
on the Delesse principle (Delesse 1847) which states
that the fractional volume (Vi) of a component (i) is
0
proportional to its fractional cross sectional area (Ai).
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1 000
An underlying assumption of this principle is that the
Oocyte diameter (µm)
component is distributed randomly and evenly through
Fig. 2. Ooctye size frequency distribution of previtellogenic the tissue.
oocytes and vitellogenic oocytes of mature hake,
Merluccius merluccius, female in stage 3 of The abundance of a specific type of oocyte, defined
maturation (Source: Murua and Motos, 1998).
by morphological criteria, per unit volume is measured
using the stereological method developed by Weibel
The advantage of using an automatic particle and Gomez (1962) (Fig. 4). The following equation
counter to determine the number of oocytes in a describes the relationship between fecundity (F) and
subsample is that the counting procedure is less time its dependent variables (Weibel et al., 1966):
consuming. Hence, the sample sizes are commonly
much larger or the whole ovary could be analyzed where:
compared to manual counts. This enables greater F=Ov × K × N a3/ 2
precision in weight measurements of the compara- β Vi1/ 2
tively large counted oocyte fraction. However, the
precision of automated particle counters must be
β is a shape coefficient, i.e., ratio between the
evaluated according to the prevailing operating
conditions (count rate, debris or bubbles in the carrier longest and shortest axis of the oocytes transected
fluid) and compared to manual counts. For example, Ov is the ovary volume,
a Hiac optical particle sensor and pulse height Na is the number of oocyte (previtellogenic,
analyzer have been shown to count samples with a vitellogenic, atretic) transections per unit area,
CV of 0.6% (Witthames and Greer Walker, 1987). A
Vi is the partial area of oocytes (previtellogenic,
similar precision was obtained using a Macro-
vitellogenic, atresia) in the histological section,
Flow-Planctometer (Hüller et al., 1991) with a CV of
K a size distribution coefficient,
0.4% for repeated counts and an average deviation of
POF
g POF
L
t g L
Fig. 3. Histological sections of ovaries showing postovulatory follicles (POFs) at various stages of resorption (for
further details see Appendix 2).
Fig. 4. Example of a Weibel grid overlaid on the image of section showing point
counts to determine the partial volume (V i) of vitellogenic oocytes in the
ovary. In this grid there are 84 bars and the end of each bar represents a
test point (n = 168). (Vi = 97/168) The parameter N a is the number of
vitellogenic oocytes transected in the grid area and includes oocytes
overlapping the green borders but not the red borders of the grid.

0  size range of vitellogenic oocytes measured (Greer
. =   Walker et. al., 1994) (Fig. 2). However, not all species
 0  conform to this criterion. Data to correct the observed
M1 is the mean oocyte diameter oocyte diameter in a section to the real diameter are
available for mackerel (Greer Walker et al., 1994),
0 = [ ' + ' + 'Q Q] bass (Dicentrarchus labrax), cod (Gadus morhua), and
sole (Witthames, unpubl. data). Where the data are
not available, the relationship between oocyte and
M3 is the cube root of the third moment about the nucleus diameter should be determined from
mean of the oocyte distribution. histological sections. Estimation of atretic oocytes
requires special mention because it is not possible to
( ) ( )

 ( ' ) + ' + ' determine atretic oocyte size frequency and certain
Q
0 =  assumptions must be made which are discussed in the
 Q  section headed atresia.
 
The proportional area (A i ), and hence the
The parameter K is determined, using the formula proportional volume (V i), occupied by vitellogenic
of Williams (1997), by measuring the oocyte diameter, oocytes is determined by counting the number of points
as the mean of the largest and smallest diameters of (po) on the Weibel grid (which overlays the oocytes of
an oocyte, in a sample of vitellogenic oocytes each stage) and dividing this number by the total
transected through the nucleus. Emerson et al., (1991) number of points on the grid (p t). The number of
stated that measuring 50 oocytes is sufficient to give oocytes per unit area (Na) is estimated by counting
a stable value for K of an ovary. the number of oocytes of each stage within the total
area of each grid (Emerson et al., 1991). New
This estimation of oocyte size frequency assumes developments in image analysis and associated
that the nucleus diameter is constant over the whole software have reduced the required labour costs
considerably as direct or even automatic area fish. Once this calibration regression has been
measurements are now possible. established, measuring fecundity with this method is
rapid, typically requiring about 5 min for each sample.
Auto-diametric fecundity method. This method The method does not require subsample weighing,
estimates the potential fecundity from the mean which eases the collection of samples at sea. Compared
vitellogenic oocyte diameter and the total ovary weight to the gravimetric method, the auto-diametric method
using a calibration curve that relates mean oocyte requires additional electronic equipment and thus is
diameter to oocyte density (Thorsen and Kjesbu, more costly. The establishment of a standardized
2001). The mean oocyte diameter is measured from measuring procedure and a calibration curve may take
whole oocytes of preserved ovaries (buffered formalin) considerable time. In conclusion, this method would
using an image analysis system (Fig. 5). The only be a valuable rapid alternative if fecundity can
measurement technique is based on light thresholding be assessed routinely and is most appropriate for
of the dark vitellogenic oocytes using background species with group-synchronous ovaries (in these
illumination. The method has to be established for species the gap between developing yolked oocytes
each species by generating regression equations and previtellogenic oocytes is very clear, and therefore,
between mean pre-spawning oocyte diameter and the the maturing oocytes are clearly separated in size and
number of oocytes per gram ovary in pre-spawning darkness from the earlier oocyte stages).
TABLE 3. Features, advantages and disadvantages of methods used to estimate fecundity of wild collected fish species.

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species.

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Fig. 5. Whole mount picture (Source: Thorsen and Kjesbu, 2001). The auto-
diametric fecundity method is based on automatic measurements of mean
vitellogenic oocyte diameter from whole mount photographs. The image
analyzer software distinguishes single vitellogenic (dark) oocytes from small
previtellogenic (light) oocytes, clusters of vitellogenic oocytes, and bits of
connective tissue using thresholds of particle size, darkness and a roundness
factor.
Spawning Fraction any of the ovary stages associated with spawning can
be used, provided its duration is known (Priede and
In fishes with indeterminate fecundity, the annual Watson, 1993).
fecundity should be estimated from the combination
of batch fecundity, spawning fraction and the duration The postovulatory follicles (POFs, Fig. 2) are the
of spawning season. The most accurate way to estimate most common ovarian features used to estimate the
the spawning frequency (batch interval) is to follow spawning fraction. It is necessary to age the
the batch production sequences of an individual fish deterioration and resorption processes of the POF with
in tank experiments (Hunter and Goldberg, 1980; a series of distinct histological stages, where each
Kjesbu et al., 1991). If this approach cannot be stage indicates the time elapsed since spawning
applied, the fraction of females spawning per day (Hunter and Macewicz, 1985). This could be achieved
(spawning fraction) could be assessed from the by sampling spawning fish in the laboratory (Leong,
prevalence of spawning stages determined from a 1971); that is by taking ovarian samples at regular
random sample of gonads (Hunter and Goldberg, intervals from time of spawning, or by getting samples
1980; Hunter and Macewicz, 1985; Priede and from a spawning aggregation over a 24 hr cycle at
Watson, 1993) as follows: sea (Goldberg et al., 1984; Alheit et al., 1984). For
6 the latter approach, the daily spawning cycle should
6 = L
be synchronous, that is, the eggs should be released

WL
at the same time interval of the day, and the average
and hence, time of spawning for the population should be
Batch Interval = 1/S estimated (Hunter and Macewicz, 1985).
where Si is the prevalence of the i-th ovary stage and To estimate the number of egg batches within a
t i is the duration of the i-th stage in hours. In principle, spawning season, knowledge of the duration of the
individual spawning season is required, in conjunction 1985; Kjesbu et al., 1991) and the amount of time
with the spawning fraction. The duration of the from first visible signs of oocyte degeneration to
individual spawning season may be estimated as the complete yolk resorption ( α − DWUHWLF stage) is
time elapsed from 50% of females in pre-spawning relatively short (Fig. 6). The distinction of the later
stages (oocytes in advanced stages of vitellogenesis atretic stages from empty follicles becomes difficult
previous to hydration without signs of spawning) to and all studies, so far, have restricted quantification
50% of the females in post-spawning stages (spent of atresia to the α − DWUHWLF (e.g., Witthames and
and recovering) (Iles, 1964; Karlous-Riga and Greer Walker, 1995). A rapid "turnover rate" of
Economidis, 1996; Armstrong et al., 2001). An the α −VWDJH means that a number of oocyte cohorts
important underlying assumption inherent with this could degenerate from the time of advanced matur-
approach is that the distribution pattern of the ation (determination of potential fecundity) to
spawning stock is unchanged during the reproductive completion of the spawning phase. As a consequence,
season, that is, fish do not migrate into or out of the changes in prevalence and intensity of atresia over
spawning area. this period could occur due to decreasing nutritional
condition in the course of the individual's spawning
Atresia period (Kraus, 2002). Thus, a quantification of atresia
In fishes with determinate fecundity, the potential should include several estimates covering maturity
annual fecundity should be corrected for atretic losses stages from late vitellogenesis (determination of
to estimate the realized fecundity (Hunter et al., 1992). potential fecundity) to cessation of spawning
Atresia is a rapid process (Hunter and Macewicz, activities.
Fig. 6. Histological sections of ovaries showing atretic oocytes at various stages of development (for further details see
Appendix 2).
In contrast, the fecundity methods commonly smaller than the vitellogenic group so that in two
applied to species with an indeterminate fecundity, dimensional sections they are less likely to appear
for example, estimating batch fecundity with the compared to the larger oocytes. This problem becomes
hydrated oocyte or empty follicle method (Hunter et even more pronounced because the smaller
al., 1985), do not need to be adjusted for egg losses vitellogenic oocytes are also the most prone to become
due to atresia. The batch fecundity estimates refer atretic (Witthames and Greer Walker, 1995). The
either to fully developed oocytes ready to be spawned stereometric method described above reduces the bias
(hydrated oocytes) or follicles that have already caused by oocyte size in three ways: i) oocyte numbers,
released their eggs. either normal or atretic, can be estimated in three size
classes based on oocyte development stage (yolk
Several methods are available to quantify the vesicle, yolk granule/yolk vesicle, and advanced yolk
standing stock of atretic oocytes in the ovary. However, granule; Appendix 2); ii) it has only been applied to
only a few attempts to quantify atresia have been estimate atresia in the beginning of the alpha stage
carried out. Early work (Kjesbu et al., 1991) used a when little shrinkage in oocyte diameter has occurred;
gravimetric method to estimate fecundity and prepared and iii) points and profiles are accumulated in separate
histological sections to determine the proportion of oocyte classes. However, these assumptions have not
the fecundity that was atretic and hence the standing been validated or compared with the disector method.
stock of atretic oocytes. This approach has been shown The disector principle is considered the method of first
to be biased (Kurita et al., 2003) and reduced the choice as it does not require a whole cross section of
estimated proportion of atretic oocytes to 79% the ovary. It can also be applied to species with very
compared to an unbiased stereological method (Fig. 7) large ovaries such as cod or tuna because only small
based on the disector principle (applied to fish ovaries subsamples from the ovary are required for analysis.
by Andersen, 2003). The problem arises because
atretic oocytes shrink as they are resorbed and become Among the few attempts to quantify oocyte atresia
either in natural or experimental populations, the most
simple but indirect method is the comparison of
potential fecundity with the realized fecundity
utilizing tank experiments to collect the spawned eggs
A B as described by Kjesbu et al. (1991), Bleil and Oeberst
e
(1998), and Witthames et al. (MS 2000). A major
7 6 f
problem associated with this method, however, is that
g
5 the potential fecundity cannot be measured on the
h same individuals used to determine realized fecundity,
1 4 as lethal sampling is required to remove an ovary.
3 i
j Consequently, the potential fecundity of reference fish
2 of a similar size and condition has to be applied.
D C Combining experimentally obtained total egg
production rates with estimates of the intensity of
Fig. 7. Illustration of the "disector principle" for estimating atresia obtained from histological investigations,
particle number in three dimensional blocks. A plain
Kjesbu et al. (1991) estimated turnover rates of the
(points ad) cuts through 7 objects; 2 large and 5
small. An object will appear in a section (parallel
atretic stage as 10 days. Together with information
lines ej) proportional to its height running from on the duration of the individual spawning season,
the top (AB) to the bottom (DC) of the plain. these rates can be used to estimate total oocyte atresia
The ratio of small to large objects in sections ej is from histological field samples. The experimental
1 to 1 but the actual ratio is 1 to 2.5 (an design was further refined in recent work by
underestimate of 2.5 times). The bias depends on Witthames et al. (MS 2000) where frequent ovarian
the relative size of the objects. If an adjacent pair biopsies were taken during the pre-spawning and
of sections are overlaid a particle is only counted if spawning period of individual captive Atlantic cod.
it does not appear in the 'Look up' (upper section)
Histological slides were prepared from these biopsy
compared to the 'Reference' (lower section). The
repeat distance between sections ej should be 1/4
samples to determine the proportion of alpha atretic
to 1/3 of the diameter of the smallest particle to to normal developing oocytes. Using these data the
function without bias; for example, particles cannot production of spawned eggs and atretic oocytes could
fit between sections (see Sterio (1984) and be determined during the spawning period of each
Gundersen et al. (1988)). experimental fish and also the average duration of
the alpha atretic stage (alpha atretic rates) could be Estimation of Realized Fecundity
estimated. Another method used to estimate atretic In order to estimate the annual egg production of
oocyte stage duration was described by Hunter and a fish stock or population, a number of reproductive
Macewicz (1985) for northern anchovy, Engraulis parameters have to be estimated depending on whether
mordax. Experimental groups of anchovies were the reproductive modality is determinate or
starved to initiate massive oocyte atresia followed by indeterminate. For species with determinate fecundity,
an intensive feeding period that led to cessation of the realized annual fecundity may be estimated as
oocyte atresia. The time elapsed until the various described in ICES (MS 1997):
atretic oocyte stages disappeared was used to estimate
the stage duration. Frealized = Fpotential -Fatresia
In summary, direct methods to estimate oocyte Fatresia = Iatr * Patr * (D/S)

atresia require histological investigations of ovaries
where:
to determine, for example, the prevalence of individual
specimens containing atretic oocytes as well as I atr is the mean number of atretic oocytes per g
stereology (described above) to examine the intensity weight of female, excluding females without
of atresia. To convert potential into realized fecundity, oocyte atresia.
the overall atretic loss of oocytes has to be determined. Patr is the proportion of females with oocyte atresia.
This requires knowledge of atretic oocyte turnover
rates that are, as part of the metabolism (among other S is the duration of spawning.
reasons), strongly temperature dependent. D is the duration of the atretic oocyte stages.
Atretic oocyte turnover rates and the duration of However, it should be noted again that Patr and
the individual spawning season are two of the most I atr may vary over the pre-spawning and spawning
important items needed to quantify oocyte loss due to period biasing the estimates of realized fecundity.
atresia. While the intensity and prevalence of oocyte
atresia are relatively easy to obtain using stereological For species with indeterminate fecundity, the
methods, estimates of atretic oocyte turnover rates and annual realized fecundity should be estimated as
the duration of the individual spawning season can follows:
prove difficult. Both factors are influenced by the Frealized = BF * S * T
temperature fish experience during the pre-spawning where
and spawning time, as well as by their nutritional
condition. In contrast to the duration of the spawning BF is the batch fecundity,
season, which is well investigated for many fish S is the spawning fraction,
species and validated by experiments (e.g., cod: Kjesbu
et al., 1996), turnover rates of atretic oocyte stages T is the duration of spawning season.
have rarely been investigated (Hunter and Macewicz,
1985; Kjesbu et al., 1991; Witthames et al., MS 2000), Summary
and in the few studies conducted large variation was
observed. In addition to sex ratio and proportion of mature
individuals, fecundity is one of the most important
Before applying a quantitative approach we would determinants of a stock's reproductive potential. We
recommend histological screening as a first step to have described a number of methods that could be
check the relevance of oocyte atresia for the stock applied to estimate fecundity for a variety of fish
under investigation. Only if a significant proportion species. However, no universal method could be
of the fish stock shows intensive atresia, is a recommended, as different fecundity types (e.g.,
quantitative estimation recommended. In the case of determinate and indeterminate) require specific
quantitative studies, not only large-scale experimental methods to estimate the number of eggs that will be
validation of both the duration of individual spawning spawned per season. A variety of methods exist that
time and atretic oocyte stages is required, but also a could be applied to estimate the number of developing
series of field samples covering relevant maturity oocytes within an ovary (potential fecundity), and their
stages. The large effort associated with available application depends not only on the species under
methods would at present hamper any quantitative investigation, but also on the resources and laboratory
routine assessment of atresia. facilities available. However, potential fecundity is not
always a suitable measure of reproductive potential. Spawning frequency and sex ratio in the Peruvian
Potential fecundity could be a biased measure of the anchovy, Engraulis ringens. Calif. Coop. Fish. Rep.,
realized fecundity, if oocytes are continuously 25: 4352.
recruited into the pool of developing oocytes or if the ANDERSEN T. E. 2003. Unbiased stereological estimation
of cell numbers and volume fractions: the disector and
number of these is reduced by atresia. Methods, so
the principles of point counting. In: Modern approaches
far, used to scale the potential fecundity of determinate to assess maturity and fecundity of warm- and cold-water
spawners to realized fecundity, that is to account for fish and squids. O. S. Kjesbu, J. Hunter, and
atresia, are very time consuming and have only been P. R.Witthames (eds.). Institute of Marine Research,
applied to a few stocks (Greer Walker et al., 1994; Fisken og Havet, 12: 1119.
Armstrong et al., 2001). In fishes with an ARMSTRONG, M. J. P., P. CONOLLY, R. D. M. NASH,
indeterminate fecundity, none of the methods M. G. PAWSON, E. ALESWORTH, P. J. COULAHAN,
described for determinate fecundity species can be M. DICKEY-COLLAS, S. P. MILLIGAN,
used and the estimation of batch fecundity is required. M. F. O'NEILL, P. R. WITTHAMES, and L. WOOL-
NER. 2001. An application of the annual egg production
method to estimate the spawning biomass of cod (Gadus
To convert batch fecundity into realized annual morhua L), plaice (Pleuronectes platessa L.) and sole
fecundity, the duration of the individual spawning (Solea solea L.) in the Irish Sea. ICES J. Mar. Sci.,
season and the spawning frequency have to be 58: 183203.
determined. Since the duration of the individual BAGENAL, T. B. 1978. Aspects of fish fecundity. In: Ecology
spawning season and the number of batches to be of freshwater fish production. S. D. Gerking (ed.) Third
spawned, are variable and difficult to measure in the edition. FBA, Windermere Lab., Ambleside, UK.
field, these factors represent the greatest challenge to p. 75101.
successfully estimate the egg production of species BELL, J. D., J. M. LYLE, C. M. BULMAN, K. J. GRAHAM,
G. M. NEWTON, and D. C. SMITH. 1992. Spatial
with an indeterminate fecundity.
variation in reproduction, and occurrence of non-
reproductive adults, in orange roughy, Hoplostethus
In conclusion, no single method could be provided atlanticus Collett (Trachichthyidae), from south-eastern
to estimate the annual egg production of the variety Australia. J. Fish Biol., 40: 107122.
of commercially important marine fish species. The BLEIL, M., and R. OEBERST. 1998. Spawning of cod in
material described here provides an overview of captivity. Part 1: Course of spawning activities. Inf.
available methods including their strengths and Fischwirtsch., 45: 164170.
weaknesses. For each attempt to quantify fecundity DAWSON, W. A. 1986. Changes in western mackerel
and total egg production, a careful review of the (Scomber scombrus) spawning stock composition during
the spawning season. J. Mar. Biol. Assoc. U. K.,
reproductive biology of the respective species is
66: 367383.
necessary in order to determine which method can DE VLAMING, V. 1983. Oocyte developmental pattern and
successfully be applied. hormonal involvements among teleosts. In: Control
processes in fish physiology. J. C. Rankin, T. J. Pitcher,
Acknowledgements and R. T. Duggan (eds.). Croom Helm, London, p. 176
199.
We want to thank our colleagues and friends of DELESSE, M. A. 1847. Procédé mécanique pour déterminer
the NAFO Working Group on Reproductive Potential; la composition des roches. C.R. Acad. Sci. (Paris),
especially the chairman, Ed Trippel, for his helpful 25: 544 p.
comments, suggestions and revision for improvement EMERSON L. S., M. GREER WALKER, and P. R. WITT-
HAMES. 1991. A stereological method for estimating fish
of this manuscript. The authors very much appreciate
fecundity. J. Fish Biol., 36: 721730.
the comments and suggestions made by three FULTON, T. W. 1898. On the growth and maturation of the
anonymous reviewers on the original manuscript that ovarian eggs of the teleostean fishes. Ann. Rep. Fish.
greatly improved the final version. Board Scot., 16: 88124.
GOLDBERG, S. R., V. H. ALARCON, and J. ALHEIT, 1984.
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Appendix 1. Sampling Protocol
The sampling shall be undertaken using a For species of INDETERMINATE FECUNDITY:

commercial or research survey vessel spanning the
total distribution area of the stock. Sampling and data g) Spawning fraction. A random sample of only
recording should be done in the following steps: adult females in maturity stages 25 (see
Table 2) should be collected during the
Accessory data to be collected: spawning season. These fish represent the
mature stock and are necessary for the
a) Data on sample location (longitude, latitude,
estimation of spawning fraction of females
depth, etc.) with all basic data concerning the
and if possible the batch fecundity. The
hauls.
sample size is about 25 ovaries collected
b) Length, total weight (+1g), gonad weight
randomly from each trawl haul. The ovary
(+1g), gutted weight (+1g), liver weight
from the selected fish should be preserved in
(+1g), sex and macroscopic maturity stage.
the fixative solution of choice (buffered 4%
c) Otoliths for age determination of individuals formaldehyde solution*). The fixative to
from which ovaries have been sampled. ovary volume ratio should be high (4:1). The
d) In addition, stomach contents, liver and ovary membrane should be cut (superficially)
muscle samples (bioenergetic studies) should transversally at intervals to permit penetra-
be carried out on females from which the tion of fixative.
gonad has been previously sampled.
h) Batch fecundity. Additional sampling
Sampling for MATURITY OGIVE:
should be done focused to obtain hydrated
e) A length-stratified sampling scheme for females for the batch fecundity estimation.
gonads and testes should be conducted during Effort should be directed to obtaining samples
the prespawning period and around peak for batch fecundity in different parts of the
spawning. This sampling scheme should spawning area. For this purpose, where
reflect the entire distribution area of a stock, spawning fraction is low, ovaries of all fish
covering both spawning and juvenile areas. with hyaline eggs should be taken.
The "critical" transition range from immature
to mature requires a fine resolution and Preservation
sufficient sample sizes to obtain reliable Most of the fish reproductive parameters used in
proportions of mature individuals. the fecundity analysis (spawning fraction, batch
Sampling for FECUNDITY: fecundity, and maturity ogive) are based on analysis
of formaldehyde-preserved specimens taken with
For species of DETERMINATE FECUNDITY:
different sampling strategies. Clearly, attention has
f) A sample of only adult females in late to be paid to preservation techniques with the purpose
vitellogenic maturity stages (stage 3) (see of a correct measurement of these variables.
Table 2) should be selected whilst there are
pre-spawning ripe females present in the Histological analysis requires special care in
population. All sampled fish require an preservation. Post-ovulatory follicles are relatively
identification number, weight, gonad weight, subtle histological structures, and poor preservation
length and macroscopic maturity assignment. makes it impossible to stage them. Tissue should be
The sample size and the length range depends taken from freshly dead fish for the best preservation
on the investigated species, but should cover of morphological structure. Neither frozen specimens
the entire length range of the species. The (or frozen gonads) nor fish dead for some time can be
ovary from the selected fish should be used (Hunter, 1985).
preserved in the fixative solution of choice
(buffered 4% formaldehyde solution*). The *The fixative should be:
fixative to ovary volume ratio should be high
(4:1). The ovary membrane of big ovaries 100ml formalin (36% formaldehyde)
should be cut (superficially) transversally at 900ml distilled water
intervals to permit penetration of fixative 4g Na H2 PO4 H2O
unless the Weibel method is applied. 6g Na2 HPO 4
Appendix 2. Histology
All the methods discussed in this review involve, Chromatin nuclear. A large nucleus surrounded
at least to some extent, the histological inspection of by a thin layer of cytoplasm. The nucleus contains a
fish ovaries. The purpose of this Appendix is to briefly large nucleolus, and also a series of very small
present the most common procedures (for more peripheral nucleoli. The oocyte is surrounded by a few
detailed descriptions of histological procedures see squamous follicle cells.
Luna (1968), Preece (1972) or any histochemical
handbook). Perinucleolar. Bigger nucleus with several big
peripheral nucleoli. Some vacuoles appear in the
The ovaries collected for histological analyses cytoplasm. The chorion precursor material begins to
should be handled by standard histological procedures: appear in patches.
a cross section of about 57 mm is taken from the
lobule and fixed/preserved in formaldehyde. Vitellogenic oocytes
Cortical Alveoli Stage. This stage is marked by
The dehydration of tissue requires moving the
the appearance of vesicles in the cytoplasm
cross section samples through ascending concentra-
preparatory to yolk development. The presence of
tions of alcohol for a specific duration. Then the
cortical alveoli indicates that oocytes have started
embedding involves transferring the cross section
endogenous vitellogenesis. At this stage there are no
through ascending concentrations of resin/paraffin;
eosinophilic yolk granules present in the cytoplasm.
and finally, the tissue samples are polymerised into
Oil vesicles begin to accumulate in the cytoplasm (if
resin/paraffin blocks. Subsequently, histological
the species presents an oil globule). The chorion and
sections 35 µm thick are cut using a microtome
follicle layers are apparent. The occurrence of this
followed by staining using the various methods as
stage means that the oocyte has started the maturation
described below.
process and, under normal conditions, will develop
Regarding the staining, the standard procedure within the current breeding season.
for Harri's Hematoxylin and Eosin is quick, easy and
the cheapest standard method that most commercial Yolk Granule or Vitellogenic Stage. Developing
laboratories offer. The results of this staining are good yolky oocytes of large diameter with eosinophilic yolk
for identifying the different oocyte classes and the granules present in the cytoplasm. Numerous oil
postovulatory follicles. Other stains as Mason's droplets or vesicles should be present (if the species
Trichrome, Heidenhain's iron hematoxylin, Periodic presents an oil globule) and the nucleus is still central.
acid-Schiff reagent (PAS), PAS-Mallory Trichrome or
Toluidine Blue instead of standard Hematoxylin and Final maturation. The start of the final matura-
Eosin staining also could be applied. tion phase is indicated by the migration of the nucleus
to the animal pole. When the nucleus has completed
Samples analyzed will be scored for presence or its migration, the first meiotic division takes place.
absence of different oocyte stages according to the The hydration phase begins in many species at the
following criteria (maturity ogive) and for prevalence end of the maturation stage, just prior to ovulation;
of atresia. this stage consists of a rapid uptake of fluid by the
oocyte through its follicle (Fulton, 1898). This process
Definitions of Histological Oocyte Stages. is especially pronounced in species which spawn
(following Wallace and Selman description, 1981) (for pelagic eggs and produce hyaline oocytes.
additional information see De Vlaming (1983);
Guraya (1986); Wallace and Selman (1990); Tyler and Post Ovulatory Follicles
Sumpter (1996)).
After ovulation, the structure or follicle that
Previtellogenic oocytes surrounds each oocyte collapses away from the
opening formed for the release of the hydrated oocyte,
Primary Growth. This is the initial phase of and remains in the ovary as an evacuated follicle.
oocyte growth, since the formation of the oocyte from
the germinal vesicles (oogonia). It can be divided in Early Post-Ovulatory Follicles. The structure of
two stages: the follicle is very well maintained. The granulosa
and thecal epithelial layer nuclei are clearly completely resorbed. Atresia has been divided into
distinguished. No signs of deterioration exist and the different stages. The classification of the α -atretic
lumen of the POF is still open and the membranes stage is based mainly on the breakdown of the chorion
still unfolded, i.e., very recently ovulated. and yolk resorption, but other changes also occur. The
follicular layer becomes much more developed and
Late Post-Ovulatory Follicles. The POFs rapidly the chorion appears to move in toward the center of
deteriorate and are resorbed, but still the organization the oocyte (Hunter and Macewicz, 1985; Witthames
of the POF is recognizable. Post-ovulatory follicles and Greer-Walker, 1995). At the end of the α -atretic
are recognized by the presence of rows of nuclei stage, the yolk is completely resorbed and the
arranged along the axis of the degenerating follicular distinction of the degenerating follicle from POF's
membranes. becomes difficult. At later atretic stages the complete
Atresia follicle gets resorbed. To estimate the intensity of
Oocytes under degeneration and oocytes that are oocyte atresia the α -atretic stage is best suited.

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