Witthames et al.: Advances in methods for determining fecundity in marine fishes 
149 
rua and Saborido-Rey, 2003): 1) group synchronous 
determinate total spawners (Atlantic herring [Clu- 
pea harengus], deep water redfish [Sebastes mentella ] 
also known as "beaked redfish [FAO, Fisheries and 
Aquaculture Dept., www.fao.org/fishery/statistics/ 
programme/3.1.1. Accessed Jan., 2009], and golden 
redfish [Sebastes marinus ]); 2) group synchronous de- 
terminate batch spawners (Atlantic cod [Gadus morhua ] 
and European plaice [Pleuronectes platessa ]); and 3) 
asynchronous types (European hake [Merluccius mer- 
luccis\ and Atlantic mackerel [Scomber scombrus ]) that 
may not be determinate (Greer-Walker et al., 1994). 
Fecundity in the first two groups included all follicles 
in the advanced mode to the right of a gap in the fol- 
licular size frequency (Hunter et al., 1992) whereas in 
the latter case the follicular distribution is continuous. 
Although fecundity may be enhanced during maturation 
in asynchronous spawning types (indeterminate spawn- 
ing strategy), it is of practical and theoretical value to 
study fecundity proliferation whatever classification is 
applied to the spawning process. 
Recent work has shown that not all the fecundity 
develops into eggs (realized fecundity) and follicular 
atresia may account for a substantial part of the fe- 
cundity in a process referred to as down regulation (see 
reviews Murua et al., 2003; Thorsen et al., 2006; Kjesbu 
and Witthames, 2007). In addition, it is also important 
to differentiate whether an individual female has en- 
tered the spawning cycle, thus reducing her fecundity, 
and how long a POF persists to indicate a previous 
spawning event (Hunter and Macewicz, 1985a). The lat- 
ter information is used to assess whether a female still 
contains her full complement of oocytes for application 
of the annual egg production method to assess spawn- 
ing stock biomass applied to fish with a determinate 
spawning strategy (Armstrong et al., 2001). 
To date no single approach has been successful in 
quantifying follicular stages associated with fecundity 
development and regression and each has one or more 
disadvantages. Fish that are very fecund, perhaps con- 
taining ovaries weighing more than a kilogram and 
with millions of follicles, will have to be subsampled for 
fecundity estimation. In this case, quantitative histolog- 
ical methods (Emerson et al., 1990) requiring sections of 
the whole ovary are not feasible — meaning only relative 
proportions of each follicular class can be measured (An- 
dersen, 2003). This approach, however, needs additional 
information on the fecundity count preferably coupled 
with measurement of follicular size frequency to exclude 
smaller PVFs that are not committed to maturation in 
the current reproductive year. Although it is feasible to 
release follicles by digesting the ovary in strong acid 
solutions (either Gilson’s fluid (Simpson, 1951) or a less 
toxic nitric acid formulation (Friedland et al., 2005)), 
such media have several adverse consequences. These 
consequences include 1) considerable follicular shrink- 
age (Witthames and Greer-Walker, 1987), 2) likely loss 
of atretic follicles and POFs (Klibansky and Juanes, 
2007), and 3) incompatibility with histological methods 
(Hunter and Macewicz, 2003). In view of the need to 
identify fecundity based on follicular size, there is a 
need to measure large numbers of follicles greater than 
a specified lower size limit even if the ovary is subsam- 
pled using the gravimetric method (Bagenal and Braum, 
1968). Manual measurement of follicular size frequency, 
even using video technology, is just too demanding on 
manual labor unless there is some way of automating 
the collection of data. Although an automatic particle 
analyzer can provide such data (Witthames and Greer- 
Walker, 1987), the method requires large quantities of 
Gilson’s fluid and is subject to all the problems listed 
above. More recently image analysis methods have been 
adopted to automate collection of size frequency data 
in Atlantic cod ( Gadus morhua) (Thorsen and Kjesbu, 
2001; Klibansky and Juanes, 2008). In each case the 
mean fecundity (the independent variable) can be used 
to estimate the number of follicles per gram (g) of ovary 
by fitting a power relationship based on a calibration 
from a data set containing the two variables (the auto- 
diametric method). Fecundity is then determined by 
raising the number of follicles per g of ovary by the 
ovarian weight. Although an alternative image analysis 
method applied to American shad ( Alosa sapidissima) 
(Friedland et al., 2005) has advantages as a cost ef- 
fective method to estimate fecundity, it also has two 
significant drawbacks: 1) relatively low resolution, and, 
2) it uses acid hydrolysis to separate follicles. Thus, the 
autodiametric method has more general utility because 
it uses neutral buffered formaldehyde solution (NBF) to 
fix tissue that is fully compatible with histology. Also 
Hunter et al. (1992) studying Dover sole ( Microstomas 
pacificus) and Oskarsson et al. (2002) studying Atlantic 
herring ( Clupea harengus ) have shown it possible to 
identify atretic follicles in NBF-fixed dispersed ovarian 
samples (whole mounts) suggesting it might be pos- 
sible to also estimate numbers of different follicular 
classes. 
Accordingly, our first objective is to report on the 
utility and precision of the autodiametric method to 
determine fecundity in several species including At- 
lantic cod, European hake, Atlantic herring, Atlantic 
mackerel, redfish species (deep water redfish and golden 
redfish), and European plaice. To emphasize the utility 
of the method several laboratories 1) AZTI [A] (Pasaia, 
Spain), 2) Cefas [B]( Lowestoft, UK), 3) CSIC [C] (Vigo, 
Spain), and 4) IMR [D] (Bergen, Norway) used different 
configurations of image analysis equipment. In order to 
complete this work, four other objectives were identi- 
fied linked to the application of fecundity determina- 
tion using Atlantic cod as the main example and to a 
lesser extent European hake: 1) ovarian sampling, and 
follicular homogeneity, 2) evaluate three stains (eosin, 
rose bengal, and periodic acid-schiff [PAS]) to improve 
the accuracy of follicular size measurement and count- 
ing in relation to the autodiametric method, 3) compare 
interpretation of NBF-fixed whole mounts with respect 
to histology to assess maturity, spawning status and 
quantify the standing stock of atretic follicles, and 4) 
consideration was also given to the effect of ovarian 
maturation on down regulation of fecundity in Atlantic 
